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27% of pregnant women are affected from preeclampsia (pe) which occurred in the second half of pregnancy and is defined mainly by the symptoms hypertension and proteinuria. 58% of these women develop hellp syndrome (hemolysis, elevated liver enzymes, and low platelet count). preeclampsia is one of the leading causes of maternal and fetal mortality worldwide and a main cause of preterm labour. women with a history of preeclampsia are at elevated risk for cardiovascular diseases later in life. to reduce morbidity and mortality resulting from this disease life - style changes and prevention the early detection for a high risk to develop preeclampsia has the potential to be a predictive tool also for other health disorders with meaningful consequences for the mothers, their offspring, and health care systems. the placenta plays a key role in the pathogenesis of preeclampsia since the symptoms of pe can occur in molar pregnancies which lack a fetus and the disease disappeared once the placenta is delivered. it is meanwhile well accepted that undernutrition results in intrauterine growth restriction which seems to program coronary heart disease and hypertension later in life [35 ]. strikingly the human placenta is only a transient organ, but its effect on the offspring is conserved throughout life. appropriate function of the placenta requires the correct differentiation of the trophectoderm to establish a nutrition route between embryo and mother. despite many years of research, a complete understanding of the molecular pathogenesis of pe is still missing. the current theory of the pathogenesis of pe as reviewed by christopher redman and ian sargent is thought to occur as a 2-stage process with poor placentation in the first half of pregnancy resulting in the maternal response in the second half of pregnancy [7, 8 ]. anatomic placental examination reveals that the basal plate is most affected by this disease, the site where cytotrophoblast (ctb) invasion occurs. in pe, interstitial ctb invasion and endovascular invasion the second stage of pe is thought to be the maternal response to abnormal placentation resulting from endothelial dysfunction and an imbalance in circulating angiogenic / vasculogenic factors such as soluble vascular endothelial growth factor receptor-1 (vegfr-1, sflt-1), placental growth factor (plgf), and the transforming growth factor - beta receptor endoglin (cd105) (reviewed by [9, 11 ]). in 2011 the role of angiogenic proteins in developing preeclampsia there is mounting evidence that a nonphysiological hypoxic environment later in pregnancy may result in this deregulation of angiogenic factors at the maternal - fetal interface. recently it has been shown that early preeclampsia is associated with abnormalities in oxygen sensing since early preeclamptic placentas are unable to regulate hif1- (hypoxia - inducible factor 1-) alpha levels. chronic exposure to nonphysiological oxygen levels in preeclampsia decreases vegf (vascular endothelial growth factor) whereas sflt-1 is highly upregulated. it is well known that secreted sflt-1 binds to vegf and plgf with high affinity and thereby decreasing their ability to bind to their receptors. these changes act like an antiangiogenic therapy which has been shown in clinical trials leading to similar clinical symptoms such as impaired angiogenesis especially maturation of vessels, hypertension, proteinuria and edema [14, 15 ]. verlohren. reported that the sflt-1/plgf ratio is important to identify women at risk for delivery and is a reliable tool to discriminate between different types of pregnancy - related hypertensive disorders. in women with suspected preeclampsia at 35 kg / m) and elevated diastolic blood pressure > 80 mm hg. further risk factors are positive family history of preeclampsia, multiple pregnancy, pregnant women over 40 years, preexisting renal disease, and clotting disorders [30, 31 ]. particularly common clotting disorders associated with an increased risk for preeclampsia are factor v leiden mutation, homozygous mthfr mutation, hyperhomocysteinemia, presence of antiphospholipid antibodies, and the combination of multiple thrombophilias. immunological causes can be attributed to the increased risk, for example, the first pregnancy with a partner. in contrast, multiparity with the same partner reduces the risk. only regarding history, 30% of women with pe are detected early with a false positive rate of 5%. regarding the pregnancy - induced hypertension without preeclampsia, the maternal history is of a much greater importance than the serum parameters and the pulsatility index of the uterine arteries. mean arterial blood pressure in the first trimester can be used in combination with maternal risk factors as a predictive marker of pe in the first trimester which has a detection rate of 76% for early - onset pe. systolic blood pressure is already significantly different in the first trimester in view of the early- and late - onset pe and pregnancy - induced hypertension. the arterial supply to the uterus occurs mostly through uterine arteries, which turn into circular running arteriae arcuatae. here radial arteries branches, the spiral arteries, penetrate deeply into the myometrium and supply the decidua and fetus during pregnancy. abnormal placentation and incomplete cytotrophoblast invasion characterized by inadequate formation and vasodilation of the spiral arteries have long been known as one of the main risk factors for development of preeclampsia [36, 37 ]. based on these morphological changes, an abnormal uteroplacental circulation is typically characterized by a persistence of the postsystolic (notch) and high resistance indices. a prediction of the severe form of pregnancy - induced hypertension and preeclampsia is possible by examining the uteroplacental vessels in the first and second trimesters. various publications showed that in first trimester screening, doppler examination of the uterine arteries identified a certain percentage of pregnant women that later develop preeclampsia with elevated uterine resistance indices and postsystolic incisions [3840 ]. about 40% of pregnant women can thus be detected at a false - positive rate of 5% [34, 41 ]. however, the sensitivity for the prediction of preeclampsia is significantly lower than that in second - trimester ultrasound measurements. higher rates of sensitivity regarding the discovery of a late onset preeclampsia can be achieved in the second trimester of pregnancy. several doppler studies in second trimester yielded detection rates of 7080% [42, 43 ]. the problem of the doppler examination alone, however, lies in the low predictive value. only in combination with biochemical markers, the combination of doppler sonography and angiogenic factors such as plgf / sendoglin (seng) and sflt-1 is a valid prediction of preeclampsia. in order to intervene preventively, papp - a was first identified as a predictive marker (see below,). further promising targets for first trimester screening are pp-13, soluble endoglin, inhibin a, activin a, pentraxin 3, p - selectin, igfbp-1 and 3, adiponectin, resistin, l - arginine, asymmetric dimethylarginine (adma), and homoarginine. the aim of scientific papers on the subject of preeclampsia is to develop a test that can predict preeclampsia in the first trimester of pregnancy and can be applied in clinical routine. in the following section, plgf belongs to the vegf family, is secreted by trophoblast cells, and has proangiogenic function. preeclampsia occurs due to an impaired placentation with subsequent ischemia resulting in an increased secretion of antiangiogenic factors such as sflt-1 (soluble fms - like tyrosine kinase-1) and seng (soluble endoglin) in the maternal circulation. pigf was in an early focus of the research groups in the search for a suitable prediction factor. it turned out that the concentration of plgf in a preeclamptic pregnancy did not increase to the extent as would be expected in a normal pregnancy, as shown by us [46, 47 ]. others could show that in first trimester, there are already significant differences between plgf concentrations in maternal blood of pregnant women with normal pregnancy and those that develop preeclampsia during pregnancy [34, 4850 ]. since 2011, the first conventional test of the company alere allows the quantitative detection of pigf in anticoagulated edta plasma in the first trimester with fluorescence immunoassay (sensitivity and specificity 95%). the detection rate of preeclampsia using plgf alone for the early - onset preeclampsia is between 41% and 59% and for late - onset preeclampsia 33%. research on anti - angiogenesis factors such as sflt-1 failed to convince as the exclusive marker for the prediction of preeclampsia in the first trimester. verlohren. showed that the combination of angiogenesis and antiangiogenesis factors, at least in the second and third trimesters, may offer the possibility of a risk classification by an sflt / plgf ratio. it was found that patients with preeclampsia had a significantly increased sflt / plgf ratio compared to patients with a normal pregnancy. papp - a (pregnancy - associated plasma protein a), an insulin - like growth factor binding protein protease, is secreted by the syncytiotrophoblast. as part of the first - trimester screening we could show that patients with decreased levels of papp - a in maternal blood during the first trimester develop preeclampsia, especially an early - onset preeclampsia as revealed also by others [34, 53, 54 ]. several studies exhibited that both inhibin a and activin a are increased in the first trimester in maternal blood of patients who later develop preeclampsia compared to pregnant women with normal pregnancies [55, 56 ]. however, no association is found between impaired trophoblast invasion and subsequent endothelial dysfunction and increased concentration of activin a. impaired placentation in the presence of preeclampsia, there is an increased secretion of pp13 in the first trimester of pregnancy [5761 ]. pentraxin 3 is a secreted protein as part of an inflammatory immune response and is increased as an acute phase protein molecule. both with manifestations of pe as well before clinical symptoms, there is an increased secretion of ptx 3 in the maternal circulation [54, 63, 64 ]. as a cell adhesion molecule, p - selectin plays a role in endothelial dysfunction. the consequence of placental ischemia in the context of preeclampsia is endothelial dysfunction and thus increased secretion of p - selectin. this is already detectable in the first trimester of pregnancy [54, 63, 64 ]. both, in early- and late- onset preeclampsia, igfbp-1 is decreased in the first trimester. such changes are detected by secretion of igfbp-3 only in late - onset preeclampsia. in both cases, there is no correlation to a disturbed trophoblast invasion [66, 67 ]. in the case of early - onset pe, resistin levels in the first trimester are higher in patients who develop preeclampsia than controls. l - arginine and l - homoarginine are increased in the first trimester at later - developing early - onset preeclampsia, as well as the ratio of adma /l - arginine and adma /l - homoarginine. this is not the case for late - onset preeclampsia and for the isolated analysis of adma. meanwhile it is clear that a single diagnostic marker does not have the strength to safely predict subsequent preeclampsia. for this reason, it seems to be promising to use history, biophysical, and several biochemical parameters to ensure the best possible detection rate achieved. finally, one must distinguish between early- and late- onset preeclampsia in order to classify the present results correctly. the early - onset preeclampsia is defined as the onset before 34 weeks of pregnancy, the intermediate - onset preeclampsia between the 34 and 37 weeks and the late - onset preeclampsia after 37 weeks. the late - onset pe seems to follow a different pathogenetic mechanism, since the serum parameters differ significantly as a marker of disturbed placentation in terms of predictive power. the placentation disorder, according to previous data, is a feature of early preeclampsia. the addition of biochemical markers in the first trimester is therefore particularly suitable for detection of early preeclampsia. poon. pioneered the evaluation of a few serum parameters and maternal factors in order to achieve a good predictive power of early preeclampsia. the detection rate of early - onset pe is 93.1% in the first trimester by an algorithms from maternal risk factors, mean arterial blood pressure, pulsatility index of the uterine arteries, papp - a, and plgf. the detection rate for the late - onset pe with an appropriate algorithm is 44.9%. akolekar. in 2011 found that the detection rate of preeclampsia in the first trimester by a combination of several markers (pigf, papp - a, pp13, inhibin a, activin a, sendoglin, ptx3, p - selectin, blood pressure, dopplersonography, and history) is increased significantly to a detection rate of 91% at a fixed 5% false - positive rate for early - onset pe, 79.4% for intermediate - onset pe (34th37th weeks of gestation), and 60% for late - onset pe. the addition of these parameters allows a better predictive power of all forms of preeclampsia compared to the above - described relatively simple algorithm, having particular effect on a high detection rate for early - onset preeclampsia. further studies are expected, that show which of the biochemical markers are really useful in clinical practice. finally, the question arises that how far it may succeed in establishing the first - trimester screening tests with the consecutive possible prevention by aspirin and/or low - molecular - weight heparin, as a screening in a large, unselected collective. since prevention is simple and inexpensive, the obstacle is much more on a personal and cost intensive screening tool. the investigation regarding chromosome abnormalities will depend on the basis of the consequences of abnormal test results of many factors and is always carried out only in a preselected collective. screening for preeclampsia should be for a much larger collective of pregnant women, not at least because of the higher risk to get preeclampsia as a chromosomal abnormal baby and the ease of prophylaxis. another important reason for early preeclampsia risk calculation is the fact that women with preeclampsia have a higher life - time risk for getting cardiovascular disease. better observation of this collective of patients, changing of life - style factors, and health education could be an important step to reduce morbidity and mortality according to cardiovascular problems worldwide. not only the mother, also the offspring bear the consequences of preeclamptic pregnancy with mostly intrauterine growth restriction like elevated risk for cardiovascular diseases and behavioural disorders, for example. it would be desirable in the future to integrate preeclampsia risk calculation to the regular prenatal care in first trimester. further studies on large collectives have to determine to what extent the false - positive and false - negative findings can lead in relation to health and economic disadvantages. even an early screening should not replace careful pregnancy monitoring. finally, pregnancy is not only a short time in a woman 's life with the aim to deliver a baby but it is also an important time giving insights in a women 's health status. as we already know pregnancy may positively influence women 's health future as could be shown by studies which detected a reduced risk of developing breast cancer after pregnancy. as an indicator of risk factors, pregnancy is not only the beginning of taking care for a family, but also for a better self - care. | preeclampsia is one of the leading causes of maternal and fetal morbidity and mortality. new molecular insights offer new possibilities of early diagnosis of elevated maternal risk. maternal risk factors, biophysical parameters like doppler examination of the uterine arteries and biochemical parameters allow early risk calculation. preventive and effective therapeutic agents like acetylsalicylacid can be started in the early second trimester. this article reviews the diagnostic possibilities of early risk calculation to detect women having high risk for preeclampsia and the potential benefits for them, the offspring and health care systems. we provide risk calculation for preeclampsia as an important and sensible part of first trimester screening. |
after its composition was firstly described by billroth1 in 1856, tcherkoff and sedlis2 reported lesions of the same type in the uterine cervix. later baggish and woodruff3 recognized it as a distinct type of cervical neoplasm from adenoid cystic carcinoma. the incidence of adenocarcinoma of the uterine cervix is reported to account for less than 1%. although the origin is debatable, it is considered derived from multipotential cells of the basal layer or reserve cells of cervical epithelium. clinically, adenoid basal carcinoma is differentiated from other types of cancer for the rare metastasis and the excellent prognosis.. twenty - two - year - old young female with adenoid basal carcinoma of the uterine cervix have been rarely reported in the literature. in korea where carcinoma of uterine cervix is one of the most common malignancy, adenoid basal carcinoma of the uterine cervix is considered relatively rare. the patient was a 22-year - old korean woman who presented with a history of abnormal genital bleeding for 3 weeks. no gross lesion was noted on the cervix and the pap smear was reported to be high - grade squamous intraepithelial lesion (hsil). the serum level of the tumor marker carbohydrate antigen (ca) 125 was elevated (48 u / ml ; normal < 35 u / ml), whereas the serum levels of carcinoembryonic antigen (cea), ca 19 - 9, a - fetoprotein and squamous cell carcinoma antigen were within the normal ranges. concerned with the patient 's hsil results, the clinician performed a cervical loop electrosurgical excision procedure (leep) and observed multiple erosions of inflammation overlying the cervix. the clinician was able to discover hsil with superficial glandular extension and report adenoid basal carcinoma. the patient is being closely followed up and has shown no evidence of recurrence within 24 months after the operation. macroscopically, the tumor size of the lesion was 1.0 cm in the largest dimension. microscopically, tumor cells were arranged in small nests or cords, with focal squamous differentiation, however, cystic change was not noted. the adenoid basal carcinoma was adjacent to the hsil lesion, but no transition between the two lesions was observed. stains for p16 and ki-67 showed positive staining, whereas staining for cytokera - were negative (fig. cervical cancer is the second most common cancer among women worldwide and is one of leading causes of death by cancer in women.4 it is generally considered that adenoid basal carcinoma of the cervix is a rare lesion which occurs mostly among postmenopausal african - american women. however, recently there have been reports that the tumors can also occur in asian women. in korea, there were four cases reported of adenoid basal carcinomas of the cervix. the rare form of mucinous adenocarcinoma of the cervix, adenoma malignum, requires differential diagnosis. especially because it is histologically and radiologically similar to the benign form and often causes confusion upon diagnosis.5 adenoid basal carcinoma is located below the epithelium. with naked eyes, it is observed as normal cervix without clear lesion. histologically, it is composed and proliferates in the form of nests of small round cells. the cells are characterized by relatively a large dense nucleus and the light cytoplasm (fig. the most important differential diagnosis is adenoid cystic carcinoma because of the local invasion and remote metastasis. as its name suggests, the histological aspects of the two tumors include basaloid cell proliferation, squamous and granular differentiated filament. brainard and hart7 proposed the use of the term basal cell epithelioma as adenoid basal carcinoma with typical histological structure is not malignant. we summarize that both adenoid basal carcinoma and adenoid cystic carcinoma originate from the reserve cells in the uterine cervix. the tumor is often observed in salivary glands, sometimes in respiratory organ, skin, head and neck mucosa, and breast. it is found rarely in the female genital organs, if found, mostly in the cervix, bartholin 's gland, and endometrium. a common symptom of adenoid cystic carcinoma of the uterine cervix is postmenopausal menorrhagia. in many cases more than half of the patients are diagnosed with clinical stage i with unfavorable prognosis. in contrast to adenoid basal carcinoma, adenoid cystic carcinoma appears as a polyp at the cervix. histologically, it shows an increase in cell size, the number of cell colonies, the number of mitotic cells, and organic reaction.8~10 immunohistochemically, adenoid basal carcinoma of the uterine cervix typically shows positive staining for ki-67 and p16. grayson.11 observed that immunohistochemical analysis of the adenoid cystic carcinoma revealed positive staining for epithelial membrane antigen (ema), collagen iv and laminin, while adenoid basal carcinoma revealed positive staining for ema and negative staining for collagen iv and laminin. ferry and scully6 reported one patient, a 67-year - old female, who died in 3 months as a result of metastatic lung cancer from adenoid basal carcinoma. it is treated with hysterectomy, chemotherapy, and radiation therapy. in conclusion, for treatment and clinical management of patients, it is important to understand adenoid basal carcinoma differently from other kinds of uterine cervix cancer. it is also critical to distinguish adenoid basal carcinoma of low metastatic potential and favorable prognosis from adenoid cystic carcinoma of similar shapes and unfavorable prognosis. in this paper, we have discussed a pimipara young woman under close observation after conization. for young female cases have been rarely reported, the case provides a clinical insight into diagnosis of adenoid basal carcinoma. | adenoid basal carcinoma of the uterine cervix is uncommon neoplasia mostly occurring in postmenopausal women. it has excellent prognosis and a favorable clinical course. in addition, adenoid basal carcinoma is differentiated from adenoid cystic carcinoma by histologic and cellular morphologies, and immunohistochemistry. in this paper, we present the case of a 22 year old korean female. she initially had a high - grade squamous intraepithelial lesion (hsil) on pap smear and a subsequent cervical loop electrosurgical excision procedure (leep) specimen revealing adenoid basal carcinoma. the lesion showed the histologic characteristics of adenoid basal carcinoma. because of the lesion 's low potential for recurrence and metastasis, the young primipara had a conization procedure performed and has been under close observation. |
nicotine is a chemical that is present in the mount of about 1% of the weight of cigarette tobacco. nicotine is the main active ingredient of tobacco and is also the main factor that leads to smoking addiction or dependence producing. cotinine, a major degradation product of nicotine metabolism, has been an important recognized specific biomarker for evaluating cigarette smoke exposure. urine specimens are commonly employed in biological monitoring because urine collection is noninvasive and poses minimal infectious disease risk to participants and researchers. continuous and complete 24 h urine collection yields more accurate results, because spot urine sampling may not provide a valid overview of the entire toxicant exposure profile. urine sample integrity and completeness is essential to exposure assessment research, and absence of compliance with the collection protocol is a fundamental concern to the researcher. because creatinine is excreted in urine at a relatively constant rate through glomerular filtration, its measurement is an evaluation of sample integrity and completeness [6, 7 ]. in addition, urinary creatinine is commonly used in a ratio format to normalize analyte quantification for specimen concentration [8, 9 ]. the normalization process involves dividing the concentration of the analyte of interest by the creatinine concentration obtained in the same urine sample, with the result reported as the concentration of target analyte per millimol of creatinine. recently, as a normalization basis, urinary creatinine was used to consider the excretion of a variety of xenobiotics related to smoking, ranging from cotinine to mercapturic acids. there are numerous papers published about the determination of creatinine in human fluids, including the jaffe method [11, 12 ], enzymatic method, flow injection analysis, high - performance liquid chromatography [1522 ], capillary electrophoretic [2325 ], zone electrophoresis, gas chromatography - mass spectrometry, or liquid chromatography combined with mass spectrometry (lc - ms - ms) [2830 ]. recent determination of creatinine, delta - aminolevulinate, and tyrosine in biological fluids with a direct injection by lc - ms - ms was performed, and isotope dilution tandem mass spectrometry was used to assess the accuracy of creatinine determination in serum, plasma, or mouse plasma [28, 29, 3133 ]. developed a rapid method for the analysis of creatinine in urine by solid - phase extraction ms - ms. though a time - consuming solid - phase extraction was used for sample preparations, the selectivity of tandem mass spectrometry can not eliminate all interferences in urine. another modified lc - ms - ms method was introduced by park. which allowed direct analysing creatinine in 24 h urine after diluted with methanol. however, the analyte was eluted from the column at 0.59 min which can not be separated from the water dissolved urinary proteins and macromolecules (retention time < 1 min on c18 column). the methods based on the color reactions and enzymatic assay are confined by the lack of selectivity. the round robin results revealed considerable and unsatisfying variations between laboratories and methods. in order to eliminate the interferences from different instruments for urinary creatinine and use urinary creatinine to normalize smoking related biomarkers in human biological fluids, a sensitive and selective lc - ms - ms method for determining creatinine in urine was developed and verified with enzymatic colorimetric assay. the data was applied to adjust cotinine values and was undertaken to explore whether any improvement occurred in the concordance with tar to relate it to tobacco exposure. creatinine was obtained from the united states pharmacopeial convention (rockville, md, usa). creatinine - d3 (n - methyl - d3 ; purity : 98% ; isotopic purity : 99%, toronto research chemicals inc., primary stock solutions of creatinine and creatinine - d3 for the preparation of standard and quality control (qc) samples, were prepared by weighing separately. the primary stock solutions (0.21 and 0.1 mg / ml) of the creatinine and creatinine - d3, respectively, prepared in water and stored at 80c were found to be stable for three months (data not shown). appropriate dilutions were made in water to produce the working stock solutions of 100, 1,000, and 10,000 ng / ml for creatinine for the preparation of calibration curve. calibrators (1, 2, 5, 10, 20, 50, 200, 500, and 2,000 ng / ml) were freshly prepared by the addition of different aliquots of the working stock solution of the analytes and 25 ng / ml of creatinine - d3 to water. quality control samples for creatinine at three different concentrations (50, 200, and 400 ng / ml) were also prepared with human urine. frozen urine samples were thawed to room temperature and mixed to suspend any settled precipitate. a 10 l formic acid was added to 1 ml aliquot of human urine sample, stirred, and centrifuged at 10000 rpm for 10 min. the mixture was filtered through a 0.22 m polyethersulfone membrane and a 5 l urine aliquot was transferred to an amber volumetric flask and brought to a total volume of 10 ml with water after being spiked with 100 l of creatinine - d3 internal standard solution (1 g / ml). a 5 l aliquot was injected on - column for lc - ms - ms. the enzymatic colorimetric method was performed in a hitachi modular automatic analyzer (roche). enzymatic method is based on the enzymatic degradation of creatinine and its reaction products by creatininase, creatinase, and sarcosine oxidase. all samples were analyzed using an agilent 1200 liquid chromatograph (agilent technologies, wilmington, de, usa) coupled with an api 4000 triple quadruple mass spectrometer equipped with a turboionspray source (applied biosystems, foster city, ca, usa). esi was performed in the positive ion mode (ionspray voltage 4500 v) with nitrogen as nebulizing (gas 1), heater (gas 2), curtain, and collision gas. gas flow parameters were optimized (nebulizer 40 psi, heater 40 psi and curtain gas 30 psi) by making successive flow injections while introducing mobile phase into the ionization source at 200 l / min. the declustering potential (73 v), entrance potential (10 ev), collision energy (29 v), and cell exit potential (8 v) were optimized for creatinine by integrated springe pump at a constant flow rate of 10 l / min. the turbo ion spray temperature was set at 480c. quantitative analysis was performed in the multiple reaction monitoring (mrm) mode with a dwell time of 100 ms. an agilent zorbax eclipse xdb - c18 column (2.1 150 mm, 3.5 m particle size, agilent technologies, wilmington, de, usa) was used with a flow rate of 200 l / min at ambient temperature. isocratic separation was performed with 50% solvent a (0.1% formic acid in water) and 50% solvent b (0.1% formic acid in acetonitrile). solvents were filtered through a 0.45 m membrane and degassed by a vacuum before use. aliquots (5 l) of the standard or diluted urine samples containing internal standard were injected onto the lc - ms - ms system. total urinary cotinine among smokers and nonsmokers was analyzed according to a previously published lc - ms - ms method. several performance parameters were tested to validate the proposed method according to food and drug administration (fda) guidelines for bioanalytical methods. these were linearity of calibration plots, goodness of fit of calibration plots to the linear regression model, specificity, selectivity, thawing stability, recovery, matrix effects, and precision. 246 24 h - urine samples from 82 smokers in three separate days and 57 blank 24 h - urine samples of nonsmokers were obtained at baseline from ongoing studies (urinary biomarkers related to smoke exposure) in the institute of clinical pharmacology of zhengzhou university. during the initial course of method development and validation, several different lc columns and relevant solvent systems were evaluated for the best chromatographic separations of analytes from background interference. poor peak shapes were observed on most columns, except those with xdb c18 column. the initial mobile phase was chosen as water and methonol (v / v), while poor peak shapes were also observed. in order to get good peak shapes and separation, solvent a (0.1% ammonium acetate in water) and solvent the detection parameters of ms were optimized using a syringe pump at a flow rate of 10 l / min. an esi mass spectrum of creatinine was shown in figure 1. under the conditions of esi, the protonated molecules ([m + h ]) of creatinine and creatinine - d3 collision - induced dissociation of both compounds yielded one major fragment ion at m / z 86 for creatinine and m / z 89 for creatinine - d3, respectively, corresponding to the neutral loss of co [m + h co ]. at the same time, the fragment ion [m + h co]could yield another main product ion at m / z 44 for creatinine and m / z 47 for creatinine - d3 (figure 1). for each analyte these ion pairs are 114/86 and 114/44 for creatinine for the confirmation and quantification and 117/47 for creatinine - d3. no significant interfering peaks from endogenous compounds were observed at the retention times of creatinine and creatinine - d3. the retention time of creatinine and creatinine - d3 was 1.4 and 1.3 min, respectively. a typical mrm chromatogram of creatinine - d3 and creatinine dissolved in water was presented (a1), (a2), and (a3) in figure 2. both compounds were detected in a diluted urine sample ((b1), (b2), and (b3) in figure 2). urine matrix underwent a thaw / refreeze cycle during each validation experiment, accumulating six such cycles by the end of the validation. the average values over the six validation experiments for the concentrations of creatinine were 10.09 0.31 mmol / l (cv = 2.1%). the cvs were of similar magnitude to interday cvs, indicating that fast thawing of urine in chilled water did not influence the concentration of analytes. the effect of urine constituents over the ionization of analytes and is was determined by comparing the responses of the postextracted urine standard qc samples (n = 6) with the response of analytes from neat samples at equivalent concentrations. matrix effect was determined at same concentration of analyte and is as in recovery experiment. reported that pretreatment of urine with solid - phase extraction was not a necessary step for urinary creatinine measurement and that simple dilution of urine without pretreatment provided high selectivity for creatinine. on account of this, a simple dilution with water and methanol was applied in this study. as shown in figure 3, urine samples dilution with water could get clear chromatograms of creatinine. the signal - to - noise ratio in urine diluted with water is higher than with methanol for the narrow creatinine chromatogram peek and low noise. creatinine was easily detected in all urinary specimens and it also could be better analyzed when the urine samples were diluted to 2000-fold in our assay. in addition, applying a diluted urine sample to the lc - ms - ms could reduce the impact of the matrix effects and thus allow more samples to be processed before cleaning. analytical recovery rates were obtained by spiking a nonsmoker pool urine sample with three concentrations of creatinine (50, 200, and 400 ng / ml). recoveries ranged from 98.6 to 106.0% (n = 6) (table 1). the peak - area ratios of creatinine to is were plotted versus creatinine concentration to construct calibration curves. the calibration curve created using creatinine dissolved in water was linear (y = 0.0106x + 0.00615, r = 0.9995) in the analytical range from 1 to 2000 ng / ml. lod and loq were determined based on the instrument response with the integrated function of the analyst 1.5 software (applied biosystems). these calculations were based on signal / noise ratios of 3 and 10 for lod and loq, respectively. the corresponding concentrations were calculated from the ratio to the internal standard area on the calibration curve. the lod and loq for creatinine dissolved in water were 0.30 and 0.99 ng / ml. the intraday precision (rsd) of the methods was established by replicate analyses (n = 10) of samples containing low, medium, and high concentration of creatinine. the interday precision (rsd) was established by replicate analyses of the same samples on 10 separate days. intra - day and interday precisions determined were 1.01.8% and 1.52.9%, respectively (table 2). published hplc method, the presence of protein in the injected samples can cause modification of the column end in biased analytical results. thus, extensive sample cleanups including liquid - liquid (l - l) extraction and spe were needed to get low sample matrix effects and good hplc separation for the target compound. however, solid - phase extraction was not a necessary step for urinary creatinine measurement and that simple dilution of the urine sample without pretreatment provided high selectivity for creatinine. in this experiment, a simple dilution with water was used after acid precipitation, centrifugation, and filtration. the creatinine concentration could be well determined and the matrix effect could be extraordinaire light after diluting 2000-fold with water. compared with published lcms / ms methods, the new methods required fewer samples (5 l compared to 50 l urine) thanks to the lower limit of detection (0.3 ng / ml compared to 22.8 ng / ml, 1 ng / ml, 6 ng / ml, and 3.7 ng / ml). in addition, with the lower limit of detection, this method can be used to measure creatinine concentrations as low as 1 ng / ml. to check the effectiveness of lc - ms - ms, 28 24 h - urine samples (16 smokers and 12 nonsmokers) were measured by using the lc - ms - ms with a simple one - step dilution and the enzymatic colorimetric method (for details for enzymatic colorimetric method see table 1 in supplementary material available at doi:10.1155/2012/245415) for the same set of urine samples run. the creatinine values measured by the colorimetric and lc - ms - ms methods were positively associated (pearson r = 0.984, r = 0.968, p < 0.0001, figure 4) (for original data see table 1 in supplementary material). however, lc - ms - ms has advantage of low detection limits and high selectivity compared with enzymatic colorimetric method. data from the same instrument could provide more accurate and stable results for creatinine normalization technique. cotinine, a major metabolite of nicotine, is the most appropriate parameter to evaluate tobacco exposure and smoking status due to its higher stability and half life when compared to nicotine [9, 39 ]. urinary creatinine and cotinine concentrations were determined in 24 h - urine samples (n = 246) from 82 smokers and 24 h - urine samples (n = 57) from 57 nonsmokers (lc - ms - ms method for the determination of cotinine see supplementary material). the normalization process involves dividing the concentration of cotinine by the creatinine concentration obtained in the same urine sample, and the result was expressed as the concentration of cotinine per millimol of creatinine. the difference between creatinine normalized and nonnormalized cotinine values and the correlation of total cotinine in 24 h - urine and cotinine creatinine ratio was evaluated. the normalized cotinine values are statistically significantly correlated with nonnormalized concentration (pearson r = 0.858, p < 0.0001). total cotinine in 24 h - urine and cotinine creatinine ratio were also positively associated (pearson r = 0.942, p < 0.0001) (original data see tables 25 in supplementary materia). 24 h urine cotinine as a means of assessing exposure to xenobiotics is considered the gold standard, which presumably represents the best information on urinary cotinine excretion. 24 h urinary cotinine was positively correlated with tar as in figure 5(a) (table 3). adjusting cotinine values was undertaken to explore whether any improvement occurred in the concordance with tar, and the result was showed in figure 5(b) (table 3). it is obvious that the corresponding bubbles in figure 5(a) were concentrated more than that in figure 5(b), except 8 mg : 10 mg. there was no use in improving the concordance, except 8 mg : 10 mg (0.716 versus 0.722). a simple and specific method was developed for the determination of urinary creatinine by the lc - ms - ms. the sample preparation only involves centrifugation and filtration of diluted urine, which not only allows a high sample throughput but also reduces creatinine background noise and urine salts concentration. in addition, urinary creatinine was used in a ratio format to normalized cotinine concentration. the normalized cotinine values are statistically significantly correlated with total cotinine in 24 h - urine and cotinine creatinine ratio were also positively associated. because cotinine : creatinine ratio varied significantly across smoking groups for the difference of individual, 24 h - urinary cotinine was more appropriate for expressing correlation with tar than cotinine : creatinine ratio. | a simple and sensitive high performance liquid chromatography - tandem mass spectrometry (hplc - esi - ms - ms) method was developed and validated for the quantification of creatinine in human urine. the analysis was carried out on an agilent zorbax eclipse xdb - c18 column (2.1 150 mm, 3.5 m). the mobile phase was 0.1% formic acid in water and 0.1% formic acid in acetonitrile (50/50, v / v). linear calibration curves were obtained in the concentration range of 12000.0 ng / ml, with a lower limit of quantification of 0.99 ng / ml. the intra- and interday precision (rsd) values were below 3%. the method was successfully applied to a bioequivalence study of creatinine in chinese smokers and nonsmokers. the total cotinine in 24 h urine and cotinine : creatinine ratio were also positively associated (pearson r = 0.942, p < 0.0001). however, cotinine : creatinine ratio varied significantly across smoking groups for the difference of individual. 24 h urinary cotinine was more appropriate for expressing correlation with tar than cotinine : creatinine ratio. |
animals : adult male wistar - imamichi strain rats (body weight (bw), 220250 g) were obtained from the imamichi institute for animal reproduction (tsuchiura, japan). the animals were housed at constant ambient temperature of 24c with controlled lighting (lights on, 05001900 hr) and given free access to food and water. the experiments were conducted according to the guidelines for the care and use of laboratory animals, graduate school of agricultural and life sciences, the university of tokyo. stress conditions : on the day of the experiment, the rats were moved to the experimental room and allowed an adaptation period of at least 2 hr. three different types of stresses, namely infectious (lps ; sigma, st. louis, mo, u.s.a. ; 100 g / kg bw, ip), hypoglycemic (2dg ; sigma ; 400 mg / kg bw, ip) or restraint (1 hr) stresses were applied to rats. animals were divided into 2 groups and given a selective cox-2 inhibitor (ns-398 ; cayman chemical, ann arbor, mi, u.s.a. ; 10 mg / kg bw, ip) or vehicle (dmso ; sigma) 30 min before stress application. just before stress application (0 min) and 30 and 120 min after the start of stress application, animals were decapitated, and the whole brain was removed and fixed in cold 4% paraformaldehyde in phosphate - buffered saline (pbs ; 0.02 m, ph 7.2). immunohistochemistry for c - fos : brains were further fixed for 48 hr at 4c in the fixative paraformaldehyde solution and then submerged in 10% sucrose solution overnight, 20% sucrose solution for 24 hr and 30% sucrose for 48 hr until the brain completely sank. brain sections of 30 m thickness were cut using a cryostat (microm hm550, thermo scientific, waltham, ma, u.s.a.) and collected serially in 24-well plates containing pbs (0.02 m, ph 7.2). one out of every eight slices was used for immunostaining for c - fos protein. sections were then incubated with 0.3% h2o2 in pbs for 30 min at room temperature and rinsed with pbs for 10 min three times. thereafter, sections were incubated in blocking solution (block ace, snow brand milk products co., sapporo, japan) for 2 hr. tissue sections were incubated in rabbit anti - fos primary antibody (1:5,000 dilution with 0.02 m pbs containing 1% bsa and 0.3% triton x-100 ; ab-5, oncogene research product, calbiochem, ma, u.s.a.) at 4c for 60 hr, washed three times with 0.02 m pbs containing 0.03% triton x-100 (0.03% pbst), and then, the sections were incubated with the biotinylated goat - anti - rabbit secondary antibody igg (1:800 dilution with 0.02 m pbs containing 0.3% triton x-100 ; vector laboratories, burlingame, ca, u.s.a.) at room temperature for 2 hr. sections were then rinsed three times with 0.03% pbst and incubated at room temperature for a further 2 hr with an avidin - biotin - horseradish peroxidase complex solution (vectastain abc kit, vector laboratories). finally, sections were treated for approximately 1.5 min in 0.5 mg / ml diaminobenzidine tetrahydrochloride (sigma, ; dissolved in 0.02 m pbs with 0.01% hydrogen peroxide and 0.25% nickel chloride). the sections were mounted on slides, air dried, dehydrated in ethanol solutions and xylene, and coverslipped. quantification of c - fos immunoreactive cells : brain regions were identified using the rat brain in stereotaxic coordinates. the number of c - fos - immunoreactive (ir) cells in the pvn and son was counted using an olympus optical system (bx-51, olympus optical co., tokyo, japan), and the digitized images were analyzed with iplab image analyzing software (scanalytics corp. a threshold was set to delineate c - fos - positive stained nuclei from background, and only cells above the threshold were included. up to four sections per animal for each region were quantified bilaterally and then averaged. statistical analyses : all statistical analyses were performed in spss 11.5 version (spss inc., data were analyzed with two - way analysis of variance (anova), followed by tukey s test. induction of c - fos expression in the brain under stress conditions : in order to determine the brain regions responding to acute stress stimuli, immunostaining for c - fos protein in the forebrain was performed. before the application of stresses (0 min), almost no c - fos - immunoreactive (ir) cells were observed, while few scattered c - fos - ir cells were seen in the paraventricular nucleus (pvn) and the supraoptic nucleus (son). as shown in figs. 1, 2, 3fig. 1.immunohistochemical staining for c - fos protein in the brain 0, 30 and 120 min after lps injection (100 g / kg bw, ip). representative photomicrographs of c - fos - immunoreactive cells in the paraventricular nucleus (pvn, a c), supraoptic nucleus (son, d f), central amygdaloid nucleus (cead, g 2.immunohistochemical staining for c - fos protein in the brain 0, 30 and 120 min after 2dg injection (400 mg / kg bw, ip). representative photomicrographs of c - fos - immunoreactive cells in the paraventricular nucleus (pvn, a c), supraoptic nucleus (son, d f), central amygdaloid nucleus (cead, g 3.immunohistochemical staining for c - fos protein in the brain 0, 30 and 120 min after restraint stress (1 hr). representative photomicrographs of c - fos - immunoreactive (c - fos - ir) cells in the paraventricular nucleus (pvn, a c), supraoptic nucleus (son, d f), central amygdaloid nucleus (cead, g scale bars : 100 m., stress stimuli induced c - fos immunoreactivity in many brain regions. after 30 min of stress stimuli, there was a small increase in the number of c - fos - ir cells in the pvn, son, ventromedial hypothalamic nucleus (vmh) and piriform cortex (pir) in the lps and restraint stress models, while an increase in c - fos - ir cell number was found in the pvn, son and lateral hypothalamic area (lha) in the 2dg stress model. strong increases in c - fos - ir cells were observed in the pvn and son at 120 min after the application of all the three different acute stresses. considerable increases in c - fos - ir cell number were also seen in other brain regions, including the central amygdaloid nucleus, medial amygdaloid nucleus, arcuate nucleus, dentate gyrus and pir. in addition, lps and restraint stresses increased c - fos - ir cells in the vmh, while 2dg injection increased those in the lha at 120 min as well as 30 min. these results suggest that the two hypothalamic regions, the pvn and son, have similar expression patterns of neuronal activation in response to all three different acute stresses applied in this study and that the vmh is sensitive to lps and restraint stresses, while the lha is sensitive to hypoglycemic stress. immunohistochemical staining for c - fos protein in the brain 0, 30 and 120 min after lps injection (100 g / kg bw, ip). representative photomicrographs of c - fos - immunoreactive cells in the paraventricular nucleus (pvn, a c), supraoptic nucleus (son, d f), central amygdaloid nucleus (cead, g immunohistochemical staining for c - fos protein in the brain 0, 30 and 120 min after 2dg injection (400 mg / kg bw, ip). representative photomicrographs of c - fos - immunoreactive cells in the paraventricular nucleus (pvn, a c), supraoptic nucleus (son, d f), central amygdaloid nucleus (cead, g immunohistochemical staining for c - fos protein in the brain 0, 30 and 120 min after restraint stress (1 hr). representative photomicrographs of c - fos - immunoreactive (c - fos - ir) cells in the paraventricular nucleus (pvn, a c), supraoptic nucleus (son, d f), central amygdaloid nucleus (cead, g effects of cox-2 inhibitor on the expression of c - fos in the pvn and son : to investigate the involvement of cox-2-related signaling in activation of the hpa axis responding to the acute stress stimuli, we evaluated the effect of cox-2 selective inhibitor ns-398 on the number of c - fos - ir cells in the pvn and son under the three different acute stress conditions. c - fos expression in the pvn is shown in figs. 4, 5, 6fig. 4.effect of cox-2 selective inhibitor ns-398 on the expression of c - fos protein in the paraventricular nucleus (pvn) after lps injection. vehicle (a c) or cox-2 selective inhibitor ns-398 (10 mg / kg bw, ip, d f) was injected to male rats 30 min before applying lps (100 g / kg bw, ip). quantification of the number of c - fos - ir cells in the pvn of vehicle - treated (closed column) or ns-398-treated (open column) rats after an injection of lps (g). values with different letters are significantly different (p0.05). in the pvn of vehicle - injected animals, the numbers of c - fos - ir cells at 120 min after stress stimuli were significantly larger than those of other 2 time points under all three acute stress conditions. there was no significant difference between the numbers of c - fos - ir cells in the pvn in vehicle- and ns-398-treated rats at any time points under three different stress conditions. these results suggest that all three acute stress conditions induce the neuronal activation in the pvn and cox-2-related signaling does not affect the total number of neurons activated in this nucleus. effect of cox-2 selective inhibitor ns-398 on the expression of c - fos protein in the paraventricular nucleus (pvn) after lps injection. vehicle (a c) or cox-2 selective inhibitor ns-398 (10 mg / kg bw, ip, d f) was injected to male rats 30 min before applying lps (100 g / kg bw, ip). quantification of the number of c - fos - ir cells in the pvn of vehicle - treated (closed column) or ns-398-treated (open column) rats after an injection of lps (g). values with different letters are significantly different (p<0.05, two - way anova followed by tukey s test). effect of cox-2 selective inhibitor ns-398 on the expression of c - fos protein in the paraventricular nucleus (pvn) after 2dg injection. vehicle (a c) or cox-2 selective inhibitor ns-398 (10 mg / kg bw, ip, d f) was injected to male rats 30 min before applying 2dg (400 mg / kg bw, ip). quantification of the number of c - fos - ir cells in the pvn of vehicle - treated (closed column) or ns-398-treated (open column) rats after an injection of 2dg (g). values with different letters are significantly different (p<0.05, two - way anova followed by tukey s test). effect of cox-2 selective inhibitor ns-398 on the expression of c - fos protein in the paraventricular nucleus (pvn) after restraint stress. vehicle (a c) or cox-2 selective inhibitor ns-398 (10 mg / kg bw, ip, d f) was injected to male intact rats 30 min before applying restraint (1 hr) stress. quantification of the number of c - fos - ir cells in the pvn of vehicle - treated (closed column) or ns-398-treated (open column) rats after restraint stress. values with different letters are significantly different (p<0.05, two - way anova followed by tukey s test). effect of cox-2 selective inhibitor ns-398 on the expression of c - fos protein in the supraoptic nucleus (son) after lps injection. vehicle (a c) or cox-2 selective inhibitor ns-398 (10 mg / kg bw, ip, d f) was injected to male intact rats 30 min before applying lps (100 g / kg bw, ip). quantification of the number of c - fos - ir cells in the son of vehicle - treated (closed column) or ns-398-treated (open column) rats after an injection of lps (g). values with different letters are significantly different (p<0.05, two - way anova followed by tukey s test). effect of cox-2 selective inhibitor ns-398 on the expression of c - fos protein in the supraoptic nucleus (son) after 2dg injection. vehicle (a c) or cox-2 selective inhibitor ns-398 (10 mg / kg bw, ip, d f) was injected to male intact rats 30 min before applying 2dg (400 mg / kg bw, ip). quantification of the number of c - fos - ir cells in the son of vehicle - treated (closed column) or ns-398-treated (open column) rats after an injection of 2dg (g). values with different letters are significantly different (p<0.05, two - way anova followed by tukey s test). effect of cox-2 selective inhibitor ns-398 on the expression of c - fos protein in the supraoptic nucleus (son) after restraint stress. vehicle (a c) or cox-2 selective inhibitor ns-398 (10 mg / kg bw, ip, d f) was injected to male intact rats 30 min before applying restraint (1 hr) stress. quantification of the number of c - fos - ir cells in the son of vehicle - treated (closed column) or ns-398-treated (open column) rats after restraint stress (g). values with different letters are significantly different (p<0.05, two - way anova followed by tukey s test). on the other hand, the number of c - fos - ir cells in the son of vehicle - injected animals was significantly larger at 30 min compared with that at 0 min only in 2dg - injected animals, but not in lps - injected or restraint - stressed ones. the number of c - fos - ir cells in the son at 120 min was significantly larger than those of other 2 time points under all three acute stress conditions. treatment with ns-398 significantly increased the number of c - fos - ir cells at 30 min, but not at 0 and 120 min, under both lps and restraint stresses, while the effect of ns-398 was not discernible under 2dg stress at any time points. these results suggest that all the three different acute stresses induce the neuronal activation of the son, on which cox-2-related signaling has a negative effect at least at 30 min under infectious and restraint stress conditions, but not under hypoglycemic stress condition. the present study showed that all the three acute stresses applied, namely infectious, hypoglycemic and restraint stresses, induced neuronal activation in some restricted populations of neurons in the brain, especially in the pvn and son. in addition, activation of the son was significantly enhanced by cox-2 inhibitor under the infectious and restraint stress conditions, but not under the hypoglycemic stress condition. these results appear to have some relationship with the data of our previous study, which suggests that activation of the hpa axis is dependent on cox-2-related signaling under infectious and restraint stresses, but less dependent on it under hypoglycemic stress. in the parvocellular region of the pvn, there is a population of neuropeptide - secretory neurons synthesizing corticotrophin - releasing hormone (crh) or both crh and arginine vasopressin (avp) [3, 27 ], while avp and oxytocin (oxt) neurons are mainly located in the magnocellular regions of the pvn and son [11, 12 ]. a previous research showed that acute stresses increased c - fos - ir cells, which were mainly colocalized with crh - ir perikarya in the pvn. in addition, c - fos - ir nuclei were also increased in both avp- and oxt - containing neurons of the pvn and son after acute stresses [16, 20, 32 ]. the present study demonstrated that many regions of the brain respond to acute stresses and especially, the pvn and son have similar expression patterns of neuronal activation in response to three different acute stresses, which are consistent with these previous reports. it is well known that crh and avp play a key role in mediating activation of the hpa axis [18, 21 ], while some researches suggested that intracerebroventricular infusion of oxt suppresses stress - induced activation of the hpa axis [28, 29 ]. in the present study, there was no significant difference between the number of c - fos - ir cells in the pvn between vehicle - injected and ns-398-treated animals at any time points under three different acute stress conditions, suggesting that cox-2-related signaling is not directly involved in stress responses in the pvn. this is consistent with a previous report showing that cox-2- or mpges-1-deficient mice did not show any attenuation of the c - fos expression in the pvn after lps administration, though the other research group reported that lps strongly reduced the number of c - fos positive nuclei in the pvn in mpges-1-deficient mice. as to the son, on the other hand, the numbers of c - fos - ir cells in ns-398-treated animals were significantly higher than those in vehicle - injected animals at 30 min under lps and restraint stresses, but not under 2dg stress condition. reported that injection of pge2 induced c - fos expression in the pvn and son. in the parvocellular division of the pvn, c - fos was mainly expressed in crh- and oxt - ir neurons and very rarely expressed in avp - ir neurons, while in the magnocellular part of the pvn and son, c - fos was mainly colocalized in oxt - ir neurons and some expression was also detected in avp - ir neurons. it is therefore likely that, in the present study, c - fos - ir neurons in the pvn are mainly crh and oxt neurons, and those in the son are mainly oxt neurons. taken together, it is suggested that, in the son, ns-398 increased the number of oxt neurons expressing c - fos and thereby decreased serum corticosterone levels under infectious and restraint stress conditions as observed in our previous study. this may at least partially account for the differences in cox-2-dependency of the activation of the hpa axis among stresses. interestingly, in the present study, infectious and restraint stresses increased c - fos expression levels in the vmh, while hypoglycemic stress increased those in the lha, which are consistent with previous researches reported separately [1, 2, 10, 22 ]. bilateral electrolytic lesions of the vmh or lha were reported to inhibit corticosteroid feedback or extinguish the role of serotonin (5-ht) on activity in the hpa axis. in addition, involvement of the vmh and lha in the regulation of energy metabolism has been well documented. the vmh and the lha have been regarded as the satiety center and hunger center, respectively. infectious stress is known to induce anorexic symptoms, in which condition the satiety center should be activated, while 2dg as an inhibitor of glucose uptake would stimulate the hunger center. taken together, these results suggest that infectious and restraint stresses or hypoglycemic stress differently activates neurons in the vmh or lha mediating the regulation of not only the hpa axis activity but also food intake. in conclusion, the present study demonstrated that many regions of the brain, especially the pvn and son, respond to acute stresses and work as common mediators that generate potent autonomic and neuroendocrine responses. in addition, it is also suggested that cox-2-related signaling decreases neuronal activity in the son under infectious and restraint, but not hypoglycemic, stresses, which may be involved in the suppression of the hpa axis. this difference in the role of cox-2-related signaling in inhibiting son neurons among stresses may at least partially account for the difference in the role of cox-2-related signaling in activating the hpa axis among stresses observed in our previous study. | abstractwe have previously suggested that activation of the hypothalamic - pituitary - adrenal (hpa) axis is dependent on cyclooxygenase (cox)-2-related signaling under infectious and restraint stresses, but less dependent on it under hypoglycemic stress. in the present study, we evaluated the neuronal activity in the brain to elucidate the possible mechanisms underlying a stress - specific relevance between cox-2-related signaling and activation of the hpa axis under infectious (lipopolysaccharide, lps), hypoglycemic (2-deoxy - d - glucose, 2dg) and restraint (1 hr) stress conditions. the number of c - fos - immunoreactive (ir) cells in several brain regions including the paraventricular nucleus (pvn) and supraoptic nucleus (son) was increased at 120 min after application of all stress stimuli. the number of c - fos - ir cells at 30 min was increased only by 2dg in the son, but not in the pvn. in the pvn, a selective cox-2 inhibitor (ns-398) did not affect the number of c - fos - ir cells at any time points. on the other hand, in the son, ns-398 increased c - fos - ir cells at 30 min after lps and restraint stresses, but not after 2dg injection. these results suggest that, among the brain regions responding to acute stresses, the pvn and son are commonly activated under three acute stresses. in addition, it is also suggested that cox-2-related signaling decreases neuronal activity in the son under infectious and restraint, but not hypoglycemic, stresses, which may be involved in the suppression of the hpa axis. |
this study included 25 patients of both sexes with mandibular fractures in the age group of 18 - 48 years, presenting no medical contra indications for the planned procedure. data collected from the patients included pre and postoperative panoramic radiographs, age at the time of injury, sex, and site of madibular fracture. the sites of fractures in the mandible consisted symphysis, parasymphysis, body, and angle and excluded condylar fractures, all of which were treated with open reduction and internal fixation with conventional stainless steel miniplates (2 and 2.5 mm, 4 and 6 holes plates). the patients were clinically and radiologically evaluated during preoperative, immediate postoperative, 6 weeks postoperative and during regular follow - ups. all the patients were administered intravenous ampicillin 500 mg, metronidazole 400 mg, and intramuscular diclofenac sodium 50 mg postoperatively, which varied between 5 and 7 days. the following clinical parameters were used : mobility of the tooth involved in the fracturevitality of the tooth involved in the fracture. mobility of the tooth involved in the fracture vitality of the tooth involved in the fracture. the following radiological parameter was used : teeth stabilizing the fracture segment. teeth stabilizing the fracture segment the anatomic location of mandibular fractures was 80% in the angle region and 20% in the parasymphysis region. 10 patients had grade ii and 10 patients had grade i mobility of the teeth in the line of fractures [graph 1 ]. pre - operative mobility totally, 15 patients with teeth in the line of fracture showed no response to cold pulp testing (nonvital) preoperatively. rest 10 patients showed positive response for cold pulp testing (vital) [graph 2 ]. 13 patients with their teeth in the line of fracture showed positive response (vital) and 12 patients showed no response to cold pulp testing during 1 week postoperative follow - up [graph 3 ]. pre - operative vitality 1st week post - operative vitality sixteen patients with teeth in the line of fracture showed positive response to cold pulp testing during the 6 week postoperative follow - up. there was a significant improvement in the vitality of the teeth in the line of fracture during the 6 week postoperative evaluation. the rest 9 patients with their teeth in the line of fracture showed no response to the cold test (nonvital) [graph 4 ]. 6th week post - operative vitality twenty patients panoramic radiographs showed minimally displaced fractures due to the presence of teeth in the line of fractures. in 5 patients, the panoramic radiographs showed displaced fractures due to the fully erupted grade iii mobile teeth [graph 5 ]. 10 patients had grade ii and 10 patients had grade i mobility of the teeth in the line of fractures [graph 1 ]. totally, 15 patients with teeth in the line of fracture showed no response to cold pulp testing (nonvital) preoperatively. rest 10 patients showed positive response for cold pulp testing (vital) [graph 2 ]. 13 patients with their teeth in the line of fracture showed positive response (vital) and 12 patients showed no response to cold pulp testing during 1 week postoperative follow - up [graph 3 ]. pre - operative vitality 1st week post - operative vitality sixteen patients with teeth in the line of fracture showed positive response to cold pulp testing during the 6 week postoperative follow - up. there was a significant improvement in the vitality of the teeth in the line of fracture during the 6 week postoperative evaluation. the rest 9 patients with their teeth in the line of fracture showed no response to the cold test (nonvital) [graph 4 ]. twenty patients panoramic radiographs showed minimally displaced fractures due to the presence of teeth in the line of fractures. in 5 patients, the panoramic radiographs showed displaced fractures due to the fully erupted grade iii mobile teeth [graph 5 ]. some studies have demonstrated that it is really common to observe teeth in the line of fractures. others authors mentioned that the presence of the teeth can be one of the determinant factor of the fracture location. the management of teeth in the line of fracture had changed within the past years. in the past, it was thought that teeth in the line of fractures should be immediately removed. although recent studies support the vision that non infected teeth in the line of fracture can be preserved. this study demonstrated that in 16 patients the vitality of the teeth in the line of fracture with grade i and ii mobility was gradually improved and where as in 9 patients the teeth in the line of fracture remained non vital and out of which 5 pts showed signs of infection due to grade iii mobility which had to be extracted to prevent further complications. macan. in his study concluded that one third of the teeth were reinnervated within 6 weeks after injury and a year after the injury 81% were reinnervated. the study demonstrated that 20 patients with teeth in the line of fractures favoured the treatment as they contributed in stabilizing the fractured segments. correct repositioning of fractured fragments is made quicker and easier if the tooth in the line of fracture is conservatively managed. if extracted, they increase the risk of fracture contamination and may sometimes be difficult to suture. study supports the vision that noninfected teeth in the line of fracture can be preserved. the maintenance of these teeth can favour the treatment in some cases ; therefore they contribute for the stability of the fracture. its removal can be harmful, once that can diminish the contact between fragments, cause additional trauma to the region, increase the risk of contamination of the fracture through the empty alveolus, convert a closed fracture into an open fracture and cause the loss of the bony bunch in the zone of tension. each case must be evaluated individually, for maintaining or not the teeth in the fracture line, depending on the clinical and radiographic findings. it is generally accepted by most surgeons that antibiotic therapy should be administered when teeth are left in the line of fracture because of open nature and contamination of the oral cavity. conservative treatment of teeth involved in the line of mandibular fractures has a favourable prognosis, especially if optimal reduction of the jaw fragments is achieved. the study demonstrated that the teeth presence in the line of mandibular fracture is not a limiting factor for the treatment. teeth associated with mandibular fracture should not be removed on a prophylactic basis to reduce the risk of infection of fracture sites just if there is an absolute indication for removal and when retained, they should be followed - up clinically and radiographically for at least 1 year with a view to endodontic treatment if indicated. patients with teeth in the fracture line showing no response on pulp vitality testing should be advised extraction to avoid further complications. despite the risk of rate of complications, tooth in the fracture line both clinical and radiological should be mandatory for at least a period of 1 year. each case must be evaluated individually, for maintaining or not the teeth in the fracture line, depending on the clinical and radiographic findings. as our sample size consisted of only 25 patients, a larger sample size with long - term follow - up | introduction : the purpose of this study was to evaluate teeth involved in the line of fracture, clinically and radiographically, and their associated complications so as to indicate if they should be managed conservatively or extracted.materials and methods : data were collected from patients records treated of mandibular fractures. it was included pre and postoperative panoramic radiographs, information such as demographic data, age, and sex, fracture location, mobility, and vitality of teeth in the line of fracture, teeth stabilizing the fracture segment.results:the sample presented 25 patients with teeth in the line of mandibular fractures. a total of 16 patients teeth in the line of fracture were vital during the 6th week postoperative follow - up and 9 patients with their teeth in the line of fracture were nonvital of which 4 were endodontically treated and the rest 5 patients teeth in the line of fracture were extracted as they showed signs of infection.conclusion:this study demonstrated that the presence of teeth in the line of fracture is not a limiting factor for the treatment. despite the risk of complications, tooth in the fracture line should be preserved for its merits. a regular clinical and radiological follow - up should be mandatory for at least a period of 1 year. |
there is strong epidemiological and genetic evidence that genetic factors play a major, though variable role, in its pathogenesis. migraine patients are characterized between attacks on various cns - evoked responses by a deficit of habituation [1, 2 ], which may have a familial character [35 ]. by contrast, other mild abnormalities have been identified that are unlikely to play a pathogenic role : a decreased safety factor at the neuromuscular junction on single fiber emg (sfemg) [79 ] and subclinical cerebellar hypermetria in the horizontal plane on opto - electronic analysis of upper arm - reaching movements. if this is correct, one may expect that in the same individual they be of a similar degree. we have therefore compared in the same migraine patient the sfemg and 3d - movement analysis. thirteen migraine patients (ichd - ii) were recruited from the headache clinic of the headache research unit in lige, belgium. eight patients suffering from typical aura with migraine headache (mta ichd - ii code 1.2.1 ; three women and five men ; median age 28.5 years ; range 1367) and five patients with migraine without aura (mo ichd - ii code 1.1 ; three women and two men ; median age 40.0 years ; range 3151) underwent both stimulation - sfemg and 3d - movement analysis. none of them had any other medical condition detectable by history and clinical examination ; none was taking drugs on a regular basis, nor had taken any drug within 3 days before the recordings. the recordings took place at least 3 days after and before an attack (checked by telephone - interview). the study was conducted after approval of our institution s ethics committee and performed in accordance with the ethical standards of the 1964 declaration of helsinki, with the understanding and consent of each involved subject. a nicolet viking iv device (nicolet biomedical, madison, wisconsin, usa) was used for stimulation single fiber electromyography. single muscle fiber activity was recorded with 25-mm - long single fiber needles (medelec neurodiagnostic accessories, ref : 16829, witney, oxfordshire, uk), and the motor nerve was stimulated with nicolet teflon - insulated monopolar needles. we stimulated suprathreshold the motor branch of the radial nerve and assessed the variability in latency, i.e. the jitter (fig. 1), of single fiber action potentials in m. extensor digitorum communis (edc) of the right arm. off - line analyses of recordings were performed, and on average 18 artifact - free edc muscle fibers per patient were selected to assess their mean mcd. 1figures representing a normally (on the top) and an abnormally (on the bottom) jittered fibers figures representing a normally (on the top) and an abnormally (on the bottom) jittered fibers movements were recorded at 100 hz in three dimensions (3d) using an infrared optoelectronic - tracking - system (elite, milan, italy) with a reflective marker attached to the tip of the index finger and another marker to the movement target. participants were seated with the target in the medio - sagittal plane on eye level. they were instructed to start with the right arm extended to the right, to touch the target with high precision (but fast) without any trunk movement, to go back to the starting position and to repeat above movements, in a given pace over 15 s (one trial) to result in 810 movements (fig. 2). the used cartesian coordinate system is head - fixed with a nasooccipital, a horizontal and a vertical axis and the origin in the target. since in our previous study abnormalities in migraineurs were most pronounced in the horizontal plane, we limited ourselves to an analysis of the mean deviation in the horizontal plane (in millimeters), measured over four trials, each consisting of 810 arm movements. 2experimental setup for the movement task experimental setup for the movement task quantitative variables in each group of migraine patients (mo and mta) were expressed as medians. the spearman rank order correlation test was used to compare the values of the mean mcd on sfemg and the mean horizontal deviation on 3d - movement analysis. thirteen migraine patients (ichd - ii) were recruited from the headache clinic of the headache research unit in lige, belgium. eight patients suffering from typical aura with migraine headache (mta ichd - ii code 1.2.1 ; three women and five men ; median age 28.5 years ; range 1367) and five patients with migraine without aura (mo ichd - ii code 1.1 ; three women and two men ; median age 40.0 years ; range 3151) underwent both stimulation - sfemg and 3d - movement analysis. none of them had any other medical condition detectable by history and clinical examination ; none was taking drugs on a regular basis, nor had taken any drug within 3 days before the recordings. the recordings took place at least 3 days after and before an attack (checked by telephone - interview). the study was conducted after approval of our institution s ethics committee and performed in accordance with the ethical standards of the 1964 declaration of helsinki, with the understanding and consent of each involved subject. a nicolet viking iv device (nicolet biomedical, madison, wisconsin, usa) was used for stimulation single fiber electromyography. single muscle fiber activity was recorded with 25-mm - long single fiber needles (medelec neurodiagnostic accessories, ref : 16829, witney, oxfordshire, uk), and the motor nerve was stimulated with nicolet teflon - insulated monopolar needles. we stimulated suprathreshold the motor branch of the radial nerve and assessed the variability in latency, i.e. the jitter (fig. 1), of single fiber action potentials in m. extensor digitorum communis (edc) of the right arm. stimulations off - line analyses of recordings were performed, and on average 18 artifact - free edc muscle fibers per patient were selected to assess their mean mcd. mean value of consecutive differences (mcd) of successive interpotential intervals, as usual in sfemg studies. 1figures representing a normally (on the top) and an abnormally (on the bottom) jittered fibers figures representing a normally (on the top) and an abnormally (on the bottom) jittered fibers movements were recorded at 100 hz in three dimensions (3d) using an infrared optoelectronic - tracking - system (elite, milan, italy) with a reflective marker attached to the tip of the index finger and another marker to the movement target. participants were seated with the target in the medio - sagittal plane on eye level. they were instructed to start with the right arm extended to the right, to touch the target with high precision (but fast) without any trunk movement, to go back to the starting position and to repeat above movements, in a given pace over 15 s (one trial) to result in 810 movements (fig. 2). the used cartesian coordinate system is head - fixed with a nasooccipital, a horizontal and a vertical axis and the origin in the target. since in our previous study abnormalities in migraineurs were most pronounced in the horizontal plane, we limited ourselves to an analysis of the mean deviation in the horizontal plane (in millimeters), measured over four trials, each consisting of 810 arm movements. 2experimental setup for the movement task experimental setup for the movement task quantitative variables in each group of migraine patients (mo and mta) were expressed as medians. the spearman rank order correlation test was used to compare the values of the mean mcd on sfemg and the mean horizontal deviation on 3d - movement analysis. on sfemg, the median value for mean mcd was not significantly different between mo (16.05 s ; range 9.5022.93) and mta (18.91 s ; range 11.5024.55). the median value of mean horizontal deviation was 4.01 mm (range 3.25 to 18.12) in mo and 10.74 mm (range 1.6017.03) in mta, a nonsignificant difference. in mo by contrast, in mta, both variables were positively correlated (r = 0.71, p = 0.046) (fig. 3). 3scatter plot relating mean mcd on sfemg (x axis, s) and mean horizontal deviation on 3d analysis of a reaching arm movement (y axis, mm), and linear regression lines. migraine without aura patients (mo) : squares, dashed line. migraine with aura patients (mta) : triangles, continuous line scatter plot relating mean mcd on sfemg (x axis, s) and mean horizontal deviation on 3d analysis of a reaching arm movement (y axis, mm), and linear regression lines. our within - patient analysis shows that, in migraine with aura, the mean mcd on sfemg increases significantly with the degree of horizontal deviation in a visually guided reaching movement. this suggests that, in subgroups of migraine patients, neuromuscular transmission (nmt) performance and control of ballistic movements by the lateral cerebellum are similarly influenced by a common biochemical and/or neural mechanism. although no gene mutations have been identified till now, the genetic load is thought to be higher in the pathogenesis of migraine with aura than that without aura. in familial hemiplegic migraine 1 (fhm1), mutations have been found in the cacna1a gene, which codifies for the main subunit of p / q - type ca channels, heavily concentrated at the neuromuscular junction and in the cerebellum [1518 ]. thus, we initially hypothesized that the nmj and cerebellar abnormalities found in some migraine patients might be due to dysfunctioning ca channels. however, sfemg studies were normal in fhm1, and mutations in the cacna1a gene are not found in patients suffering from the common forms of migraine, with or without aura. some evidence that the cacna1a gene may be involved in migraine with typical aura, and the facts that nmt impairment can be found in episodic ataxia type 2, an allelic disorder of fhm1, and normalizes after treatment with acetazolamide are still in favor of a possible involvement of cachannels, particularly in migraine with aura. other proteins implicated in migraine pathophysiology may constitute a common link between neuromuscular junctions (nmj) and the cerebellum. this is unlikely for the alpha-2 subunit of the na - k atpase, of which the gene atp1a2 is mutated in fhm2, because the alpha-2 isoform is found at intracellular membranes and not at the nmj, where the major role is played by the alpha-3 isoform. by contrast, a different group of calcium channels, the r - type, influence neuromuscular transmission and can compensate for dysfunctioning p / q channels. moreover, they seem to play a role in cerebellar functions ; thus, their activity may influence in parallel nmj and cerebellar performances, similarly to what we found in the subgroup of migraineurs with aura. the cacna1e gene, which codes for the alpha-1e subunit of r - type channels, is precisely located on locus 1q31, for which significant linkage was found in the common forms of migraine, and a single nucleotide polymorphism was more frequent in subgroups of patients affected by migraine with aura. in conclusion, the correlation between nmj and cerebellar performances that we have found in migraine with aura patients might be due to a common genetically determined molecular mechanism, possibly influencing ion channels functions, but this has still to be proven by appropriate genetic studies. | in previous studies, we described subclinical abnormalities of neuromuscular transmission and cerebellar functions in migraineurs. the aim of this study was to search if these two functions are correlated in the same patient. thirteen migraineurs [five without aura (mo) and eight with aura (ma) ] underwent both stimulation - sfemg and 3d - movement analysis. single fiber emg (sfemg) results were expressed as the mean value of consecutive differences (mean mcd). precision of arm - reaching movements (measured with an infrared optoelectronic tracking system) was expressed as the average deviation in the horizontal plane. median values of mean mcd and mean horizontal deviation were not different between mo and ma. however, in ma, but not in mo, both variables were positively correlated. thus, we conclude that neuromuscular transmission and cerebellar functions are correlated in the same patient when affected by migraine with aura. we suggest that this correlation might be due to a common molecular abnormality. |
bladder cancer is the second most common genitourinary malignancy and the sixth most common cancer in the world. in china, the incidence of bladder cancer also increases with age, and the peak is reached approximately at the age of 60 years ; bladder cancer is three times more common in men than in women. among the newly diagnosed cases of transitional cell carcinomas, approximately 75% to 80% of these cases present superficial tumours ; 50% to 70% of these superficial tumours relapse within five years ; and roughly 10% to 20% progress to a more aggressive disease. bladder cancer is a multifactorial disease mediated by genetic abnormalities, environmental factors, and chronic irritation. although many individuals are exposed to these risk factors, only a fraction of exposed individuals develop bladder cancer in their lifetime, suggesting that genetic variations may participate in bladder carcinogenesis. thrombospondin-1 (tsp-1) is an adhesive glycoprotein with a size of 450 kd initially discovered in platelets, where tsp-1 is sequestered in a platelet -granule. tsp-1 has been implicated in regulating numerous biological activities, including cell adhesion, cell migration, proliferation, angiogenesis, inflammation, and wound healing [712 ]. tsp-1 also elicits different effects on tumour growth and progression depending on the tumour type, leading to cancer progression and inhibition in different instances [1118 ]. in bladder cancer, low tsp-1 expression is significantly associated with an increased risk of disease recurrence and decreased overall survival [17, 18 ]. studies have been conducted to assess the association between polymorphisms in candidate genes and bladder cancer risk. to date, genetic polymorphisms in tsp-1 gene are possibly associated with coronary artery disease and myocardial infarction [20, 21 ]. the transcriptional initiation site of the tsp-1 gene was identified by laherty.. tsp-1 - 1223 a / g polymorphism is located in the 5 near - gene region of the tsp-1 gene, which may influence the transcriptional activity of tsp-1. furthermore, tsp-1 - 1223 a / g polymorphism may participate in the aetiology and development of bladder cancer. in the present study, tsp-1 - 1223 a / g polymorphism (rs2169830), we genotyped the polymorphism and determined its association with the risk of bladder cancer in our ongoing, hospital - based, case - control study in a chinese population. the study was approved by the institutional review board of the first affiliated hospital of nanjing medical university, nanjing, china. during recruitment, the present study included 609 patients with bladder cancer and 670 age - matched control subjects from han population living in jiangsu and anhui provinces in eastern china. the patients with bladder cancer were recruited from july 2006 to july 2012 in the department of urology, the first affiliated hospital of nanjing medical university. the patients were excluded in this study according to the following : previous cancer, metastasised cancer from other or unknown origins, and previously subjected to radiotherapy or chemotherapy. cancer - free control individuals were frequency matched with the cancer patients in terms of age (5 years) and gender. all of the control subjects were recruited from healthy individuals who were scheduled for a physical examination in the outpatient department at the same hospital. the control subjects were excluded if they manifested symptoms of bladder cancer, such as haematuria. prior to recruitment, all of the subjects were personally interviewed to collect demographic data and clinical characteristics, including age, gender, race, tobacco use, alcohol use, and self - reported family history of cancer. according to the tumour, node, and metastasis classification of cancer stages (2002 international union against cancer), the clinical stage at the time of diagnosis was classified into two subgroups : nonmuscle invasive group (pta - pt1) and muscle invasive group (pt2-pt4). according to histopathological grade (who 1973, grading of urothelial papilloma), the patients were classified into three subgroups : grades 1, 2, and 3. individuals who smoked daily for > 1 year were defined as smokers and the rest were considered as nonsmokers. individuals who drank alcohol at least three times per week for more than 6 months were defined as drinkers and the rest were considered as nondrinkers. for survival analysis, 260 patients were followed up. among these follow - up cases, 24 (24/260, 9.2%) survival time was calculated from the date of confirmed diagnosis until the date of the last follow - up or recurrence. the date of recurrence was obtained from inpatient and outpatient records or from patients ' families via follow - up telephone calls. the patients who did not suffer from recurrence on the last follow - up date were considered as non - recurrent. genomic dna of each individual was extracted from 150 l of edta - anticoagulated peripheral blood samples by using a dna extraction kit (tiangen biotech, beijing, china) according to the manufacturer 's instructions. the tsp-1 - 1223 a / g polymorphism was genotyped using taqman single nucleotide polymorphism (snp) genotyping assay (applied biosystems, foster city, ca, usa). the primers, probes, and reaction conditions of each snp are available upon request. for quality control, four negative controls were included in each plate, and 5% of the samples were randomly selected for repeated genotyping to verify the results ; all of the results were 100% consistent. total rna from 66 bladder cancer tissues was extracted using trizol reagent (invitrogen, carlsbad, ca) according to the manufacturer 's protocol. total rna (1 g) was used for cdna synthesis with oligo - dt primers (invitrogen, karlsruhe, germany) and superscript ii reverse transcriptase (takara bio, shiga, japan). pcr was performed using pcr master (roche, mannheim, germany) with the following primers : for tsp-1 mrna, 5-actgtccattccattacaacccagc-3 (forward) and 5-tgtcacactgatctccaaccccatcca-3 (reverse) ; and for -actin, 5-actggaacggtgaaggtgac-3 (forward) and 5-agagaagtggggtggctttt-3 (reverse). fold changes were normalised based on -actin expression, and each assay was conducted in a 384-well abi 7900ht real - time pcr system (applied biosystems, foster city, ca, usa). the frequency distributions of the selected demographic variables as well as each allele and genotype of the tsp-1 - 1223 a / g polymorphism between the cases and the control subjects were evaluated using -type distribution. hardy - weinberg equilibrium (hwe) was determined using a goodness - of - fit test. unconditional univariate and multivariate logistic regression analyses were conducted to calculate the crude and adjusted odds ratios (ors) and 95% confidence intervals (cis) to determine the risk of bladder cancer. interaction was investigated using a multiplicative interaction term included in the multivariate model. to investigate potential interactions between the polymorphism and tobacco smoking, we assessed a multiplicative gene - environment interaction by logistic regression analysis, including the main effect variables and their product terms. hazard ratios (hrs) and 95% cis of the hrs were derived from univariate and multivariate cox proportional hazard models. survival curves were generated using the kaplan - meier method and compared using the log - rank - mantel - cox test. the frequency distributions of the selected characteristics of the cases and the control subjects are shown in table 1. the cases and the control subjects were adequately matched in terms of age and gender (p = 0.486 for age and p = 0.091 for gender). a higher number of smokers were found among the cases than the control subjects (47.9% versus 36.4% ; p < 0.001). no significant difference in drinking status was found between the cases and the control subjects (p = 0.196). in addition, the frequency of the first - degree relatives with cancer was higher in the cases than in the control subjects (27.8% versus 7.2% ; p < 0.001). these variables were further adjusted in the multivariate logistic regression analysis to assess the main effect of the tsp-1 - 1223 a / g polymorphism on bladder cancer risk. the genotype and allele frequency distributions of the tsp-1 - 1223 a / g polymorphism among the cases and the control subjects as well as their associations with the risk of bladder cancer are presented in table 2. the genotype frequencies in the control subjects are consistent with those of hwe (p = 0.851). the frequencies of aa, ag, and gg genotypes were 47.1%, 40.2%, and 12.6% among the cases and 43.9%, 44.5%, and 11.6% among the control subjects, respectively (p = 0.307). no correlation was observed between tsp-1 - 1223 a / g polymorphism and the risk of bladder cancer. we further assessed the effect of tsp-1 - 1223 a / g polymorphism on the risk of bladder cancer risk stratified by age, gender, smoking status, drinking status, self - reported family history of cancer, and tumour grade and tumour stage (tables 3, 4, and 5). logistic regression analysis also showed no association between tsp-1 - 1223 a / g polymorphism and bladder cancer risk. a significantly increased risk was observed in smokers with aa / ag genotypes compared with nonsmokers exhibiting aa / ag genotypes (p = 0.001 ; ors = 1.58 ; 95% ci = 1.20 to 2.10 ; table 6). we then evaluated whether or not an interaction between smoking status and tsp-1 - 1223 a / g polymorphism status occurs. we did not observe a multiplicative interaction effect between polymorphism and smoking status (p = 0.823 ; table 6). we further investigated whether or not tsp-1 - 1223 a / g polymorphism is associated with the recurrence of bladder cancer. the demographic and clinical characteristics at primary diagnosis and the genotype distribution in patients with bladder cancer are shown in table 7. the median follow - up duration for the 236 patients was 19 months (range, 1 month to 68 months). the mean time - to - recurrence was 20.8 months (95% ci = 17.2 to 24.3 months). kaplan - meier curves showed statistical difference between tsp-1 - 1223 a / g polymorphism and time - to - recurrence in bladder cancer (aa 20.5 months, 95% ci = 14.7 to 26.3 ; ag 22.2 months, 95% ci 15.6 to 28.7 ; gg 18.8 months, 95% ci 12.725.0 ; log - rank test, p = 0.017, figure 2). compared with aa + ag genotypes, the gg genotype exhibited a significant recurrence risk of bladder cancer (hr = 2.07, 95% ci = 1.23 to 3.49, p = 0.006), and the recurrence risk was more prominent among patients with the gg genotype (hr = 2.63, 95% ci = 1.43 to 4.83, p = 0.002). the cases exhibiting ag + gg genotypes were also associated with an increased recurrence risk compared with the aa genotype (hr = 1.95, 95% ci = 1.20 to 3.19, p = 0.007 ; table 8). to evaluate the association between tsp-1 - 1223 a / g polymorphism and recurrence of bladder cancer, we examined whether or not the tsp-1 - 1223 a / g polymorphism was associated with an altered tsp-1 mrna expression. no significant difference was observed in the relative tsp-1 mrna expression level between patients with aa (n = 23) and ag (n = 28) genotypes (p = 0.103). the patients with the gg genotype exhibited lower tsp-1 mrna expression levels than those with aa genotype (n = 15 ; p = 0.002), although a significant overlap among the three groups was observed (figure 1). in the present study, the association between -1223 a / g polymorphism in tsp-1 gene (rs2169830) and risk of bladder cancer was investigated. the results do not indicate that the genotypes of the tsp-1 - 1223 a / g polymorphism are associated with an increased bladder cancer incidence. however, the time - to - recurrence was significantly shorter in g allele carriers than that in individuals with a homozygous aa genotype. patients with the ag / gg genotypes have 1.95 times greater risk of the recurrence of bladder cancer than those with the aa genotype (p = 0.007). furthermore, individuals with the gg genotype exhibited a higher risk of recurrence than those with the aa genotype (or = 2.63, p = 0.002). to the best of our knowledge, this study is the first to investigate the function of the tsp-1 - 1223 a / g polymorphism in the aetiology of bladder cancer. tsp-1 has been implicated in tumour growth and progression by regulating cell adhesion, motility, proliferation, and angiogenesis. as a multifunctional protein involved in tumour growth regulation, tsp-1 elicits an inhibitory effect on the growth of various malignancies such as melanoma, prostate cancer, cutaneous squamous cell carcinoma, and glioblastoma. strong evidence has also suggested that tsp-1 may stimulate lung cancer and breast cancer. a previous study identified that tsp-1, secreted by normal urothelium cells, is responsible for antiangiogenic activity. the downregulation of tsp-1 in bladder cancer is considered as a primary factor contributing to specific changes, for instance, from an antiangiogenic to an angiogenic phenotype during cancer development and recurrence [17, 18, 33 ]. most of the tsp-1 snps have only been observed in cardiac disease [20, 21 ] ; no studies have been shown to provide a relationship between tsp-1 gene polymorphism and cancer. in our study, the gg / ag genotypes of the tsp-1 - 1223 a / g polymorphism in bladder cancer were associated with a shorter time - to - recurrence than the aa genotype. individuals carrying the lower production genotype of tsp-1 - 1223 a / g polymorphism possibly possess an enhanced ability to promote the recurrence of bladder cancer. using the program tfsearch, we found that the g allele of tsp-1 - 1223 a / g polymorphism was located in the consensus dna sequence tgtggt. the dna sequence was a potential transcription regulation region, which can be recognised by aml1-a [35, 36 ]. however, any transcriptional factor was not bound to the sequence tgtgat containing the a allele of the tsp-1 - 1223 a / g polymorphism. furthermore, aml1-a participates as a transcription inhibitor by suppressing the transcriptional activation of aml1-b. thus, we cautiously speculated that aml1-a binds to the consensus sequence (tgtggt) of the g allele in the tsp-1 - 1223 a / g polymorphism, thereby suppressing the transcription of tsp-1. a previous study revealed that tsp-1 can suppress vegf mobilisation from the extracellular matrix by inhibiting the activity of mmp-9. the possible mechanism between angiogenesis and bladder cancer recurrence has been related to an increased vegf expression level, which serves as a major factor in angiogenesis. the increased expression level of vegf was implicated in the pathogenesis of bladder cancer recurrence by promoting the growth and implantation of a bladder cancer cell via angiogenesis. conversely, decreased tsp-1 may be associated with an increased risk of bladder cancer recurrence via the inhibitory effect of tsp-1 on vegf - mediated tumour angiogenesis. in the current study, the expression level of tsp-1 mrna in bladder cancer samples was lower in patients with heterozygous g allele than those with homozygous a allele. the expression level of tsp-1 mrna was the lowest in individuals with homozygous g allele. as such, the shorter time - to - recurrence in patients with bladder cancer exhibiting a g allele may be attributed to a decreased tsp-1 expression, which has been associated with an increased risk of disease recurrence and a decreased overall survival by promoting tumour neovascularization [17, 18, 34 ]. our results partly supported our hypothesis ; however, further functional experiments should be conducted to validate the specific mechanism. first, our sample size was relatively small, which may limit the statistical power of our study, particularly for gene - environment interaction analyses. second, our study was a hospital - based case - control study ; hence, we can not rule out the possibility of selection bias for subjects who may have been associated with a particular genotype. in conclusion, our study shows that g allele in tsp-1 - 1223 a / g polymorphism may modulate the risk of recurrence in bladder cancer. a lack of association between tsp-1 - 1223 a / g polymorphism and risk of bladder cancer was observed in our population. nevertheless, a possible function of this polymorphism in other cancers or in other bladder cancer populations should be considered. hence, additional studies should be conducted to confirm the specific function of tsp-1 - 1223 a / g polymorphism in bladder cancer development. | backgrounds. tsp-1 is a glycoprotein that functions in the biology of bladder cancer. we investigated the relationship between the distribution of tsp-1 - 1223 a / g polymorphism (rs2169830) and the clinical characteristics of bladder cancer. materials and methods. taqman assay was performed to determine the genotype of 609 cases and 670 control subjects in a chinese population. logistic regression was used to assess the association between the polymorphism and the risk of bladder cancer. quantitative real - time polymerase chain reaction was performed to determine tsp-1 mrna expression. survival curves were generated using the kaplan - meier method. results. no significant differences were detected in the genotype frequencies of healthy control subjects and patients with bladder cancer. by contrast, the time until the first recurrence differed significantly between genotypes (p = 0.017). the expression of tsp-1 mrna in bladder cancer tissues was lower in patients with an ag genotype than in those with an aa genotype. the lowest expression was observed in patients with a gg genotype. conclusions. in conclusion, tsp-1 - 1223 a / g polymorphism may contribute to the recurrence of bladder cancer in chinese population. |
a 38-year - old male patient with a 40-pack - year history of smoking initially presented with sudden - onset right - sided facial nerve palsy which was diagnosed as idiopathic bell 's palsy and was treated with prednisolone. three weeks later, the patient developed a new left - sided facial palsy in addition to his unrecovered right - sided facial palsy. outside of the facial nerve paralysis, he was neurologically intact, and his physical examination revealed no abnormalities. further workup was carried out to rule out autoimmune, metabolic, infectious, and vasculitic causes and revealed no abnormality. a chest x - ray showed a right upper lobe opacity which prompted a computed tomography (ct) scan of the chest which showed a well - defined, suspected soft - tissue lesion in the right upper lobe with extensive hilar and ipsilateral mediastinal lymphadenopathy (fig 1a, b). at this point, the differential diagnosis included sarcoidosis and primary lung cancer given the presenting complaint was bilateral facial nerve paralysis. because the intrapulmonary lesion was too distal to be reached by flexible bronchoscopy, video - assisted mediastinoscopy was performed and revealed gross right - sided mediastinal invasion. multiple biopsies from lymph nodes were taken and revealed metastatic poorly differentiated adenocarcinoma of the lung. accordingly, positron emission tomography (pet)/ct and magnetic resonance imaging (mri) of the brain were performed to stage the cancer. brain mri with contrast showed normal enhancement of the seventh and eighth cranial nerves with neither brain metastasis nor leptomeningeal metastasis. the pet / ct showed a metabolically active right upper lobe lesion with extension into the mediastinum and active ipsilateral mediastinal lymph nodes but no distant metastasis. due to our high clinical suspicion of leptomeningeal involvement, a lumbar puncture was performed. at this point, his clinical stage was considered ciiib ; ct4 (extension) n2 m0. the patient received platinum - based doublet chemotherapy with a total of 60 gray of radiation. he tolerated the chemotherapy and radiation well but had no resolution of his facial paralysis. nine months later, the patient presented to the emergency department with severe headache and generalized tonic - clonic seizures. on physical examination an abdominal and pelvic ct scan was obtained which showed newly developed liver metastases as well. given the patient 's disease progression and deteriorating condition, palliative care was offered, and the patient succumbed to his disease. the incidence of leptomeningeal carcinomatosis has been reported to be between 0.8 and 8% in many autopsy studies, though the true incidence is difficult to determine. among all known solid tumors, leptomeningeal metastasis is more common in breast cancer, lung cancer, and melanoma [1, 2 ]. with improvements in survival rates among patients who have cancer, the incidence of leptomeningeal carcinomatosis in patients with non - small - cell lung cancer is increasing. however, small - cell lung cancer is more likely to cause leptomeningeal metastases with a frequency of 1025%, as compared to 1% in the case of non - small - cell lung cancer. though leptomeningeal metastasis is a late complication of cancer, it can be the initial presentation in some cases. our case is one of these cases where neurologic symptoms were the first and only presentation of pulmonary adenocarcinoma. reviewing the literature, and to the best of our knowledge, our case is the only case describing bilateral facial nerve palsy as the only manifestation of lung adenocarcinoma. however, as demonstrated here, patients may present with a single isolated neurological sign or symptom. among these facial weakness was detected as a sign of an underlying leptomeningeal metastasis in about 13% of cases at the time of presentation. multiple cases in the literature reported unilateral peripheral facial nerve palsy as the first presentation of an underlying cancer. in some of these cases, the underlying cancer was pulmonary adenocarcinoma. csf analysis has been considered the gold standard method to diagnose leptomeningeal metastases ; however, the false - negative rate seen in many cases (even after repeating the lumber puncture) makes the diagnosis of leptomeningeal metastases challenging, causing many to suggest using mri as well as csf analysis. csf findings suggestive of leptomeningeal metastases are high cell count, high protein, low glucose, and high csf opening pressure. mri of the brain is considered more sensitive in leptomeningeal metastases from solid tumors, while csf analysis is superior to mri in cases of hematological malignancies [5, 7 ]. csf samples are more likely to have positive findings from either clinically or radiographically diseased areas. our patient 's second csf sample was drawn directly from the ventricles after his disease had progressed, which may have played a role in the positive result of the csf cytology. all authors have been personally and actively involved in substantive work leading to the manuscript and will hold themselves jointly and individually responsible for its content. the authors declare that no financial or other conflict of interest exists in relation to the content of this paper. | leptomeningeal carcinomatosis is rare, and its precise incidence is unknown. it is associated with a wide spectrum of solid and hematological malignancies. to complicate its diagnosis, the clinical presentation of leptomeningeal carcinomatosis can be variable. we report a case of a 38-year - old male with bilateral facial nerve paralysis as first presentation of lung adenocarcinoma. to our knowledge, this is the only case describing bilateral facial nerve palsy as the first and only manifestation of lung adenocarcinoma. |
since the advent of the hiv - aids pandemic across the globe, pneumocystis jiroveci pneumonia has been one of the most common defining illnesses of this condition. idiopathic cd4 lymphocytopenia is one such condition where the patient presents with such aids defining illnesses but with negative serological marker for this dreaded disease. here, we describe a case where a young man presented with pneumocystis infection but was having negative markers for hiv and thus the diagnosis of icl was concluded. a 32-year - old adequately nourished male was admitted for complaints of shortness of breath for 1 month which had exacerbated for the past 2 days. he used to work as a tailor with a mixed diet usually on his platter. examination revealed a well built and nourished male with a body mass index of 25.3 kg / m who was tachypneic with the presence of tachycardia and mild fever. oral thrush was present and scrapings were sent for fungal culture. there were no apparent risk factors for the presence of hiv or any other immunosuppressive states. chest x - ray revealed few scattered inspiratory crackles and the saturation monitor was showing a spo2 of 87%. high flow o2 was initiated and the patient was then shifted to the intensive care unit for further management. given the clinical picture of the patient, pneumocystis jiroveci pneumonia (pjp) was high on cards, given the apparent immunosuppressed state. empirical antibiotics, fluconazole and oral trimethoprim - sulphomethoxazole [tmp - smx ], was started along with steroids. u / l (high) and high - resolution computed tomography of the thorax revealed ground glass opacities in both lung fields, more so being perihilar. thus, the diagnosis of pjp was confirmed. to identify the cause of the immunosuppressed state, since the clinical suspicion of hiv was so high, a western blot and a hiv - rna polymerase chain reaction were ordered as well. cd4 and cd8 counts were ordered, as a suspicion for idiopathic cd4 lymphocytopenia (icl) was high. the cd8 counts were 312/mm (normal) with a cd4:cd8 ratio, inverted (0.35 ; normal : 1.41.7). he denied any history of immunosuppressive drug intake, did not experience repeated infections in the childhood, his immunoglobulin and sugar levels were normal, and the titers for htlv1 and 2 were negative. thus, idiopathic cd4 lymphocytopenia was labeled as the cause of his immunodeficiency and was given a prophylaxis of tmp - smx and fluconazole post - treatment. the centre for disease control in the usa in 1993 defined a new entity in the ever expanding world of immunology, known as the idiopathic cd4 lymphocytopenia. the hiv - aids pandemic had just began to shape in those days and defining a disease, so close and yet so far from it, was a challenge that the centers for disease control and prevention had to face. they defined this entity as the presence of cd4 counts < 300/mm or < 20% of the total lymphocyte count on more than one occasion, at least 6 weeks apart, with the absence of hiv infection or any other condition that might cause cd4 cytopenia. icl is a heterogeneous condition which is usually diagnosed in middle age and has a slight male preponderance. the spectrum of the presence of opportunistic infections in icl is the same when compared to a patient with hiv infection. cryptococcosis is the most common infection associated with this disease, but candida, cytomegalovirus, nontuberculous, and tuberculous infections are common. icl appears to be due to a physiological response where there is an altered environment of cytokines and inflammation that leads to decreased production of t - cell precursors and clonogenic capacity of the bone marrow. this appears due to decreased interleukin-2 and increased tumor necrosis factor alpha in the internal mileu. in addition, decreased t - cell response and increased t - cell activation have been noted. one study had found that there is a profound defect in cxcr4 expression on cd4 cells and an abnormal intracellular accumulation of cxcr4 and its ligand cxcl12. loss of this cxcr4 was postulated to cause disturbance in the priming of t - cells in the secondary lymphoid organs and its improper antigen reception and subsequent activation. barr virus, adenovirus, parvovirus b19, and some other viral infections can cause lymphopenia in their acute stages, but it is highly unusual for them to cause an exclusive isolated cd4 cytopenia - like syndrome. there are not any published guidelines for managing these cases, and most centers advocate using a cd4 cell count level of below 200/mm to start antimicrobial prophylaxis. however, few case reports do suggest that icl may not be at an identical risk of infection as a patient with hiv at the same cd4 count, these data are too less to refute such claims and deny the patient of these measures. if the cd4 cytopenia is very low, i.e. below 100 and there is a presence of recalcitrant infections, then treatment with interleukin-2 is warranted ; however, this therapy has its own set of side effects and needs an informed written consent, depicting the investigational nature of the treatment protocol. hematopoietic stem cell transplant also has shown promise in some reports to be an effective mode of treatment for this condition. pjp is regarded as an aids - defining illness, but its presence in our patient without the serological evidence of hiv in any form leads us to investigate for this rare but important disorder. icl is a rare disease which warrants a high index of suspicion to be diagnosed. it can come with the same opportunistic infections as in a hiv patient, but the negative serology gives way the diagnosis. | idiopathic cd4 + lymphocytopenia (icl) is a rare disorder characterized by the presence of depleted cd4 cell line without the presence of hiv infection. slight male preponderance is noticed and is usually seen in the middle age group. opportunistic infections are the reason for their discovery and here we describe a case where a man was diagnosed as having pneumocystis jiroveci pneumonia and oral candidiasis. |
diabetes is a metabolic disease with the main symptom of chronic hyperglycemia caused by insufficient insulin action. most symptoms can not be noticed when the degree of the metabolic disorders caused by insufficient insulin action is mild. however, thirst, polydipsia, polyuria, and weight loss are observed in the metabolic state where the bloodsugar level is significantly higher. moreover, this more advanced condition leads to acute complications such as disturbance of consciousness and coma, and may also lead to death if effective therapy is not provided. the risk of developing characteristic longterm complications (retinopathy, nephropathy, and neuropathy) is high even when mild metabolic disorders continue for a long time. moreover, arteriosclerosis of the whole body is accelerated with diabetes, and this can cause myocardial infarction, cerebral infarction, and arteriosclerosis obliterans of the lower extremities. the goal of diabetes therapy is to prevent the onset and exacerbation of shortterm and longterm complications, to maintain quality of life (qol), and to achieve a life span that is comparable to that of a healthy person. it is evident from some epidemiological analyses that the risks of onset and progression of microangiopathy and macroangiopathy are reduced with better glycemic control. there is no clear standard indicating the degree to which glycemic control has to be improved to prevent the onset of complications. evidence has been reported in japan that the onset and progression of microangiopathy could be prevented with hba1c (jds) levels of 8.0%10. the onset of microvascular complications was significantly high in the conventional therapy group of ukdps for hba1c (ngsp) with a median value of 7.9%11, and hence, an hba1c value of 8.0% was set as one of the delimiters (figure 1). the main objective value of hba1c was set to < 7% from the perspective of preventing complications. the objective when aiming to normal glycemia was set to < 6%, and the objective when intensification of therapy considered difficult was set to < 8%. moreover, the earlier evaluation was mainly from the perspective of the risk of microangiopathy. the decode study12 revealed that higher blood glucose levels after the 75 g oral glucose tolerance test (ogtt ; 2h test) is a risk factor that is independent of blood pressure and lipids in cardiovascular diseases. the necessity of strict glycemic control during pregnancy (during the period before pregnancy up to the delivery) also has to be kept in mind. in the evidencebased practice guideline for the treatment for diabetes in japan 2013, a new concept of the glycemic control in patients with diabetes in japan has been declared. the main objective value of hba1c was set to < 7% from the perspective of preventing microvascular complications. on the other hand, the objective when aiming to normal glycemia was set to < 6%, and the objective when intensification of therapy considered difficult was set to < 8%. treatment objectives should be established in each subject, considering the age, duration of disease, organ damage, risk of hypoglycemia, support structure, and etc. eiichi araki has received honoraria for lectures from drug company astellas pharma inc., and honoraria for manuscripts from medical review co., ltd., and total clinical research grants from astellas pharma inc., mitsubishi tanabe pharma corporation, daiichi sankyo company, limited, taisho pharmaceutical holdings co., ltd., masakazu haneda has received honoraria for lectures from drug companies mitsubishi tanabe pharma corporation, boehringer ingelheim gmbh, taisho toyama pharmaceutical co., ltd., kohjiro ueki has received honoraria for lectures from drug company msd, and courses endowed by companies, etc. from drug companies msd, novo nordisk pharma ltd., and boehringer ingelheim gmbh, masato kasuga, takeshi nishikawa, tatsuya kondo, and takashi kadowaki have no conflict of interest. | abstractin the evidencebased practice guideline for the treatment for diabetes in japan 2013, a new concept of the glycemic control in patients with diabetes in japan has been declared from the japan diabetes society. the main objective value of hba1c was set to < 7% from the perspective of preventing microvascular complications. on the other hand, the objective in cases where objectives can be attained by appropriate dietary or exercise therapy, or during pharmacotherapy without the occurrence of side effects such as hypoglycemia was set to < 6%, and the objective in cases where intensification of treatment was considered difficult due to side effects such as hypoglycemia or for other reasons was set to < 8%. treatment objectives should be established individually, in consideration of age, duration of disease, organ damage, risk of hypoglycemia, support structure, and etc. |
urethritis in males that is caused by sexual transmission is a major health - care concern worldwide. gonococcal urethritis (gu) is a common sexually transmitted disease (std) that is caused by neisseria gonorrhoeae. common signs and symptoms in males are purulent discharge from the urethra with dysuria, perimeatal erythema, and edema. positive test for intracellular gram - negative diplococci from urethral discharge establishes definitive clinical diagnosis of gu. in contrast, non - gu (ngu) is a nonspecific infection of the urethra with many infectious etiologies. ngu is confirmed in symptomatic men when staining of urethral secretions indicates inflammation without gram - negative diplococci. gu and ngu coinfections are common. according to the 2014 annual epidemiological surveillance report on sexually transmitted infection conducted by the bureau of epidemiology, ministry of public health, thailand, 6,184 (17.6%) of 35,073 patients with stds age ranges of std - infected individuals were as follows : 1524 years (57.9%), 2534 years (18.8%), and 3544 years (8.7%). the percentage of infection in 1524 years age group continues to grow on a year - over - year basis. as a result, gu and ngu the gonococcal isolate surveillance project from the united states reported 0.3%, 0.4%, 1.1%, 13.5%, and 22.5% of gonococcal isolates resistant to ceftriaxone, azithromycin, cefixime, ciprofloxacin, and tetracycline, respectively. the gonococcal resistance to antimicrobials surveillance program from england and wales reported 0.2%, 0.8%, 5.7%, 2.2%, 25.2%, and 75.0% of gonococcal isolates resistant to those same five antibiotics, respectively. loss to follow - up and poor compliance rates among gu and ngu patients are an increasingly serious problem in thailand. these two factors could result in an increase in the prevalence of antimicrobial resistance as well as an increase in the spread of infection to sexual partners. studies regarding treatment outcomes and loss to follow - up rate in gu and ngu in thailand have been limited. this information is essential for policy and strategic planning regarding diagnostic tools and treatment regimens. accordingly, the aim of this study was to determine treatment outcomes and loss to follow - up rate of male patients with gu and ngu at an std clinic at thailand 's largest university - based tertiary care hospital. clinical manifestations, risk behavior, laboratory investigations, and treatment were also evaluated between gu and ngu patients. this retrospective chart review of male patients diagnosed with gu and/or ngu during january 2007 to december 2014 study period at std clinic, siriraj hospital, thailand, was approved by the siriraj institutional review board. data regarding prevalence, clinical manifestations, risk behavior, laboratory investigations, and treatment were collected and evaluated. gu was diagnosed based on urethral swab that found 5 intracellular gram - negative diplococci per oil - immersion field. ngu was diagnosed from urethral swab with white blood cell 5 per oil - immersion field but without gram - negative diplococci. nucleic acid amplification testing (naat) was not routinely used for diagnosis in our center 's std clinic. treatment outcome in our study was based on clinical signs and symptoms of patients when they visited for follow - up. normally, patients were appointed to follow - up at 2 weeks after treatment to check for treatment adherence and symptoms. retest or urethral swabs were performed only in persistent symptomatic or suspected recurrent patients. descriptive statistics, such as mean standard deviation (sd), median and range, and number and percentage, were used to describe demographic data, clinical characteristics, laboratory investigations, medications, outcome of treatment, and loss to follow - up rate. chi - square test or fisher 's exact test was used to compare categorical variables. continuous variables with and without normal distribution were analyzed by student 's t - test and mann whitney u - test, respectively. descriptive statistics, such as mean standard deviation (sd), median and range, and number and percentage, were used to describe demographic data, clinical characteristics, laboratory investigations, medications, outcome of treatment, and loss to follow - up rate. chi - square test or fisher 's exact test was used to compare categorical variables. continuous variables with and without normal distribution were analyzed by student 's t - test and mann whitney u - test, respectively. mean age (sd) was 29.5 (11.6) years, with a range of 1582 years. there were 29 (12.2%) patients that were men who have sex with men (msm) and 10.6% were hiv - infected patients. 59% had multiple sex partners, 37.6% had engaged in sex with a prostitute within the previous 5 years, and 2.3% worked as a commercial sex worker. regarding clinical characteristics, 73.1% presented with urethral discharge, of which 62.1% had purulent discharge, 4.4% had mucoid discharge, and 6.6% had clear fluid discharge. regarding other symptoms, 7.5% had regional lymphadenopathy and 1.8% had associated fever. concerning diagnosis, 120 (52.9%) patients were diagnosed as gu and 107 (47.1%) as ngu. overall prevalence of gu and ngu among total patients in the std clinic during the study period was 9.1% and 8.1%, respectively. the prevalence of gu and ngu was 11.0% and 9.7% in 2007, 10.9% and 8.7% in 2008, 9.6% and 7.2% in 2009, 7.9% and 10.8% in 2010, 6.2% and 8.7% in 2011, 7.8% and 4.5% in 2012, 7.6% and 8.6% in 2013, and 11.8% and 7.7% in 2014, respectively. almost all gu patients were diagnosed by gram stain of intracellular gram - negative cocci organism. collection of intraurethral discharge for aerobic culture was performed in 45 of 120 (37.5%) gu patients depending on patient 's health insurance coverage and aimed to determine drug resistance organism. thirty - six (80%) patients were positive for n. gonorrhoeae that was sensitive to ceftriaxone and resistant to ciprofloxacin, penicillin, and tetracycline. only one culture specimen demonstrated beta - lactamase - negative n. gonorrhoeae with sensitivity to ceftriaxone, penicillin, and tetracycline but resistance to ciprofloxacin. regarding treatment, gu patients were prescribed with dual therapy consisting of ceftriaxone 250 mg intramuscular in a single dose with doxycycline 100 mg orally twice a day for 7 days for 102 patients (85%), ceftriaxone intramuscular alone for ten patients (8.3%), ceftriaxone intramuscular with azithromycin 1 g orally in a single dose for three patients (2.5%), azithromycin orally alone for two patients (1.7%), doxycycline 100 mg orally twice a day for 7 days for one patient (0.8%), azithromycin with doxycycline orally for one patient (0.8%), and azithromycin with ciprofloxacin 500 mg orally a day for 7 days for one patient (0.8%). for ngu, patients were treated with doxycycline 100 mg orally twice a day for 7 days for 67 patients (62.6%), ceftriaxone 250 mg intramuscular 250 mg in a single dose with doxycycline orally for 27 patients (25.2%), ciprofloxacin 500 mg orally a day for 7 days for six patients (5.6%), azithromycin 1 g orally in a single dose for three patients (2.8%), ceftriaxone intramuscular alone for two patients (1.9%), and ceftriaxone intramuscular with azithromycin orally for two patients (1.9%). among patients who complied with all average time to cure for both gu and ngu (sd) was 13.3 (6.6) days. ninety - six patients (42.3%) were lost to follow - up as shown in table 1. mean age of loss to follow - up patients was 29 years. among these patients, the rate of hiv infection was 11.6%. compared to patients that came to follow - up as appointed, msm patients had a significantly lower rate of loss to follow - up (p = 0.012). comparison of demographic data and clinical characteristics between male patients who were lost to follow - up and patients with good compliance demographic and clinical characteristics of gu and ngu patients are presented in table 2. shorter median time to onset, more purulent discharge, more dysuria, and more inguinal lymphadenopathy were found in gu patients than in ngu patients. mean age of ngu patients tended to be higher than that of gu patients, but the difference was not statistically significant. mucoid discharge, clear fluid discharge, and pruritus were presenting symptoms more often in ngu patients than in gu patients. a 2013 study conducted in men in the united states reported the number of gonorrhea and chlamydia urethritis cases to be 19.8% and 17.2%, respectively. from a 2011 study conducted in india, the prevalence of gu and ngu in an std clinic was 5.97% and 4.97%, respectively. in thailand, study of young thai men in northern of thailand reported percentage of gu and ngu among stds in 19931995 was 20.2% and 15.2%, respectively. the present study identified increases in the prevalence for both gu and ngu over the 8-year study period in thailand. antimicrobial resistance in n. gonorrhea is increasing and is becoming a public health crisis. a previous survey of n. gonorrhea antibiotic susceptibility in six southeast asian countries from 2009 to 2012 reported that 84%, 59.5%, and 87% of n. gonorrhea isolates were resistant to penicillin, tetracycline, and ciprofloxacin, respectively. in the present study, almost all n. gonorrhea isolates (97.2%) produced beta - lactamase and were resistant to penicillin, tetracycline, and ciprofloxacin. in addition, the patient loss to follow - up decreases a physician 's ability to evaluate treatment outcomes. if the prescribed treatment protocol fails, the spread of infection (especially resistant strains) to sex partners becomes a major concern.. this may be due to a higher awareness of health - care problems among msm. moreover, clinical management, including timely and accurate diagnosis and effective treatment, remains a challenge in resource - limited countries. as a result, diagnostic tests that are sensitive and that provide an accurate and rapid result are a necessary tool in an std unit setting. however, gram - stain test for urethritis in males still has an essential role due to its high specificity, with interpretable results available within a few minutes to a few hours. furthermore, the cost of gram - stain testing is very low and is available in every hospital. however, because of its lower sensitivity, a negative gram stain should not be considered sufficient for ruling out infection in asymptomatic men. additional testing to identify etiology is recommended to prevent complications, reinfection, and transmission because definitive diagnosis improves treatment compliance, delivery of risk reduction interventions, and partner notification. naat is preferred for detection of chlamydia trachomatis and n. gonorrhoeae, with urine being the preferred specimen in males. treatment modalities in our clinic were subject to physicians ' decisions according to uncertainty treatment guideline and patient 's history of drug allergy. appropriate treatment is becoming more challenging due to increasing antimicrobial resistance, particularly in gonorrhea and mycoplasma genitalium infections. having acknowledged the increase in antibiotic resistance, the effectiveness of antibiotic treatment remains high. by way of example, a previous study reported that only 0.3% of n. gonorrhea isolates in thailand were resistant to azithromycin. the current 2015 centers for disease control and prevention treatment guideline recommends ceftriaxone 250 mg intramuscular in a single dose plus azithromycin 1 g orally in a single dose. this dual therapy is considered to be the first - line regimen for gu treatment. from the findings of our study, the high loss to follow - up rates should raise awareness regarding the importance of patient compliance. moreover, because of the higher rate of gonococcal resistance to tetracycline and high rate of azithromycin susceptibility of m. genitalium, the use of azithromycin should be encouraged in thailand. to maximize compliance with recommended therapies, the administration of medications should be directly observed in the clinic or at least the first dose should be dispensed on site and directly observed in patients prescribed with multidose regimens. direct observation of single - dose therapy with azithromycin should be considered the first best choice in patients for whom adherence to multiday dosing is a concern. almost half of patients with gu or ngu were lost to follow - up, and one - quarter had recurrent urethritis. according to cure rate, even though high cure rate was demonstrated in patients who came to follow - up, we could not assume good outcomes in patients who lost to follow - up. good counseling at the first visit is essential to enhance the patient 's education and to improve the compliance. fast and easy access to services that provide accurate diagnostic testing and effective treatment should be a public health priority to prevent complications and reduce rates of disease transmission. almost half of patients with gu or ngu were lost to follow - up, and one- quarter had recurrent urethritis. fast and easy access to services that provide accurate diagnostic testing and effective treatment should be a public health priority to prevent complications and reduce rates of disease transmission. | background : poor follow - up compliance of patients with infectious urethritis is a recognized and serious public health problem in thailand.aim:the aim of this study was to determine treatment outcomes and loss to follow - up rate of male patients with gonococcal urethritis (gu) and non - gu (ngu) at a sexually transmitted disease (std) clinic at thailand 's tertiary hospital.methods:this retrospective chart review of male patients who sought treatment at stds clinic, siriraj hospital, and who were diagnosed with gu and/or ngu was conducted during january 2007 to december 2014 study period.results:two hundred and twenty - seven male urethritis patients were included in this study with a mean age was 29.5 years. gu and ngu were found in 120 (52.9%) and 107 (47.1%) of patients, respectively. overall prevalence of gu and ngu during the 8-year study period at std clinic, siriraj hospital, was 8.6% and 7.8%, respectively. ninety - six patients (42.3%) were lost to follow - up. recurrent urethritis was found in 23.8% of patients, and hiv infection was identified in 11.6%. mean age of patients lost to follow - up was 29 years. compared with patients who attended every scheduled follow - up visit, men who have sex with men had a significantly lower rate of loss to follow - up (p = 0.012).conclusion : almost half of patients with gu or ngu were lost to follow - up, and one - quarter had recurrent urethritis. fast and easy access to services that provide accurate diagnostic testing and effective treatment should be a public health priority to prevent complications and reduce rates of disease transmission. |
for decades, tracking studies with eels have been conducted to reveal the oceanic migration routes to their mysterious spawning areas6,000 km from the european coast by using acoustic and archival tags (tesch 2003). recently, the use of pop - up satellite tags (psats), developed for tracking large animals (> 50 kg), were used in several studies for tracking eels (aarestrup. although the oceanic migration routes were partially obtained this way, the assumed spawning areas have never been reached. results showed a much lower travel speed than required for reaching the spawning areas in time. the minimal speed for european silver eels is 0.4 m s, 6,000 km within 6 months the time between leaving the coast and the occurrence of the first larvae (tesch 2003). 2009) found an average horizontal migration speed of 13.8 km day, which corresponds to ca. the effect of psats on the swimming efficiency and swimming behaviour of eels has not been tested before. eels have been shown to be very efficient swimmers ; they are some five times more efficient than salmonids with respect to the energy cost of swimming (van ginneken and van den thillart 2000 ; van den thillart. particularly this extreme high swimming efficiency must have been a strong selection force during evolution ; as a consequence, any interference with shape and movement must have a serious impact on the cost of transport (cot) and thus interferes with successful spawning migration. current psats have almost the same cross section as that of an eel of 1 kg, which therefore almost doubles the hydrodynamic drag. the psat resists not only forward but also sideward motion, which must have an additional disturbing effect on the anguilliform mode of swimming. furthermore due to the positive buoyancy of the psat, there will be an additional constant pull upwards, which also impairs the swimming mode. thus, one may expect a strong interference of a psat with swimming mode, swimming capacity and swimming efficiency. in this study we tested the effect of a small psat on the swimming efficiency of female european eels of about 1 kg. swim performance tests (trial 15) were carried out in 127-l blazka - type swimming tunnels (van den thillart. 2004) with running natural seawater (35 ppt) at 18 1c under red light according to the speed test protocol of palstra. five different trials were performed on each of eight farmed female silver eels (1,026 31 g ; 76.5 1.0 cm). every trial included a 1-day swim performance test (speed test) after appropriate conditioning. the trials were performed in the following order : (1) no tag (control) ; (2) no tag, after operation (to check the effect of the operation) ; (3) positive buoyant tag ; (4) neutral buoyant tag ; (5) no tag, after removal of the tag (to test handling and training effects). the 1-day speed test was carried out in five steps from 0.4 to 0.8 m s with increments of 0.1 m s at 2-h intervals. the optimal swimming speed speed with lowest cot is within this range of speeds, as demonstrated in a previous study (palstra. it was calculated from the decline of the oxygen concentration in the closed swimming tunnel. the oxygen levels were kept between 95% and 75% air saturation. to restore the initial saturation level, the swimming tunnel was flushed with air - saturated water during the last 30 min of each interval. the swimming tunnels were calibrated with a doppler flow technique to determine the correct water flow in the tunnel (van den thillart. table 1protocol swim performance testdayaction1adaptation ; overnight at 0.4 m s2trial 1 : no tag, before operation speed test ; overnight at 0.4 m s3 + 4operation, attach base ; 2 days at 0.4 m s5trial 2 : no tag, after operation speed test ; overnight at 0.4 m s6psat attached ; overnight at 0.3 m s7trial 3 : positive buoyant tag speed test ; overnight at 0.3 m s8added metal weight to tag ; overnight at 0.3 m s9trial 4 : neutral buoyant tag speed test ; overnight at 0.3 m s10tag removed ; overnight at 0.4 m s11trial 5 : no tag, final test speed test ; endspeed test includes five 2-h intervals 0.40.8 m s in steps of 0.1 m s. after the last step, the eels were kept at 0.1 m s for 1.5 h to measure the resting rate. the oxygen consumption was measured over the first 90 min of the interval ; thereafter, the tunnel was flushed for 30 min to restore the oxygen level. before introduction to the tunnels and before the operation, the eels were anaesthetized with 1 ml l clove oil solution (10% clove oil dissolved in 96% ethanol) protocol swim performance test speed test includes five 2-h intervals 0.40.8 m s in steps of 0.1 m s. after the last step, the eels were kept at 0.1 m s for 1.5 h to measure the resting rate. the oxygen consumption was measured over the first 90 min of the interval ; thereafter, the tunnel was flushed for 30 min to restore the oxygen level. before introduction to the tunnels and before the operation, the eels were anaesthetized with 1 ml l clove oil solution (10% clove oil dissolved in 96% ethanol) the eels were introduced in the tunnels 1 day before each trial, and left overnight while swimming at 0.4 m s. after trial 1, a 20 9-mm teflon plate (1.5 mm thick) was placed under the skin of the experimental animal about 30 mm in front of the dorsal fin, the same location as used by jellyman and tsukamoto (2002). a silk line was pulled through the plate and skin on either side ; the line was left outside the body. after the operation, the eels were placed back into the swimming tunnels and swam for 2 days at 0.4 m s. trial 2 was carried out 2 days after the operation. thereafter a psat was attached to the teflon plate, leaving about 20 mm between the fish and the tag. the eels were placed back in the tunnel and left swimming overnight at 0.3 m s. the lower speed was necessary, as the eels could not swim faster overnight with a psat attached. at the end of trial 3, a small metal weight (10.9 g) the eels were placed back in the tunnel and left swimming overnight at 0.3 m s. after trial 4 the psat was removed and the eels were introduced in the tunnel and left overnight swimming at 0.4 m s. the last trial (5) was carried out to control whether the handling and previous swim tests changed the swim performance. at the end of each trial, the speed was set at 0.1 m s for 1.5 h to measure resting conditions. this low speed was necessary to keep the water well mixed, while low enough for the eels to stay at rest. the optimal swimming speed (cot) and critical swimming speed were calculated according to brett (1964). according to this method, the swimming speed is increased in intervals of > 30 min, in 810 equal steps up to collapse ; the critical speed is then interpolated from the last two speeds. dimensions of the psat were : body length 115 mm, diameter first part 20 mm, diameter second part non - functional 40 mm, length of antenna 170 mm and weight in air 53 g. the used tags were not functional but corresponded in size and buoyancy with minipat from wildlife computers. the experiments were carried out according to the dutch law on animal experimentation with approval # dec-10089. in this study, female silver eels swam at 0.10.8 m s, with and without a psat. the swimming performance was tested with the 1-day speed test at five different conditions (table 1). there were no significant differences in oxygen consumption rates (at all speeds) between the conditions without tag ; i.e. trials 1, 2, 5 (fig. therefore, we can infer that there was no negative effect of the operation on the swimming efficiency of the eels. thus the results of trial 1, 2 and 5 can be considered as controls. in contrast, when a psat was attached to the eels (trials 3 and 4), the oxygen consumption during swimming was more than twofold higher compared to the control groups (p 0.05), the results suggests that the drag more than the lift of the psat may have been the most crucial factor impairing swimming performance. although, the vertical migration as observed for eels in the wild (aarestrup. 2009) could not be simulated during this study. fig. 1effect of pop - up satellite tags on a oxygen consumption (in milligram o2 per kilogram per hour) and b cost of transport (in milligram o2 per kilogram per kilometer) for swimming 1 kg european eels (n = 8). treatment levels were : no tag, before operation (filled square, black) ; no tag, after operation (filled triangle, blue), with positive buoyant tag (multiplication sign, green) ; with neutral buoyant tag (filled diamond, orange) ; no tag, final test (filled circle, red). asterisk indicates significant difference from control at p < 0.05 effect of pop - up satellite tags on a oxygen consumption (in milligram o2 per kilogram per hour) and b cost of transport (in milligram o2 per kilogram per kilometer) for swimming 1 kg european eels (n = 8). treatment levels were : no tag, before operation (filled square, black) ; no tag, after operation (filled triangle, blue), with positive buoyant tag (multiplication sign, green) ; with neutral buoyant tag (filled diamond, orange) ; no tag, final test (filled circle, red). asterisk indicates significant difference from control at p < 0.05 also the cot was significantly higher when eels were swimming with a psat (p < 0.001, fig. 1b), i.e. a change from 25 to 75 mg o2 kg h. eels with a psat showed irregular swimming at 0.5 m s and fatigued after a few minutes when the speed was raised to 0.6 m s. as eels have a very regular swimming mode with a nearly constant wave frequency, irregular swimming was immediately visible. the calculated critical swimming speed with a psat was significantly lower compared to the control groups ; i.e. 0.48 0.02 and 0.73 0.02 m s respectively (p < 0.001). in contrast, during the trials without psat, all eels were able to swim up to 0.8 m s. the results of trials 1, 2, and 5 (without psat) were comparable to those published recently (palstra. in a recent study, steinhausen (2006) observed that an external tag increased the oxygen consumption of cod at high swimming speeds, while optimal and critical swimming speeds were significantly decreased. in our study, with the much bigger psat, we observed severely impaired swimming already at low speeds. the difference between the two studies may be due to the different mode of swimming, i.e. subcarangiform vs anguilliform, but more likely due to the rather large difference in extra drag (corresponding to the cross section surface of the tag, i.e. 2 vs 13 cm respectively). although tag technology is advancing, tags show negative effects in several studies on swimming performance, survival, behaviour and growth rates (bridger and booth 2003 ; makiguchi and ueda 2009). negative effects of external tags on swimming performance are also found in other animals like penguins (saraux. 2011) and seals (hazekamp. 2010). our results show a dramatic effect of the smallest available psat on the swimming efficiency of european eels : a more than twofold higher oxygen consumption at all tested cruising speeds and a severely reduced swimming performance. hence, smaller psats with much less interference on swimming performance are required to unravel the still mysterious journey to the spawning areas of european eels. | the journey of the european eel to the spawning area in the sargasso sea is still a mystery. several trials have been carried out to follow migrating eels with pop - up satellite tags (psats), without much success. as eels are very efficient swimmers, tags likely interfere with their high swimming efficiency. here we report a more than twofold increase in swimming cost caused by a regular small satellite tag. the impact was determined at a range of swimming speeds with and without tag in a 2-m swimming tunnel. these results help to explain why the previous use of psats to identify spawning sites in the sargasso sea was thus far unsuccessful. |
over the past years in italy, the current affairs press has produced ample information on the subject of haccp (hazard analysis and critical control points) with the goal of providing support to those in charge of food companies and to allow these companies to conform to that provided for by italian legislative decree no.155/97, which introduced the self - monitoring system. after an initial haccp system planning period, marked on the legislative level by the different derogations to the sanctions, we have now reached complete actuation of self - monitoring. during this phase, the evaluation of self - monitoring plans plays an important role, as it is an important activity for interlocution between the person in charge of the food industry and the health authority. the evaluation investigates the concreteness of the self - monitoring plans with reference to the hygienic - sanitary safety of foods. food companies essentially - in addition to having the piece of paper of the self - monitoring plan (and in many cases, they are substantial, well laid out volumes with various colours) must adopt all the procedures indicated in the plans themselves in order to knock down or reduce potential hazards [25 ]. panunzio. introduced and used an evaluation sheet that took into consideration the most important aspects of self - monitoring from the point of view of specificity, simplicity, feasibility and adherence. in this manner, different self - monitoring plans were evaluated using these four profiles ; a score was determined and for each profile. the question we asked ourselves during the study of this work was what evaluation of the self - monitoring plans can be made in the light of adherence to the haccp method ? adherence was taken into consideration because it is the most critical aspect of the self - monitoring plans that contain haccp principles. when a plan is drawn up based on photocopies of other non - specific plans, the haccp method is generally almost non - existent because it was not based on the individual company [710 ]. therefore, we evaluated a sample of self - monitoring plans drawn up by just as many food companies located in the lha territory of foggia. during the period from january december 2006, 116 self - monitoring plans were taken under examination and evaluated, which had been drawn up by food companies whose production, distribution and/or sales offices were located within the territorial area of the lha of foggia. using randomization and the technique of layered sampling, we proceeded with identifying the food companies. the sample extracted in this manner, even if not representational, can supply a measure of the most common methodological mistakes. the evaluation aspects focused their attention on adherence to the haccp method, as it was felt to be the main measure of credibility for a self - monitoring plan - self - monitoring that explicitly refers to this method. metaphorically, adherence is the neck of a bottle that contains specificity, simplicity and feasibility of haccp plans. therefore, beginning with the evaluation sheet, errors were classified as general and specific while the plans were divided into classes of activities of the food companies, as indicated in enclosure no. 1 of italian ministerial decree no. table 1 indicates the distribution of the companies per activity class, whose self - monitoring plans were evaluated. based on the evidence of the application of the evaluation sheet, the errors were classified as general and specific. the general errors were linked with three types : terminology, voluminosity and redundancy. concerning terminology, a very frequent error was that of using the name self - monitoring manual for what was the self - monitoring plan thus confusing a non - specific instrument such as the manual, which can be written by public and private bodies whose effectiveness depends on the validation of the same by the ministry for health and which can be of support only in the identification phase of potential hazards and procedures of correct hygienic practice. voluminosity refers to the voluminosity of the self - monitoring plans ; it would almost seem that the validity of the plans is directly proportional to their paper weight. well - packaged self - monitoring plans were examined with good typographical layout and coloured sheets, but that were filled with superfluous elements such as the legislation, philosophy and the history of haccp. these elements invalidated rapid consultation of the plan, making the plan lack in the inspiring motto only write what you have to do, do what you have written. concerning redundancy, a few plans did not follow a precise table of contents for their subjects but rather many things were repeated in different parts of the plan. this resulted in rather difficult specific, immediate and unambiguous comprehension of the procedures to be followed. the specific errors refer to : the plan for transversal phases, confusion of critical limits, hazard non - specificity and lack of a time plan for the control. in the self - monitoring plans for transversal phases, the sheets were not drawn up following the production flow chart but rather by homogeneous phases of the production process. this way for example, instead of a few sales businesses having a plan concerning frozen products that included the receiving, storing and sales phases, a plan for the receiving phase, one for the storing phase and finally one for the sales phase were designed for all food products for sale. in this manner, the decision tree is a diagram indicating a few questions / answers, built on the flow of the production activity. its use is an essential tool to remove the inherent subjectivity in self - monitoring. non - specificity of the hazard refers to the generic wording such a biological, physical or chemical contamination. if the hazard is non - specific, it goes without saying that the rest of the plan can only be generic and therefore useless. finally, a few plans did not indicate a time plan for the controls to carry out, therefore there was no precise agenda to follow for hazard self - monitoring measures. during the period january december 2006, the servizio di igiene degli alimenti e della nutrizione (food and nutrition health service) of lha evaluated 116 haccp plans of food companies. as can be seen, almost two - thirds of the plans examined contained general errors. the most interesting are : hotels, restaurants and coffee bars in 85% of the cases against one - third of the food industries. terminological errors involve half of the haccp plans containing general errors and 47% involve superfluous elements, while 60% of the cases include subject redundancy. table 4 indicates the percentages of each type of general error based on the number of plans that contained these errors. subject redundancy is the general error that haccp plans contained the most of for all activity classes of food companies. with the exception of terminology errors in the food and beverage class, table 6 indicates the frequency of the specific errors in relation to corporate activity classes. the lack of procedures is the least frequent error, while the other three types of errors involve the different activity areas in a different manner. the area containing the most specific errors, in the four types considered, in haccp plans, is that of retail trade, hotels, restaurants and coffee bars. the most frequent specific errors are the transversal plan and the critical limits, both concerning the retail trade area. in table 7, specific errors are layered on the frequency of the haccp plans that contain errors. frequencies are high for all types of errors and for the different activity classes, except for the food and beverage industry, where there are only three haccp plans containing specific errors, which all involved erroneous critical limits. in the same manner, the non- specificity of the hazard involved all incorrect haccp plans in the canteen area. table 8 illustrates the performance relative to the general and specific errors per corporate activity class. performance is obtained from the relation between the error percentage and the percentage of the corporate industry taken into consideration. the emerging results indicate that the canteen area obtained the worst score (13.92 in general error performance ; 18.561 in specific error performance), followed by wholesale businesses and business intermediaries (the two performances respectively : 5.44 and 5.87) and then at a distance, the other areas were distributed on the same level. the goal of this work is to examine the errors contained in the self - monitoring plans of the food industry that are modelled on the haccp method, in compliance with italian leg. even if the sample examined is not representational of the entire universe of food companies present in the territory of the lha of foggia, it can nevertheless be considered a preliminary study concerning the evaluation of methodological and formal errors of haccp plans. an analysis of the results does not indicate a reassuring picture of self - monitoring. in our opinion, the error with the most significance is the non - specificity of the plans that involve all activity classes, with the exception of the food and beverage industry. even if it is difficult to be able to attribute the meaning of this last data to a sampling fault or to a specific reality, the high frequency of the non - specificity of the hazard, if confirmed by further studies, could nevertheless indicate a passive acceptance of the contents of italian legislative decree no. 155/97 by food companies who have obviously understood formally conforming to the law as the adoption of a self - monitoring plan, but who have not taken enough interest concerning the possibility of using this tool to improve the hygienic - sanitary safety of foods. although it is a theory still to be proven by facts, negative feedback might be given. therefore, an important role is taken on by those professionals who, in their capacity as true and proper food company trainers, are involved throughout the different phases of making adjustments to and managing self - monitoring [1112 ]. if the output of the self - monitoring system is the recording of procedures and self - monitoring plans, the outcome of haccp is the healthiness of food. in our capacity as a public control body, we are interested in both ; however, the greatest attention is paid to outcome in order to guarantee the healthiness of foods. the evaluation of haccp plans concerning methodological errors can constitute a method of dealing with the output while not losing sight of the final goal of safeguarding the consumer [1416 ]. | with respect to food safety, many works have studied the effectiveness of self - monitoring plans of food companies, designed using the hazard analysis and critical control point (haccp) method. on the other hand, in - depth research has not been made concerning the adherence of the plans to haccp standards. during our research, we evaluated 116 self - monitoring plans adopted by food companies located in the territory of the local health authority (lha) of foggia, italy. the general errors (terminology, philosophy and redundancy) and the specific errors (transversal plan, critical limits, hazard specificity, and lack of procedures) were standardized. concerning the general errors, terminological errors pertain to half the plans examined, 47% include superfluous elements and 60% have repetitive subjects. with regards to the specific errors, 77% of the plans examined contained specific errors. the evaluation has pointed out the lack of comprehension of the haccp system by the food companies and has allowed the servizio di igiene degli alimenti e della nutrizione (food and nutrition health service), in its capacity as a control body, to intervene with the companies in order to improve designing haccp plans. |
estrogen - based therapies decrease hot flashes by up to 90%, but their use is generally discouraged in breast cancer survivors and in women with an increased risk of breast cancer.13 several hormonal and nonhormonal therapies are claimed to alleviate hot flashes. these include hormonal agents, such as progestational agents, complementary and alternative therapies and supplements, and pharmacologic agents. hormonal therapies seem to be most effective, but women at risk for or who have survived breast cancer may carry an increased risk of new or relapsed cancer.4 as such, studies have aimed to find nonhormonal agents that can decrease this bothersome symptom. many therapies have been studied, but none seem to be as effective as hormonal therapies.5 other commonly prescribed nonhormonal treatments for hot flashes are well described in a recent review,6 and include selective serotonin reuptake inhibitors (ssris), serotonin norepinephrine reuptake inhibitors (snris), clonidine, gabapentin, and others. many women take prescription and over - the - counter medication for treatment of hot flashes. there is sparse information about what women are actually taking. in a population of breast cancer patients, we aimed to describe the frequency of hot flashes and night sweats, list what treatments were employed, and explore the possible association of hot flashes and use of calcium supplements. we retrospectively reviewed clinic charts of a randomly selected sample of women with breast cancer treated at a single institution. cases were randomly selected for inclusion by arranging all breast cancer cases in alphabetical order and then choosing every fourth chart for review. the study was approved by the institutional review board at wake forest university school of medicine. information gathered included the following : demographic information including age at diagnosis and race ; adjuvant hormonal therapy use for breast cancer, defined as any record in the chart for prescription of tamoxifen or an aromatase inhibitor ; menopausal status as recorded in clinician notes ; presence or absence of hot flashes and/or night sweats as recorded in the chart ; prescribed treatment for hot flashes as indicated in the clinical records ; and any record of calcium supplementation in the medication list, although the exact amount of calcium per day and additional use of vitamin d was not available in most charts. descriptive statistics were used to describe variables. to explore the associations between variables, the chi - square test and fisher s exact tests were used for variables with larger and smaller sample sizes, respectively. menopausal status was post- in 79%, peri- in 10%, and pre- in 11%. a record of hot flashes was found in 42 (49%) charts and a record of night- time hot flashes was found in 18 (21% of the total and 43% of those with hot flashes) charts (table 2). there was no statistically significant association between presence of hot flashes and antihormonal therapy ; 42% of women not taking antihormonal therapy reported hot flashes versus 59% on antihormonal therapy (p = 0.13). treatment for hot flashes was recorded in 31 (36%) of the 86 charts. treatments for hot flashes included ssris / snris (n = 19), clonidine (n = 7), bellergal - s (n = 8), sleep - aid (n = 7), and others (n = 5). calcium supplementation was recorded in 31% of records. interestingly, we found an association between hot flashes and calcium supplementation. of women with hot flashes, 44% took calcium supplements and 56% did not ; of women without hot flashes, 18% took calcium supplements and 82% did not (chi - square p = 0.02) (table 3). hot flashes were recorded in half of this group of primarily postmenopausal breast cancer survivors. among women with hot flashes, these figures are similar to what has been reported in a review of the world literature on prevalence of hot flashes and night sweats.7 we found no relationship between use of antihormonal therapy and occurrence of hot flashes. in contrast, clinical trials and other studies find that menopausal symptoms are more common in breast cancer patients versus controls and in patients taking antihormonal therapy versus not.712 there are several possible explanations for the lack of an effect of antihormonal therapy on occurrence of hot flashes in our analysis. first, there may have been a lack of recorded information about hot flashes in the medical records, but at this particular institution, there were ongoing studies for women with hot flashes and inquiry about the symptom was common. second, the study population, although randomly selected from an existing group, may not be representative of the group or of the population of breast cancer survivors as a whole. alternatively, the finding may be real in this population of primarily postmenopausal women, whose hot flash level may not have been as significantly affected by the addition of adjuvant hormonal therapy. in this sample of breast cancer survivors, this is lower than found in a survey of a family practice, where almost 70% of postmenopausal women with moderate - to - severe hot flashes said they would want an intervention, with a desired hot flash reduction of at least 50%.13 breast cancer survivors in this study were taking a variety of treatments to manage symptoms of hot flashes. these included ssris / snris, clonidine, bellergal - s, sleep - aid, and others. none of the charts reviewed recorded use of estrogen or herbal supplements to manage hot flashes. this is in line with therapies that have been proven helpful and safe in managing hot flashes in breast cancer survivors.6 in a systematic review and meta - analysis of data on nonhormonal therapies for menopausal hot flashes, compared with placebo, the number of daily hot flashes decreased with ssris or snris (mean difference 1.13 ; 95% confidence interval [ci ] 1.70 to 0.57), clonidine (0.95 ; 95% ci 1.44 to 0.47), and gabapentin (2.05 ; 95% ci 2.80 to 1.30).5 this chart review took place in 2003, before gabapentin and pregabalin had been shown to be helpful for treatment of hot flashes.1416 in a case - control study of 73 breast cancer survivors, 29% reported use of nonestrogen therapy specifically for treatment of menopausal symptoms.12 in their study, which was published perhaps before widespread acceptance of the value of ssris / snris for alleviation of hot flashes, the most commonly reported nonestrogen treatments were deliberate increase in either dietary or supplemental soy products, vitamin e, other herbal preparations, clonidine, vitamin b6, and other miscellaneous therapies. antidepressants, including tricyclic antidepressants (used in 2/73 cases) and venlafaxine (used in 1/73 cases), were among the miscellaneous therapies. having observed reports that calcium might alleviate symptoms of premenstrual syndrome,17,18 we were interested to explore the possibility that calcium supplements might alleviate hot flashes. interestingly, we found an association between calcium supplementation and hot flashes, but it was not in the direction that we had hypothesized. women whose records indicated use of calcium supplements were more likely to report hot flashes than those not using calcium. one possible explanation for this could be as simple as the fact that women with lower estrogen levels, and thereby more hot flashes, have lower bone mass and are more frequently told to take calcium supplements. in other words, we may simply find more hot flashes in women who take calcium because of low estrogen levels. another potential explanation is that some women were taking calcium supplements to treat hot flashes, but we did not find this was the case from the records reviewed. however, this was a retrospective study, and we realize that we may not be able to detect when a patient independently decided to take calcium supplements to treat hot flashes and, if this particular therapeutic strategy was of low efficacy, we would detect a higher level of hot flashes in these women. a plausible endocrinologic explanation for calcium s relationship with hot flashes is through calcitonin gene - related peptide (cgrp). calcium stimulates secretion of two peptides, ie, calcitonin, a hormone that functions to reduce blood calcium levels by increasing renal calcium excretion and decreasing release of calcium from bone, and cgrp, a neuropeptide that may be important in vasomotor - related symptoms. cgrp is a potent vasodilator neuropeptide and has been shown to be closely related to the occurrence of menopausal hot flashes.1922 in addition, cgrp is high in postmenopausal women with hot flashes and decreases after successful treatment.23,24 although a causal explanation for the increased reports of hot flashes in women also taking calcium can not be made from this study, further exploration of the association between hot flashes and calcium supplementation is warranted. in summary, complaints of hot flashes were found in the records of approximately half of these breast cancer survivors and were life - disrupting in 17%. a variety of medications were used for hot flashes, including those that were considered effective at the time. in fact, we found an association between calcium supplementation and hot flashes, such that women taking calcium supplements were more likely to report hot flashes. because the combination of calcium and vitamin d is recommended for good bone health, the relationship of this combined supplementation and hot flashes warrants exploration | aimsin breast cancer survivors, we aimed to describe the frequency of hot flashes and night sweats, frequency and type of treatment, and the association of hot flashes and use of calcium supplements.methodscharts of breast cancer survivors were reviewed for information about hot flashes, treatment for hot flashes, and calcium supplementation. associations between variables were explored using the chi - square test and fisher s exact test.resultseighty-six charts were reviewed. mean age of the women was 58 years and 79% were postmenopausal. forty - two (49%) of women had hot flashes and 18 (21%) had night sweats. thirty - one (36%) were treated for hot flashes. treatment included selective serotonin reuptake inhibitors / serotonin - norepinephrine reuptake inhibitors (n = 19), clonidine (n = 7), bellergal - s (n = 8), sleep - aid (n = 7), and other (n = 5). calcium supplementation was recorded in 31%. of women with hot flashes, 44% took calcium supplements ; of women without hot flashes, 18% took calcium supplements (chi - square p = 0.02).conclusionhot flashes were recorded in 49% of this group of primarily postmenopausal breast cancer survivors. women with hot flashes were more likely to be taking calcium supplements. further exploration of the association between hot flashes and calcium supplementation is warranted. |
antidepressant medication is a standard treatment and the first choice for depressed patients in current psychiatric guidelines.1 approximately 60% of patients respond well to active treatment,24 but ~40%50% of patients using the full dosage and duration of antidepressant medications still have residual symptoms and can not obtain full remission.46 one reason for this may be that some depressed patients discontinue treatment because of adverse effects or higher economic burden.2 moreover, misdiagnosis, genetic inheritance, and comorbid somatic disease also influence the outcome of treatment.7,8 in a 5-year follow - up study, riihimki studied 137 major depressive disorders (mdds) and found that the patients spent 34% of the follow - up time in major depressive episodes, 24% in partial remission, and 42% in full remission. szauliska reported that obstructive sleep apnea (osa) is present in 11%18% of patients with mdds, 15%48% of patients with schizophrenia, and 21%43% of patients with bipolar disorder, but in only 5% of the general population.11 one study has shown that patients diagnosed with osa are at an increased risk of a diagnosis of depression.12 additionally, the symptoms of sleep apnea might imitate the symptoms of depression, including sleep disturbance, general fatigue, decreased volition and judgment ability, aggravated cognitive impairment, and a lower quality of life.10 in addition, osa might exacerbate symptom severity in mdds.8 participants with both mdds and osa also reported more severe and longer episodes of depression.13 meanwhile, osa might influence response to pharmacological treatment of depression as well as reduce patient adherence to antidepressants.8,14 waterman studied 400 patients with mdds and found that those with comorbid osa were 1.5 times less likely to respond to 12 weeks of treatment with the antidepressant venlafaxine than those with either depressive disorder occurring alone. kerner confirmed that the cognitive performance of mdds comorbid with severe osa is even worse than that of mild to moderate osa, while some reports suggest that depressive symptoms might be improved in at least some patients receiving continuous positive airway pressure therapy or other treatment for osa.1619 from the point of view of the physicians, this suggests that screening and treatment for osa is very important.10 in depressive disorders, comorbid osa is even higher.10 physicians should consider screening for osa when assessing patients for depression, as it may indicate more difficulty to treat depression.8,19 however, it is difficult to define who actually is at risk of osa in depressive disorders. this has prompted the need to identify osa using appropriate risk factors in clinical settings. multiple studies have found that sex, age, obesity, snoring, pharyngeal abnormalities, and cephalometric features are risk factors for osa in the general population.2022 hattori studied 32 mood disorder patients and found that appropriate osa risk factors (such as apnea, snoring, and body mass index [bmi ]) may help to better identify osa.23 nonetheless, to our knowledge, there have been few studies on risk factors for osa in depressive disorders. we screened osa patients with mood disorders presenting with depression using polysomnography (psg) and examined which or how many risk factors would contribute most to osa screening. all participants provided written informed consent to be included in the study, and the study design was approved by the ethics committee of sir run run shaw hospital, zhejiang university school of medicine. we reviewed 1,091 medical files of patients who had undergone in - room psg from december 2013 to december 2015. the inclusion criteria were as follows : 1) diagnosis of an mdd or a bipolar disorder (in a major depressive episode) through a structured mini - international neuropsychiatric interview (mini) based on the diagnostic and statistical manual of mental disorders, fourth edition (dsm - iv) ; 2) patients aged 18 years or older ; and 3) the hamilton depression rating scale (hamd ; 17 items) 10. patients with bmi 30 kg / m, underlying respiratory disease, history of taking drugs that can affect normal sleep architecture and psychotic symptoms, suicidal thoughts, restless legs syndrome, diagnosis of other sleep - related disorder, other clinical phases of bipolar disorder, and a previous history of treatment for osa were excluded. a total of 115 patients met the criteria and were divided into non - osa (n=56) and osa (n=59) groups according to psg results. patients who had apnea hypopnea index (ahi) 3%. the number of episodes of apnea and hypopnea per hour during total sleep time was calculated, and the result was the ahi. the hamd, the hamilton anxiety rating scale (hama), and the pittsburgh sleep quality index (psqi) were used for included patients when they underwent psg. to evaluate the relation between clinical factors (sex, age, and bmi, hamd, hama, psqi, diagnosis [mdd or bipolar disorder ]), and ahi, logistic regression was used., chicago, il, usa), and p 3%. the number of episodes of apnea and hypopnea per hour during total sleep time was calculated, and the result was the ahi. the hamd, the hamilton anxiety rating scale (hama), and the pittsburgh sleep quality index (psqi) were used for included patients when they underwent psg. to evaluate the relation between clinical factors (sex, age, and bmi, hamd, hama, psqi, diagnosis [mdd or bipolar disorder ]), and ahi, logistic regression was used., chicago, il, usa), and p<0.05 was considered as statistically significant. seventy - four patients were diagnosed with mdd, and 41 were diagnosed with bipolar affective disorder. on psg, 59 patients were diagnosed with osa (ahi 5) and 56 patients were diagnosed with non - osa (ahi < 5 ; table 1). when referring to the correlation between risk factors and ahi, the results showed a significant association between the ahi and diagnosis (mdd or bipolar disorder [in a major depressive episode ]), bmi, hamd, and psqi (table 2). in all, 15%30% of patients with depressive disorder were classified as having treatment - resistant depression.23 a previous study reported that osa is present in 11%18% of patients with mdds.10 from this, it is reasonable to assume that some treatment - resistant depression is comorbid with osa.24 however, of the 115 depressive disorder patients who met the inclusion criteria in the present study, 51.3% had osa. additionally, in the logistic regression analysis, the results showed a higher significant relation between ahi and diagnosis (mdds or bipolar disorder [in a major depressive episode ]), bmi, hamd, and psqi. therefore, we may consider that there were many undiagnosed osa cases in depression patients. of the 115 patients in the present study, 41 were diagnosed with bipolar depression and 74 were diagnosed with unipolar depression. of the 59 depressive disorder patients who met the osa criteria, 16 were diagnosed with bipolar depressive disorder and 43 were diagnosed with unipolar depression. meanwhile, the logistic test results showed that the risk of comorbid osa in unipolar depression is 2.72 times that of bipolar depressive disorders. it is thought that the osa diagnosis rate in mdd patients is higher than that in bipolar disorder patients, and the possibility can not be ruled out that the sample included a high proportion of patients with mdd. furthermore, a significant correlation was seen between the hamd and psqi scores and the ahi or osa diagnosis. thus, it seems that having a higher hamd or psqi score is associated with increased probability or possibility of having osa. a significant correlation was seen between ahi and depressive severity, as well as sleep quality, according to hamd and psqi scores in the present patients. some studies have indicated that risk factors for osa include sex, age, obesity, pharyngeal abnormalities, and cephalometric features.2022,25 in our results, significant associations were noted between the bmi and ahi or osa diagnosis. it would be logical to assume that a higher bmi is associated with increased probability or possibility of having osa. therefore, it may be concluded that these risk factors (bmi, hamd, psqi, and diagnosis) would greatly increase the diagnosis rate of osa in the psychiatric department. depressed patients with the above risk factors or suspected osa should be referred to a sleep disorder center for evaluation by psg to confirm the diagnosis of osa. however, others found the predictive value of excessive daytime sleepiness for osa to be low.21,25 in addition, rosenthal and dolan25 found that nonspecific daytime sleepiness may be more strongly observed in mood disorder patients than in the general population because of psychotropic drugs or the underlying disease itself. clinical factors, such as cardiovascular disease, hypertension, and diabetes, have been shown to be associated with osa.13 however, the sample size was relatively small in the present study. in the future, investigations with a larger sample should be conducted, to further investigate whether treatment for osa might achieve further gains on mood. particular attention should be paid to depressive patients who are resistant to treatment, and psychiatrists should consider calling for voluntary osa screening. the clinical risk factors (diagnosis [mdds or bipolar disorder { in a major depressive episode } ], bmi, hamd, and psqi) could predict ahi or osa diagnosis. the presence of these risk factors, including bmi, hamd, diagnosis, and psqi, is more efficient for recognizing osa in mood disorders than a single risk factor. in clinical practice it is important to screen patients at high risk of osa when assessing patients for depression, especially those with unresponsive depression. treatment of osa could improve not only the compliance to pharmacological antidepressant treatment but also the treatment response rate for depression.14 psychiatrists involved in treating mood disorders comorbid with osa should be cautious in using drugs with a muscle relaxant effect such as benzodiazepines. | objectiveoverlap of obstructive sleep apnea (osa) complicates diagnosis of depressive disorder and renders antidepressant treatment challenging. previous studies have reported that the incidence of osa is higher in patients with depression than in the general population. the purpose of this article was to investigate clinical risk factors to predict osa in depression disorders.methodsa total of 115 patients diagnosed with major depressive disorder (mdd) and bipolar disorder (in a major depressive episode), who underwent overnight polysomnography, were studied retrospectively. they were divided into two groups : non - osa and osa. the patients who had apnea hypopnea index (ahi) < 5 were defined as the non - osa group, whereas the osa group was defined as those with an ahi 5. logistic regression was used to analyze the association among ahi and clinical factors, including sex, age, body mass index (bmi), hamilton depression rating scale (hamd), hamilton anxiety rating scale, pittsburgh sleep quality index (psqi), and diagnosis (mdd or bipolar disorder [in a major depressive episode]).resultsin 115 patients, 51.3% had osa. logistic regression analysis showed significant associations between ahi and diagnosis (mdd or bipolar disorder [in a major depressive episode ]), bmi, hamd, and psqi (p<0.05).conclusionthe findings of our study suggested that the rate of depression being comorbid with osa is remarkably high and revealed that there is a high rate of undetected osa among depressive disorder patients and untreated osa among mood disorder patients. the clinical risk factors (diagnosis [mdd or bipolar disorder { in a major depressive episode } ], bmi, hamd, and psqi) could predict ahi or osa diagnosis and contribute to osa screening in depressive disorder patients. |
adverse drug reactions (adrs) occur frequently with cardiovascular drugs leading to change in therapy, compliance, and increasing morbidity and mortality and inflating the healthcare cost. angiotensin - converting enzyme (ace) inhibitors have been increasingly used for the therapeutic management of several conditions, such as hypertension, acute myocardial infarction, left ventricular systolic dysfunction, chronic renal failure, and so on. large multicentric trials have proved that ace inhibitors not only increase the life expectancy, but also improve the quality of life in high - risk patients suffering from cardiovascular events. ace inhibitors have specific effect on myocardial and vascular cell growth, also referred to as remodeling, they have a greater protective potential than any other class of antihypertensive drugs. very few studies have conducted to evaluate the occurrence of adrs due to ace inhibitors. reporting of adrs has become an important component of monitoring and evaluation activities performed in hospitals. such adr reporting programs encourage surveillance for adrs, promote the reporting of adrs, and stimulate the education of health professionals regarding potential adrs. intensive monitoring study will help in obtaining detailed data on incidence and pattern of adrs. the present study was designed to evaluate the incidence and pattern of adrs due to ace inhibitors in the cardiology department. a cross - sectional observational study was carried out for the period of 6 months (january june 2010) in an indian teaching hospital. the ethical approval was obtained from institutional ethics committee prior to study initiation (uec/19/2010). prescription from each patient during his / her hospitalization in the ward during the study period was included. the information such as demographics, complaints on admission, routine biochemical investigations, were obtained from the patient 's clinical records. details necessary for evaluations regarding previous allergies, concomitant medications, comorbidities, and others were collected. all drugs were classified with anatomical therapeutic chemical classification (atc code level 1, who, 2008). factors studied were (a) patient characteristics [gender, age (> 18 years), comorbidities and length of stay ], (b) drug characteristics [number of drugs ] and laboratory investigations. identification of adrs were done based on the regular follow of the patients by analyzing the subjective findings like cough, and objective findings like routine monitoring of electrolytes, blood pressure, and serum creatinine. data on the reported adrs were evaluated to understand the pattern of the adrs with respect to patient demographics, nature of the reaction, characteristics of the drugs involved, and outcome of the reactions. causality : in order to assess the likelihood that drugs has caused the reaction, causality assessment was done using naranjo 's adr probability scale whereby the adrs were classified into certain, probable, possible, and unlikely to be drug induced depending upon the level of association. severity : depending upon the severity, adrs were classified into mild, moderate, and severe reactions using the criterion developed by hartwig. for severity assessment. preventability : adrs were categorized into definitely preventable, probably preventable, and not preventable using the criteria of schumock and thornton modified. predisposing factors : factors which could have predisposed to the occurrence of adrs in the individual reports were evaluated. predisposing factors were generally classified for the purpose of study into age, gender, multiple and intercurrent disease state, and polypharmacy. management strategies employed for the management of adrs were categorized as drug withdrawal, dose reduction, additional treatment for adr, and no change in regimen with any additional treatment. further, categorization of the outcome of adrs was done for response after dechallenge and rechallenge as well as the final outcome of the event. frequencies with percentage were used to summarize sex, number of drugs dispensed, frequency of adrs, drugs involved in the adrs, and severity of adrs. the chi - square test was used to find the association between sex, number of drugs, and adrs. spearman 's correlation was used to find the correlation between numbers of drugs, length of stay with adrs. the information such as demographics, complaints on admission, routine biochemical investigations, were obtained from the patient 's clinical records. details necessary for evaluations regarding previous allergies, concomitant medications, comorbidities, and others were collected. all drugs were classified with anatomical therapeutic chemical classification (atc code level 1, who, 2008). factors studied were (a) patient characteristics [gender, age (> 18 years), comorbidities and length of stay ], (b) drug characteristics [number of drugs ] and laboratory investigations. identification of adrs were done based on the regular follow of the patients by analyzing the subjective findings like cough, and objective findings like routine monitoring of electrolytes, blood pressure, and serum creatinine. data on the reported adrs were evaluated to understand the pattern of the adrs with respect to patient demographics, nature of the reaction, characteristics of the drugs involved, and outcome of the reactions. causality : in order to assess the likelihood that drugs has caused the reaction, causality assessment was done using naranjo 's adr probability scale whereby the adrs were classified into certain, probable, possible, and unlikely to be drug induced depending upon the level of association. severity : depending upon the severity, adrs were classified into mild, moderate, and severe reactions using the criterion developed by hartwig. for severity assessment. preventability : adrs were categorized into definitely preventable, probably preventable, and not preventable using the criteria of schumock and thornton modified. predisposing factors : factors which could have predisposed to the occurrence of adrs in the individual reports were evaluated. predisposing factors were generally classified for the purpose of study into age, gender, multiple and intercurrent disease state, and polypharmacy. management strategies employed for the management of adrs were categorized as drug withdrawal, dose reduction, additional treatment for adr, and no change in regimen with any additional treatment. further, categorization of the outcome of adrs was done for response after dechallenge and rechallenge as well as the final outcome of the event. frequencies with percentage were used to summarize sex, number of drugs dispensed, frequency of adrs, drugs involved in the adrs, and severity of adrs. the chi - square test was used to find the association between sex, number of drugs, and adrs. spearman 's correlation was used to find the correlation between numbers of drugs, length of stay with adrs. a total of 692 patients who were prescribed with ace inhibitors were analyzed during the study period in cardiology unit and 51 (7.36%) patients had developed 60 adrs. the significant proportions of patients with adrs were female 48 (80%) than in male 12 (20%). a total of 35 (58.33%) adrs were developed mostly in the age group of > 61 years, followed by the other age group and the patients who had taken more than 7 drugs developed 54 (90%) adrs. a total of 37 (61.66%) adrs were developed with a length of hospital stay of > 14 days, followed by 23 (38.33%) with 61 years, followed by the other age group and the patients who had taken more than 7 drugs developed 54 (90%) adrs. a total of 37 (61.66%) adrs were developed with a length of hospital stay of > 14 days, followed by 23 (38.33%) with < 7 days. the age, sex, and number of drugs taken were identified as the risk factors for developing adrs (p<0.05). spearman correlation showed that there was an extremely significant linear relationship (r=0.16, 95% ci=2.4113.44, p<0.000) was observed between number of drugs taken and adrs. similarly, a significant linear relationship (r=0.08, 95% ci=1.03 - 3.82, p<0.036) between sex and the adrs in patients and linear relationship (r=0.09, 95% ci=0.300.91, p<0.022) between age and the adrs in patients. ramipril 27 (45%), enalapril 18 (30.00%), captopril 11 (18.33%), and linisopril 4 (6.64%) were the most commonly observed drugs involved and the common adrs observed were cough 23 (38.33%), hyperkalemia 19 (31.66%), hypotension 13 (21.66%), and acute renal failure 05 (8.33%). incidence of individual suspected drugs with their adrs results are summarized in the table 2. respiratory disorders due to cough were 23 (38.33%), followed by metabolic and nutritional disorders due to hyperkalemia 19 (31.66%), which is presented in table 3. in majority, 47 (78.33%) of the cases, the suspected drug was withdrawn for the management of the adrs and a specific treatment for the reaction was instituted in 20 (33.33%). in 56 (93.33%) of the cases, the patient recovered from the reaction at the time of evaluation of the adr report. an improvement in the adverse reaction was observed in majority (76.8%) of the cases, in which dechallenge or dose reduction was done [table 4 ]. upon causality assessment, majority of the reports were rated as probable 56 (93.33%) followed by possible 4 (06.66%). a total of 13 (21.66%) of reactions were mild and moderate reactions accounted for 47 (78.33%). our study revealed that the overall incidence of aceis - induced adrs in the study populations were 7.367% which is low, compared to similar studies. a total of 58.33% of adrs were observed in the age group of more than 61 years and considering the result of the chi - square test for analytical evaluation of the influence of age on occurring adrs, it appears that older patients are more likely to experience an adr. moore. found an inverse relationship between age and adrs occurring during hospital stay. altered physiology in geriatrics could be another reason for higher incidence of adrs in the population. while considering the gender distribution, there was increased number of adrs present in women than men, which is similar to some earlier reported studies. several studies have found that risk of developing adrs was more in females than males. reasons suggested for this include differences in perception of adrs, pharmacology of adrs, differences in pharmacokinetics such as volume of distribution, polypharmacy, and hormonal differences between men and women. the incidence of adrs was more in the population receiving seven or more medications concurrently, which is supported by previously published studies. our results show that drugs like ramipril 45% and enalapril 30% had the higher incidence of adrs and the common adrs observed were found to be cough 38.33%, hyperkalemia 31.66%, and acute renal failure 8.33% and these results were similar to other studies conducted in various university hospitals.[2022 ] drug withdrawal or dose reduction is usually the first step to be employed for the management of an adr. in 78.33% of the cases, the suspected drug was withdrawn and dose reduction was done in 22.2% of cases. considering the final outcome, 93.33% of patients were recovered from adrs, whereas 6.66% of patients were continuing and these findings were similar to the one observed by suh. in their study patients. on causality assessment of adrs, majority of the reactions 93.33% were categorized as probable in nature, followed by possible 6.70%. considering the severity assessment of the reactions, majority of the reactions were categorized moderate in nature followed by mild and these findings were not comparable with the spontaneous reporting study. preventability assessment of adrs showed that 8.33% of the adrs were not preventable in nature. definitely preventable adrs were not present in this study compared to the study conducted on adverse drug reaction to cardiovascular drugs, where 1.9% of adrs were preventable. drug withdrawal or dose reduction is usually the first step to be employed for the management of adrs. | background : adverse drug reactions (adrs) occur frequently with cardiovascular drugs leading to change in therapy, increasing morbidity, and mortality.aim:the study was conducted to evaluate the incidence of adrs due to angiotensin - converting enzyme inhibitors in cardiology department.materials and methods : a cross - sectional observational study was carried out for a period of 6 months. the data were assessed for the pattern of the adrs with respect to patient demographics, nature of the reaction, outcome of the reactions, causality, severity, and preventability.results:among 692 patients, 51 (7.36%) had developed 60 adrs, and majority of cases (56.66%) were in the age group of > 61 years and most of them were developed in female (80%). the common adrs observed were cough, hypotension, hyperkalemia, and acute renal failure. in 21.66% cases the dose of the suspected drug was altered and in 78.33% cases the drug was withdrawn. considering the outcome, 93.33% of cases recovered from adrs, whereas in 6.66% cases were continuing. causality assessment showed that majority of adrs was probable and were found to be moderately severe.conclusion:our study concludes geriatrics and female patients have higher incidence of adrs. so early identification and management of adrs are essential for this population. |
melioidosis, a febrile illness with disease states ranging from acute pneumonia or septicemia to chronic or localized abscess formation, was first documented by whitmore and krishnaswami (1912). the causative agent, burkholderia pseudomallei, was subsequently identified as a motile, gram - negative bacillus, which is principally a saprophyte found in soil and water in subtropical areas. the disease is acquired by inhalation, aspiration, sub - cutaneously, or occasionally via ingestion. melioidosis has become an increasingly important disease in endemic areas such as south - east asia and northern australia causing a significant number of deaths despite antibiotic treatment. furthermore, b. pseudomallei is also recognized as a pathogen of numerous animal species (reviewed in lazar adler., 2009 ; galyov., 2010 burkholderia mallei is closely related to b. pseudomallei and is the etiological agent of glanders, an infectious equine disease that can be transmitted to humans. the disease may be acute or chronic, with the chronic form usually occurring in horses and the acute form occurring in mules and donkeys. b. mallei is adapted to a narrower range of hosts than b. pseudomallei and is not considered able to persist for protracted periods in the environment. infections in humans frequently involve sepsis, pneumonia and abscess formation and have a high mortality rate. the incubation period ranges from days to weeks, and animals can die within a week of the onset of clinical symptoms (reviewed in whitlock. autotransporters (ats) are a large and diverse family of bacterial secreted and outer membrane (om) proteins that are translocated from the bacterial cytoplasm via the type v pathway, one of seven recognized secretion pathways in gram - negative bacteria. ats play diverse roles, and many such proteins influence pathogenesis and immunity, for example by acting as invasins, adhesins, proteases, or actin - nucleating factors. ats typically consist of a 20400-kda passenger domain that contains the effector functions and a 1030-kda domain facilitating translocation across the om (dautin and bernstein, 2007). ats can be further divided into classical ats and trimeric autotransported adhesins (taas), referred to as type va and type vc respectively. taas have a coiled - coil motif of unknown function, and are therefore referred to as oligomeric coiled - coil adhesins (oca). trimerization of taas is essential for their translocation and function as a single barrel is formed from the three shorter 4-stranded barrel domains which is superimposable with the classical ats 12-stranded barrel. while classical ats have diverse effector functions, all characterized taas, with the notable exception of bima from burkholderia spp., are adhesins (cotter., 2005 ; dautin and bernstein, 2007). the threefold symmetry provides the potential for a multivalent interaction to enhance avidity, which could overcome mechanical forces and provide stability against proteases and detergents within extracellular secretions (cotter., 2005). despite diversity in sequence, length, and function of the passenger domains, most ats are variations on a single structural theme : an elongated solenoid (a coil wound into a tightly packed helix) which may contribute to protein folding after transport and/or allow effector domains to traverse lps and capsule layers. this conserved shape creates a scaffold for various loops and domains without disturbing structural integrity and allows multiple interfaces to mediate interactions (wells., 2007 ; nishimura., 2010). classical ats form a right - handed helical structure while taas have a roll (two strands contributing to a single superhelical turn) ; these structural differences affect curvature and binding specificity (dautin and bernstein, 2007). however, the recent full structure for the pseudomonas aeruginosa classical at esta demonstrated an exception to the solenoid structure, with a -- globular fold (van den berg, 2010), which may relate to its function as a lipase rather than an adhesin. classical ats are then predominantly cleaved for secretion into the extracellular milieu whereas taas remain integrated in the bacterial om via their barrel domain. firstly, ats are targeted to the i m by their n - terminal signal sequence, where translocation occurs via the general secretory (sec) pathway. the n - terminal signal sequence ranges from 20 to 60 aa, with the most elongated signal peptides being associated with large (> 1000 aa) passenger domains in taas (dautin and bernstein, 2007). such extended signal peptides exhibit reduced interaction with the sec complex promoting delayed post - translational translocation across the inner membrane which is proposed to allow partial folding for subsequent om secretion (peterson., 2006 ; desvaux., once in the periplasm, ats can undergo modifications including glycosylation and lipidation and may also form disulfide bonds. as implied in their name, ats were originally believed to mediate their own transport across the om. however, recent research suggests that they are chaperoned by periplasmic proteins, such as sura and degp to the bam (omp85) complex located within the om which may assist in translocation (oomen., 2004 ; several ats have been shown to interact directly with the bam complex (ieva and bernstein, 2009 ; sauri., 2009 ; lehr., 2010), as well as periplasmic chaperones including sura and degp (ieva and bernstein, 2009 ; ruiz - perez., 2009). furthermore, depletion of the essential bama protein significantly affects at secretion (jain and goldberg, 2007). bama forms a 16-strand barrel with a pore size of ~2.5 nm which is significantly larger than the at barrel (~1.2 nm) and would allow for translocation of partially folded ats. additionally, bama oligomerizes in vitro and could possibly form oligomer pores capable of translocating folded ats. these observations have led to two hypotheses : translocation of the unfolded passenger domain through the bam - stabilized at barrel via a hairpin curvature or translocation of the entire at, possibly partially folded, via the bam complex (sauri., 2009). structural analysis indicates that sequential folding of the passenger domain may act as the driving force for translocation of at (van den berg, 2010), although evidence suggests that partially folded or misfolded ats are still capable of translocation (dautin and bernstein, 2007). therefore, the translocation of ats is dependent on periplasmic chaperones and the bam complex via a mechanism which remains to be elucidated but which appears to be mediated by interaction with the domain of the ats. this would account for the observation that the c - terminal domains of taas from diverse bacterial species are functionally interchangeable (ackermann., 2008). the b. pseudomallei k96243 genome contains 11 predicted ats, annotated as such based on sequence similarity to known ats (table 1). additionally, 10 of these ats are conserved across the other three completed b. pseudomallei genomes, while bpsl1705 is missing from both the 1106a and 688 strains. the b. mallei atcc 23344 genome has eight homologs of the b. pseudomallei ats (table 1). six of these are conserved across the three additional b. mallei completed genomes, with bma1027 being present only in b. mallei atcc 23344, while the avirulent b. mallei savpi strain also lacks bmaa0649. homologs for seven of the b. pseudomallei ats are also found in avirulent b. thailandensis (table 1). however, we caution that conservation of loci in pathogenic burkholderia and b. thailandensis does not necessarily imply that the genes play no role in virulence or host cell - interactions, as demonstrated by our analysis of the bsa type iii secretion locus shared by such strains (stevens., only two ats are predicted to be classical ats : bpss0962/bmaa1263 (a putative serine protease) and bpsl2237/bma1647 (a putative lipase / esterase). the remainder are predicted taas (tiyawisutsri., 2007). an in silico subtractive hybridization of pathogenic versus non - pathogenic burkholderia species to generate a 650 gene virulome, identified six ats (bpss0962, bpsl2237, bpsl1631, bpsl2063, bpss796, bpss0908 ; schell., 2008). furthermore, a gain of function genomic library screen identified five ats as having a possible role in invasion and survival within macrophages (bpsl1705, bpsl2237, bpss0088, bpss0796, bpss1492 ; dowling., 2010). neither approach identified a common set of dominant ats, which may indicate that the ats have a cumulative importance. however, direct experimental evidence for a role of ats in virulence or intramacrophage survival, as proposed by schell. several predicted ats are recognized by human convalescent sera, in some cases with a specificity adequate for serodiagnosis, and such data indicate that the proteins are expressed in vivo (tiyawisutsri. 2011). to date, the best characterized of the predicted ats of b. pseudomallei and b. mallei is a factor influencing intracellular motility, bima. the 11 b. pseudomallei ats and their eight b. mallei homologs are listed below alongside relevant functional information. in common with selected species of listeria, shigella, rickettsia, and mycobacterium marinum, b. pseudomallei shares the ability to induce polymerization of actin at one pole of the bacterial cell to promote its movement within and between host cells (figure 1). the formation of actin - rich bacteria - containing membrane protrusions was first described in b. pseudomallei - infected hela and j774.1 cells (kespichayawattana., 2000), but is also a feature of b. mallei and b. thailandensis infection (stevens., 2005a). the bacterial factor(s) required for actin - based motility of b. pseudomallei have been sought by screening random mutants for a small plaque phenotype (pilatz., 2006) and by targeted inactivation of candidate factors (stevens., 2005b) one such candidate (bpss1492, bima) belongs to the oca / type vc family of taas with a barrel sequence and linker region with 48% similarity to the y. enterocolitica yada c - terminal (pfam domain 03895). this was targeted on the basis that shigella and rickettsia use ats for actin - based motility (icsa and sca2, respectively). the predicted bpss1492 protein also contained domains associated with actin binding, including proline - rich motifs and wiskott aldrich syndrome protein (wasp) homology-2 (wh2) domains that suggested it may be involved in actin assembly (stevens., 2005b). actin - based motility of b. pseudomallei in j774.2 cells was abolished by inactivation of bima and could be restored by transient expression of the gene in trans (stevens., 2005b). bima is located at the pole of the bacterial cell at which actin polymerization occurs and polar localization is frequently seen in only one daughter cell at division (figure 1), implying that polar targeting of bima may rely on distinguishing the new and old poles of the cell (stevens., 2005b). actin - based motility of b. pseudomallei in a j774.2 murine macrophage - like cell 8 h post - infection. b. pseudomallei was detected with antibodies to its lipopolysaccharide (green) and bima was detected with a panel of specific monoclonal antibodies (blue). burkholderia pseudomallei bima possesses an unusually long signal sequence typical of autotransporters and a passenger domain containing several putative functional domains. within this effector domain, the two wh2 motifs have each been found to influence actin binding and polymerization in vitro (sitthidet., 2011). in addition, b. pseudomallei bima contains between 2 and 7 predicted target sites for host cell casein kinase ii (pdasx), with variation across sequenced strains (sitthidet., 2008). the pdasx repeats have since been established to act in an additive manner to enhance actin polymerization in vitro (sitthidet., 2011). it remains unclear if such repeats are phosphorylated in vivo, or what the functional consequences of post - translational modification of bima may be. interestingly a 13 aa repeat region of bima was found to be required for intercellular spread of b. pseudomallei but was dispensable for actin binding and polymerization (sitthidet., 2011). other bacterial factors involved in actin - based motility have been shown to promote intracellular survival through evasion of cytosolic killing by autophagy and further studies with defined mutants are required to determine if bima or its domains confer such activity. bima orthologs exist in b. mallei atcc23344 (bmaa0749) and b. thailandensis e264 (bth_ii0875) and can restore actin - based motility of a b. pseudomallei bima mutant (stevens. the bima passenger domain of the three species differ markedly in primary sequence resulting in differences in the number of wh2 domains, sequence of proline - rich motifs and the presence of a central and acidic (ca) domain, indicating that they may initiate actin assembly by distinct mechanisms (stevens.. many mechanisms of bacterial actin - based motility converge on activation of the cellular arp (actin - related protein) 2/3 complex. activation of the arp2/3 complex requires cellular nucleation - promoting factors (npfs) such as wasp - family members, and pathogens capable of actin - based motility often mimic the activity of npfs or recruit and activate them at the bacterial pole (reviewed in stevens., 2006). in recent years however, a number of virulence - associated factors have been identified in bacteria that initiate actin assembly in an arp2/3-independent manner. for example, the at sca2 facilitates actin - based motility of certain rickettsia species in an arp2/3-independent manner akin to cellular formin proteins (haglund., 2010 ; kleba., it is notable that truncated recombinant b. pseudomallei bima purified from e. coli polymerizes actin in vitro in the absence of any other cellular and bacterial co - factor in an arp2/3-independent manner (stevens. 2010) ; however, it remains unclear if such intrinsic activity would be adequate to propel the bacteria inside cells. in contrast, the ability of the b. thailandensis bima ortholog to assemble actin in vitro is arp2/3-dependent and requires the arp2/3-recruiting ca domain (sitthidet., 2010). little is presently known about the mechanism of action of the b. mallei bima ortholog ; however, it is considered unlikely to be arp2/3-dependent as it lacks an invariant tryptophan residue within an acidic stretch of residues required for arp2/3 binding and activation (stevens., 2005b). intra - species conservation of bima in natural populations of b. pseudomallei and b. thailandensis is high, with the exception of a geographically restricted subset of b. pseudomallei isolates harboring a b. mallei - like bima variant (sitthidet. the impact of sequence variation within bima on pathogenesis and recognition by host immunity remains to be elucidated. recently, researchers have developed pcr - based methods to discriminate between burkholderia species in a clinical setting (ulrich., 2006a, b ; sitthidet., 2011), and protein microarray studies led to inclusion of bima in a 10-protein multiplex classifier to improve diagnosis of human b. pseudomallei infection (felgner., 2009 ; suwannasaen., curiously the b. mallei bima protein was non - immunogenic in a test with serum from b. mallei - infected horses (tiyawisutsri., 2007), yet b. pseudomallei bima is one of the most immunogenic proteins recognized by sera from convalescent melioidosis patients (felgner., 2009 ; suwannasaen., the role of bima in the pathogenesis and virulence of b. mallei and b. pseudomallei also appears to differ, with b. mallei bima being dispensable for virulence in a syrian hamster model of glanders (schell., 2007) whilst b. pseudomallei bima contributes to pathogenesis in murine models of melioidosis and confers protection against homologous challenge (galyov. bima in b. mallei is regulated by the virag two - component system that is encoded nearby and which also controls expression of a virulence - associated type vi secretion system (schell., 2007). bima appears to be poorly expressed in vitro and the stimuli for its expression are ill - defined. in addition to bima, two predicted taas have been the subject of further characterization : bpss0796/bmaa0649 and bpsl1705, re - annotated as boaa and boab respectively, were expressed in a heterologous e. coli host where they were displayed on the bacterial surface and promoted attachment of the recombinant e. coli strains to epithelial cell lines (balder., 2010). in common with the role of other taa family members in adherence, b. pseudomallei and/or b. mallei mutants with disruptions in boaa or boab exhibited reduced adherence to epithelial cells and a double mutant, but neither single mutant, displayed a growth defect in phagocytic cell lines (balder., 2010). though suggestive of a role in virulence, studies with defined mutant and repaired or complemented strains in animals are required to establish the role of boaa and boab in pathogenesis. evidence of expression of selected taas has been obtained by screening of expression libraries with convalescent sera. serum from b. mallei - infected horses identified multiple independent clones for four taas (bma0840, bma1027, bma0649, and bmaa1324) suggestive of a strong antibody response (tiyawisutsri., 2007). a similar screen with convalescent serum from human melioidosis patients identified five taas (bpsl2063, bpss0908, bpss0796, bpss1439, and bpss1492) ; however, these were found at a low frequency indicative of a relatively poor immune response (tiyawisutsri., 2007). further studies are required to determine if this reflects genuine differences in immunogenicity of the predicted proteins, or the distinct hosts, nature of exposure and/or sampling times. additionally, a protein microarray probed with pooled melioidosis patients sera identified 4 ats (bpss0088, bpss0796, bpss1434/bpaa, bpss1492/bima) as having serodiagnostic potential due to their ability to interact with melioidosis - specific antibodies (felgner., 2009). the ats identified by these two studies demonstrate minimal overlap although this may reflect the variation in in vivo bacterial gene expression or the host immune response. microarray studies under 82 in vitro different conditions demonstrated that the 11 ats of b. pseudomallei are constitutively expressed (tan, personal comm.), while qrt - pcr analysis of the transcription of two at genes (boaa, boab) demonstrated low expression (balder., 2010). alignment of the c - terminal barrel domain regions of the putative b. pseudomallei and b. mallei taas demonstrates considerable conservation indicative of amplification of these adhesins through gene duplication events (figure 2a). this is potentially significant given the importance of c - terminal regions of taas in translocation and trimer stability, as evidenced by the ability of c - terminal domains of yada family members from diverse bacteria to functionally replace the cognate domain in yersinia (ackermann. a phylogenetic tree of the barrel domains is supportive of this idea as the taas form two clusters suggesting that these genes may have been amplified from two original ancestral genes (figure 2b). both clusters appear to be derived from a common ancestor to the prototypic yersinia yada taa. additionally, the two outliers in the alignment, bpss0908/bmaa1324 and bpss1434, display early separation in the phylogenetic tree. interestingly, of the three unique b. pseudomallei taas, two (bpss1434 and bpss0088) are early branches in the phylogenetic tree suggesting that their absence from b. mallei is due to gene loss as later branching genes are present. meanwhile bpsl1705, which is located within a genomic island (gi8), is very recently branched from bpss0796/bmaa0649 and has been proposed to have originated as a duplication of bpss0796/bmaa0649 (tiyawisutsri., 2007). this duplication event may explain why bpsl1705 is the only at not conserved across the four completed b. pseudomallei genomes. furthermore, the barrel domains of the b. pseudomallei and b. mallei taa homologs show 100% identity although little similarity is seen between the passenger domains. however, the effector domains of taas are generally not conserved at a sequence level and the crystal structure analysis of a section of the passenger domain of bpss1434 (re - annotated as bpaa, b. pseudomallei adhesion a) demonstrated quaternary structure similar to that of other taas (edwards., 2010). this new annotation gives bpss1434 the same name as the unrelated type v two - partner secreted bpaa (brown., 2004) for which no homolog exists in either b. pseudomallei k96243 or b. mallei atcc 23344. the b. pseudomallei and b. mallei genomes contain homologs of bama, bamb, and sura which are proposed to assist in translocation of ats across the om (ieva and bernstein, 2009 ; ruiz - perez. 2010), and further studies are needed to evaluate the role of such factors in at secretion and pathogenesis. alignment and phylogenetic analysis of the barrel domains of the putative b. pseudomallei and b. mallei taas : (a) the nine b. pseudomallei c - terminal 7072 aa barrel domains (b. mallei homologs 100% identical at this region) were aligned ; identical residues are highlighted in red, those with high homology (at least 66%) in blue while those with highly conserved substitutions in green. (b) the phylogenetic analysis of these domains demonstrates two distinct clusters both of which derived from a common ancestor to the prototypical yersinia yada taa. on the basis of homology and the precedent of phenotypes for at mutants in other bacteria, it is reasonable to infer that ats predicted to be encoded in the genomes of pathogenic burkholderia species may play a role in pathogenesis and immunity. in the short time since annotation of the genomes of b. pseudomallei and b. mallei, it has been established that several putative ats mediate bacterial interactions with host cells. for example, bima mediates intracellular actin - based motility, which in the case of b. pseudomallei is necessary for intercellular spread and full virulence, whereas boaa and boab appear to function as adhesins. one should note that significant sequence divergence in effector domains can occur even among closely related species, and within species, as we have noted for bima (sitthidet., 2008). such variation in bima can markedly alter the mode of action (sitthidet., 2011) and future studies will need to consider the extent to which the repertoire and sequence of ats in prototype strains is typical of wider populations. very few published studies have probed the role of ats in pathogenesis or immunity during b. pseudomallei and b. mallei infection in model hosts, and further studies with defined mutants and complemented strains are needed. attenuated mutants may be suitable as live - attenuated vaccines, and based on the ability of some ats to act as b- and t - cell antigens (tiyawisutsri., 2007), purified ats may have merit as subunit vaccines or as diagnostic tools. importantly, the mode of translocation of burkholderia ats is inferred only from homologous systems, and studies are needed to address outstanding questions regarding their biological role. future experiments should evaluate the role of at domains and specific residues in protein folding, oligomerization, interaction and translocation across the om, with particular reference to the requirement for periplasmic chaperones and the bam complex. analysis of the structure of burkholderia ats may also provide clues to the mechanistic basis of their secretion and function. 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. | burkholderia pseudomallei and burkholderia mallei are closely related gram - negative bacteria responsible for the infectious diseases melioidosis and glanders, respectively. autotransporters (ats) comprise a large and diverse family of secreted and outer membrane proteins that includes virulence - associated invasins, adhesins, proteases, and actin - nucleating factors. the b. pseudomallei k96243 genome contains 11 predicted ats, eight of which share homologs in the b. mallei atcc 23344 genome. this review distils key findings from in silico, in vitro, and in vivo studies on the ats of b. pseudomallei and b. mallei. to date, the best characterized of the predicted ats of b. pseudomallei and b. mallei is bima, a predicted trimeric at mediating actin - based motility which varies in sequence and mode of action between burkholderia species. of the remaining eight predicted b. pseudomallei trimeric autotransporters, five of which are also present in b. mallei, two (boaa and boab), have been implicated in bacterial adhesion to epithelial cells. several predicted burkholderia ats are recognized by human humoral and cell - mediated immunity, indicating that they are expressed during infection and may be useful for diagnosis and vaccine - mediated protection. further studies on the mode of secretion and functions of burkholderia ats will facilitate the rational design of control strategies. |
repetitive lifting and unloading as well as moving with excessive weights have been known to be major causes of various musculoskeletal disorders and low back pain1. such carrying or actions may increase the load on the spine while decreasing the pressure that protects the spine, thereby increasing the risk of low back pain and injury to the spine2. therefore, to ensure workers safety and prevent injury, cumulative fatigue induced in repetitive work environment needs to be in studies using various interventions for recovery from fatigue. in this study, muscle fatigue, tone of the erector spinae, and blood levels of ck (creatine kinase) and ldh (lactate dehydrogenase), two blood serum enzymes, were measured before and after the intervention and their results were compared to analyze the cumulative fatigue. it has been widely used to improve reduced muscle function, muscle strength, and joint range of motion, as well as the muscle activities of neurological patients. however, if long - term tens is applied to the human body, it can cause muscle fatigue and the accumulation of waste matter, and serious muscle damage. it has also been reported that tens does not have a significant effect on fatigue after high - intensity exercise3,4,5. therefore, although therapies using tens may have a variety of advantages, more study is required to conclusively determine the effect tens has on muscles with cumulative fatigue. microcurrent stimulation plays a role in restoring cells or facilitating healing by increasing the creation of adenosine triphosphate (atp) and proteins, and curing wounds through the supply of electric energy at the cell level, which restores the potential of the cell membrane to normal potential6. in recent years, a number of studies of microcurrent stimulation have been performed for in a wide range of conditions such as fracture and fatigue recovery, and various diseases involving cancers and diabetic nerve damage7,8,9,10. in particular, microcurrent stimulation is known to be effective for cumulative fatigue recovery as it reduces ck in the blood of patients with delayed onset muscle soreness11. however, a study by cheng. reported that microcurrent stimulation had no positive effect on the recovery of an arm with delayed onset muscle soreness12. in this study, microcurrent stimulation and tens were administered to subjects with cumulative fatigue to analyze their effects on fatigue recovery. thirty - two male persons in their 20s participated in this study and they were randomly divided into three groups : an mc group of 12 persons who underwent microcurrent stimulation, a tens group of 10 persons who underwent transcutaneous electrical nerve stimulation, and a con group of 10 persons who only rested. the subjects had no musculoskeletal disorders or related disease histories and were restricted to right - hand dominant males. prior to participation in this study, subjects were given a detailed description of the experimental and safety procedures, and each subject signed an informed - consent form. the following tables show the general characteristics of the subjects and the homogeneity test results for the two groups (tables 1table 1.general characteristics of the subjectsmc (n=12)tens (n=10)con (n=10)age (yr)21.81.822.41.521.51.8height (cm)173.64.9176.25.9174.85.3weight (kg)70.19.869.73.165.46.4meansd, mc : microcurrent group, tens : tens group, con : control group and 2table 2.pre-intervention values of the three groupsmc (n=12)tens (n=10)con (n=10)rmf (hz)72.610.6073.34.6073.34.7lmf (hz)74.37.6071.26.1070.55.7rmt (kg / mm)12.20.9012.40.8012.31.4lmt (kg / mm)12.51.8014.16.0012.21.2ck (u / l)119.017.4126.561.1115.332.8ldh (u / l)417.140.6421.241.2429.353.7there were no significant differences : anova, p>0.05, mc : microcurrent group, tens : tens group, con : control group, rmf : right median frequency, lmf : left median frequency, rmt : right myotonometry, lmt : left myotonometry, ck : creatine kinase lactate, ldh : lactate dehydrogenase). meansd, mc : microcurrent group, tens : tens group, con : control group there were no significant differences : anova, p>0.05, mc : microcurrent group, tens : tens group, con : control group, rmf : right median frequency, lmf : left median frequency, rmt : right myotonometry, lmt : left myotonometry, ck : creatine kinase lactate, ldh : lactate dehydrogenase before the test, subjects wore comfortable dresses and were informed about the correct motions. to maintain the same work condition, a 75-cm - high small table was prepared and a 30-cm - wide, 30-cm - deep, and 25-cm - high box was filled with a 10-kg dumbbell13. as soon as subjects had completed repeated lifting and lowering of the box 100 times during 15 minutes with a symmetrical posture in the sagittal plane14, the pre - test measurements were conducted. immediately after the pre - test, the tens group was administered transcutaneous electrical nerve stimulation (es-420, ito, japan) after connecting two rectangular radiofrequency catheters (5090 mm). the longissimus and iliocostalis were treated for 20 minutes (monophasic pulsed direct current of 80 hz with phase duration of 10 s, pulse width of 300 s and on / off time 10 s/50 s). the mc group received microcurrent from a microcurrent stimulator (es-420, ito, japan) at 100 ma and 0.3 hz on the same spot for 20 minutes. the con group took a rest for 20 minutes while lying prone on the floor. immediately after the intervention, emg, muscle tone, ck, and ldh were measured. the temperature and humidity in the laboratory were maintained at 23 c and 60%. muscle activating signal was checked by using surface emg machine (mp100, biopac, usa). the sampling rate of the signal was 100 hz and bandpass filtering was performed between 20500 hz. the digitized signal was processed with fft (fast fourier transformation) using acqknowlege 3.91 software on a personal computer to obtain the median frequency (mf). the closest two of the recorded values were averaged and used as the mvc value of the subjects. after the mvc measurement, 60% mvc was calculated for the quantification of the collected measurement values from the experiments so that measurements before and after the treatment were performed with the above method. the electrode was attached to the right and left erector spinae of the right- and left - side back and the longissimus and the iliocostalis around the hip. to measure the emg of these muscles, a pair of surface electrodes was attached horizontally in the right and left directions to the muscle belly 5 cm away from the neurocentral joints of thoracic vertebrae t10 and lumbar vertebrae l214. in order to measure muscle tone of the erector spinae, a myotonometer (missoula, mt, neurogenic technologies inc., while subjects rested on a bed, the longissimus and the iliocostalis at the waist were measured. the measurement probe consists of inner and outer cylinders. according to the resistance of the tissue, the distance between two cylinders changes and the resistance of the tissue the force applied to the cylinder was divided into eight stages (0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00 kg) so that measurement at each position of the level of displacement could be made. force - displacement curves were created using computational software and the areas under the curve (auc) were calculated from the obtained data. the area under the curve (auc) is indicating of level of muscle tone15,16,17. spss / win 18.0 was used for all statistical processing. means and standard deviations of all measurements were calculated and analysis of variance (anova) was conducted to test the homogeneity of the experimental groups. in order to analyze the effect of the treatment on muscle fatigue, muscle tone, and ck and ldh serum levels, the paired t - test was used to examine within group pre- and post - test differences, while analysis of covariance (ancova) was used to compare the mc and tens groups with the con group. microcurrent stimulation, tens, and rests were given to subjects who had fatigues and then emg, muscle tones, ck and ldh were measured and analyzed. the results are shown in tables 3table 3.the effects of intervention on the muscle fatigue indexgrouppre - interventionpost - interventionrmf (hz)con70.710.667.47.9mc 72.610.663.88.5tens73.34.7065.93.8lmf (hz)con70.811.463.79.7mc 75.47.659.110.4tens70.55.768.55.3paired t - test (p<0.05), anova, mc group con group (p<0.05), mc : microcurrent group, con : control group, tens : tens group, 4table 4.the effects of intervention on the myotonometry indexgrouppre - interventionpost - interventionrmt (kg / mm)con12.31.416.51.8mc 12.20.918.81.2tens12.40.816.21.3lmt (kg / mm)con12.21.215.22.4mc 12.51.819.31.3tens14.16.016.71.1paired t - test (p<0.05), anova, mc group con group (p<0.05), mc : microcurrent group, con : control group, ts : tens group group, tens : tens group, and 5table 5.the effects of intervention on fatigue markers in the bloodgrouppre - interventionpost - interventionck (u / l)con119.040.691.636.4mc126.561.185.729.8tens139.342.095.521.8ldh (u / l)con421.241.2374.741.1mc417.140.6355.031.1tens433.355.8395.044.6paired t - test (p<0.05), mc : microcurrent group, con : control group, tens : tens group. paired t - test (p<0.05), anova, mc group con group (p<0.05), mc : microcurrent group, con : control group, tens : tens group paired t - test (p<0.05), anova, mc group con group (p<0.05), mc : microcurrent group, con : control group, ts : tens group group, tens : tens group paired t - test (p<0.05), mc : microcurrent group, con : control group, tens : tens group all the three groups showed significant changes in muscle fatigue, muscle tone, and blood levels of ck and ldh, between before and after the intervention (p<0.05). the comparison result of the mc and con groups found a significant difference in muscle fatigue and muscle tone in the right and left erector spinae (p<0.05). however, no significant difference was found in the blood levels of ck and ldh. in addition, no significant difference was found between the tens and con groups. a study by dolan. reported that 100 times of lifting and lowering a 10-kg weight for 12.1 minutes resulted in increase of muscle fatigue14. furthermore, lee. revealed that after repetitive work of more than six times per minute with a sub - maximal weight of 15%, it took more than five minutes for the workers backs to recover from cumulative fatigue to the initial condition. when cumulative fatigue occurs, loads on the vertebrae continue to increase during the repetitive work whereas pressure that protects the vertebrae starts to reduce thereby increasing the risk of low back pain2. clarkson. reported that when subjects aged between 18 and 40 years performed repeated of lifting and lowering, 50 times, with maximum eccentric contraction, muscle damage to the biceps brachii was induced along with increases in both ck and ldh level in the blood18. the accumulation of markers of fatigue in the blood and muscle fatigue due to repetitive lifting and lowering work is known as one of the main reasons for injury during work19. an increase in muscle tone indicates an abnormal state in the musculoskeletal system and it is used as an indicator of muscle fatigue20. reported that with additional lifting and lowering of heavy goods, muscle tone of workers increased further, whereas workers without such repetitive work showed relatively less muscle tone21. in addition, muscle tone was shown to correlate with emg frequency and a decrease in muscle fatigue increases muscle tone as well22. in this study, cumulative muscle fatigue and muscle tone showed significant changes after the intervention. in particular, subjects administered microcurrent stimulation showed significant changes in muscle fatigue and muscle tone. weber. administered microcurrent stimulation to subjects with delayed onset muscle soreness and reported no significant difference in muscle fatigue reported that patients with plantar fasciitis who wore shoes that produced microcurrent stimulation for six weeks showed a significant reduction in, muscle fatigue in the tibialis anterior as assessed by the median frequency6. their result is similar to that of our present study in which muscle fatigue in the right and left erector spinae was reduced significantly. microcurrent stimulation can induce cell responses in damaged tissues through bioelectricity thereby assisting with the healing of tissues. it can also facilitate endogenous bioelectric current which would reduce resistance in the damaged portion, and promote conduction of bioelectric current assisting with the recovery of homeostasis of the human body. that is, microcurrent enhances electrical and chemical processes, which assist the healing process, returning damaged muscle tissues to a normal state23. reduction of the median frequency value indicates a reduction in muscle fatigue22, 24, and muscle tone and microcurrent stimulation is more effective for the recovery of muscle fatigue and muscle tone than only taking a rest. the microcurrent therapy increased collagen formation speed in the recovery of damaged cells more than in the tens therapy, which is why the microcurrent stimulation had a more positive effect on muscle fatigue and muscle tone. no significant differencs were found after the intervention between the tens and con groups. this result is in agreement with park s study in which an administration of 100 pps to finger muscles for 30 minutes had no effect on muscle fatigue and muscle strength25. although appropriate stimulation through tens can help to prevent fatigue accumulation by increasing the local blood flow, the temporary increase in local circulation was not sufficient enough to have a significant effect on muscle fatigue recovery. a study by lim reported that after heavy exercise, ck and ldh decreased slightly or increased for up to 30 minutes, together with aggravation of the pain in patients with delayed onset muscle soreness26, 27. furthermore, a study that can provide a generalized conclusion through a large number of subjects and various measurement tools shall be conducted in the future. | [purpose ] the aim of this study was to determine the effect of low - frequency electrical stimulation on fatigue recovery of the erector spinae with cumulative fatigue induced by repeated lifting and lowering work. [subjects ] thirty - two healthy men volunteered to participate in this study and they were randomly divided into three groups : a mc group of 12 persons who underwent microcurrent, a tens group of 10 persons who underwent transcutaneous electrical nerve stimulation, and a control group of 10 persons who only rested. [methods ] cumulative fatigue was induced and then, emg, muscle tone, ck and ldh serum levels of the erector spinae were measured. each group then underwent the assigned intervention and was re - measured. to analyze the differences in fatigue between before and after the intervention, the paired t - test was conducted, while groups were compared using analysis of covariance with a control group. [results ] the mc groups showed a significant reduction in muscle fatigue and decreased muscle tone when compared to the control group. however, no significant differences were found between the tens and control groups. [conclusion ] these results suggest that microcurrent stimulation was effective for recovery from cumulative muscle fatigue while tens had no effect. |
cemento - ossifying fibroma (cof) is considered a benign osseous tumor, very closely related to other lesions such as fibrous dysplasia, cementifying periapical dysplasia, or cemento - osseous florid dysplasia, however, creating its own entity in the 1992 who classification. consequently, one of its principal characteristics is the massive formation of cementum, cementoid substance, or calcified material in the interior of a predominantly fibrous. a 35-year - old female patient reported to the department with a chief complaint of painless swelling on the left upper side of face since one and a half years. initially, swelling was smaller in size, approximately 1 cm, and it grew slowly and has reached to its present size in last one and a half years. patient suffered from the same problem 15 years back in the left lower jaw region and was operated for the same problem. after fourteen and a half years, swelling reappeared in the left upper and right lower jaw regions. extraoral examination revealed that face of the patient was bilaterally asymmetrical with the diffused swelling roughly circular in shape measuring about 3 to 4 cm in its greatest dimension extending anteroposteriorly from the inner canthus of the eye to the angle of the mandible and superior - inferiorly from the infraorbital margin to the angle of the mouth. color of the overlying skin was normal but had ill - defined borders [figure 1 ]. on palpation, all inspectory findings were confirmed. swelling was non - tender on palpation, hard in consistency, non - fluctuant, and had diffused borders, temperature of the overlying skin was not raised, tmj was normal without any deviation, clicking sound, or tenderness. submandibular lymph nodes on the left side were palpable, non - tender, firm in consistency, and mobile. intraoral examination showed solitary dome - shaped swelling measuring about 3 to 4 cm in its greatest dimension in the left upper posterior teeth region extending anteroposteriorly from the mesial surface of 24 to the retromolar area. there was expansion of buccal and palatal cortical plates in relation to 24, 25, 26, 27, and 28. no vestibular obliteration was present in relation to 25, 26, 27, and 28 [figure 2 ]. intraoral swelling in the left maxillary alveolar ridge soft tissue examination for the lower arch revealed the presence of diffused swelling measuring about 1.5 to 2 cm in diameter in the fourth quadrant in relation to 44, 45, 46, and 47. vestibular obliteration was present in relation to 43, 44, 45, 46, and 47 regions. intraoral swelling in the right mandibular alveolar ridge hemimandibulectomy on the left side on palpation, inspectory findings were confirmed. swelling is non - tender on palpation, hard in consistency with well - defined borders. no vestibular tenderness and no pus discharge were evident. hard tissue examination showed distally displaced teeth in relation to 26, 27, 28 and missing teeth in relation to 31 - 37 and 47. other findings were attrition in relation to 41, 42, 43, and 47. the second quadrant teeth, i.e., 24 - 28, were non - tender on vertical percussion. considering the history and clinical examination, provisional diagnosis of central ossifying fibroma of the left maxillary alveolar ridge and residual cyst in relation to 46 was given. clinical differential diagnosis of florid cemento - osseous dysplasia, central odontogenic fibroma, osteoma, paget 's disease, and central giant cell granuloma was considered. investigations such as the iopar of the left maxillary teeth region showed the presence of well - defined roughly spherical - shaped radiopacity with radiolucency at certain areas, having sharp radiolucent rim surrounded by sclerotic border standing mesially up to the root of 25 and distally up to the mesial root surface of 28. lesion was applying pressure on the roots of 25, 26, 27, and 28 and had displaced the roots of 25 mesially and roots of 26, 27, and 28 distally. there was discontinuity of lamina dura in relation to 25, 26, and 27 and vertical bone loss in relation to 24 and 25 [figure 5 ]. iopar showing well radiopacity surrounded by radiolucent rim followed by sclerotic border iopar of the right mandibular teeth region revealed the presence of well - defined mixed radiolucent - radiopaque area extending mesially up to the distal root surface of 45, superiorly extending to the alveolar ridge, having thin radiolucent rim followed by thick sclerotic margin, with this margin thicker distally. iopar also showed the presence of radiolucent area around mesial surface of 47 involving the crown, and the root was suggestive of caries. horizontal bone loss was evident in relation to 43 - 45 [figure 6 ]. iopar showing mixed radiolucent - radiopaque region lateral cross - sectional maxillary occlusal radiograph revealed the expansion of buccal and lingual cortical plate [figure 7 ]. mandibular cross - sectional radiograph showed the more uniform expansion of buccal and lingual cortical plate mimicking the cystic expansion [figure 8 ]. maxillary occlusal radiograph showing expansion of buccal cortex mandibular occlusal radiograph showing expansion of both buccal and lingual cortex opg revealed the presence of well - defined roughly spherical - shaped radiopaque lesion with radiolucency at certain areas extending mesiodistally from the distal root surface of 25 to the mesial root surface of 28, having radiolucent rim surrounded by sclerotic border, which was thicker distally. lesion had displaced floor of the maxillary sinus upward and was applying pressure over the roots of 25 - 28 and had displaced the roots of 25 mesially and roots of 26, 27, and 28 distally. radiopaque lesion in relation to 43 - 47, extending mesially up to the distal root surface of 43 and distally up to the mesial surface of 47 having radiolucent margin followed by thick sclerotic border not involving the inferior alveolar canal [figure 9 ]. opg showing well radiopacity surrounded by radiolucent rim followed by sclerotic border in the left maxillary alveolar ridge and mixed radiolucent - radiopaque region followed by radiolucent rim and by sclerotic border pns view was taken to see the involvement of sinuses which was not found, and was later confirmed by ct scan. ct scan axial view showed hyper - attenuated mass measuring about 3 2 cm in diameter, involving the left maxillary alveolar ridge. coronal section showed the presence of iso - attenuated and hyper - attenuated mass roughly round in shape in relation to right mandibular alveolar ridge [figure 11 ]. axial section of ct scan showing lesion in the maxillary alveolar ridge coronal section of ct scan showing well - defined lesion in the right mandibular alveolar ridge radiographic differential diagnosis of central ossifying fibroma, florid cemento - osseous dysplasia, and fibrous dysplasia was given. fibro - cellular connective tissue is composed of thick bundles of collagen fibers and large plump, proliferating fibroblast with eosinophilic cytoplasm and dark nucleus. the calcified material is composed of few irregular trabeculae of bone and numerous spherical - shaped cementum - like materials [figure 12 ]. histopathological picture considering the history clinical examination, radiographic investigation, and histopathological finding, final diagnosis of central cementifying / ossifying fibroma of left maxillary and right mandibular ridge and chronic generalized gingivitis was given. the term benign fibro - osseous lesion has been used in the literature to describe a spectrum of lesions ranging from fibrous dysplasia to ossifying fibroma, including cementifying or cof, psammomatoid ossifying fibroma, psammo - osteoid fibroma, juvenile or young ossifying fibroma, and juvenile active ossifying fibroma. ossifying fibroma is a rare, expansile, benign tumor that predominantly involves the maxillary (approximately 1020% of cases) and mandibular (approximately 75%) bone. in rare cases, consequently, one of its principal characteristics is the massive formation of cementum, cementoid substance, or calcified material in the interior of a predominantly fibrous. in 1872, menzel gave the first description of variant of ossifying fibroma, called cof. ossifying fibroma was first reported in the jaw by montgomery in 1927. in 1968, hamner and colleagues proposed that ossifying fibroma, cement - ossifying fibroma, and ossifying fibroma are the histological variants originating from the periodontal ligament, although who designates the cementifying fibroma as odontogenic and the ossifying fibroma as nonodontogenic in origin and suggests that they are separate entities. finally, it was concluded that separation of the three conditions is arbitrary because the clinical, radiologic, and prognostic features of the lesions are identical. cof has been defined by who as a demarcated, or rarely encapsulated, neoplasm consisting of fibrous tissue containing varying amount of mineralized material resembling bone or cementum. its synonyms are cementifying fibroma, cof, fibro - osteoma, osteofibroma, benign fibro - osseous lesion of periodontal ligament origin, and benign periodontoma. the connective tissue of the periodontal membrane harbors the potential for elaboration of both bone and cementum. bernier and thompson speculated that infection with resulting inflammation and fibrosis of the periapical area might stimulate the periodontal membrane. after trauma, such as tooth extraction, the remaining periodontal tissue that is attached to the wall of the alveolus may serve as the origin of cof. the fact that this tumor is most common in the jaws is related to the vast amount of mesenchymal cellular induction into bone (lamina dura) and cementum in odontogenesis ; therefore, the probability of induction error or genetic alteration leading to a neoplasm is greater. clinically, it is most commonly seen in third and fourth decade of life (56%) with the average age of 36 years. there is striking predilection feminine sex with a ratio of 5 : 1. the mandibular premolar cof manifest it as slow - growing, asymptomatic, intraosseous masses, mostly detected incidentally during a routine radiographic survey. larger lesion grow more rapidly and extensive and could even provoke a mandibular fracture. infrequently hamner and colleagues found multiple lesions in some of their patients. in a series reviewed by hauser, 20% of the cases showed facial asymmetry, other findings included pain in four patients and numbness in two patients. displacement of teeth is also seen in some cases (as in the current case). radiographically, it has a striking predilection for the mandible ranging from 70% to 89%. lesion appears limited to tooth - bearing area with an intimate relationship to the root of the teeth or periapical region. a few have extended to angle - ramus area or encroached on the maxillary sinus. eversole and colleagues reported the following location : molar region, 52% ; premolar area, 25% ; incisor area, 13% ; and cuspid region, 11%. radiographically, the cof presents as a well - defined unilocular or multilocular lesion with smooth contours. the maturity of the lesion determines the degree of radiopacity ; the immature lesion may present as completely radiolucent, whereas the mature lesion may appear completely radiopaque. hauser. reported that 26% were lytic type, 63% were lytic with radiopaque foci, and 12% consisted of a diffuse, homogenous appearance that was mildly radiopaque (ground glass appearance). a sclerotic rim sometimes is present within the host bone at the margin ; it may be smooth and delicate or it may be slightly irregular, more diffuse, and of varying thickness up to approximately 3 mm. in some cases, lesion may be punched out with no sclerosis at the margin. divergence of adjacent roots (17%) and root resorption (11%) may also be seen. lesion enlarges equally in all direction producing expansion of buccal and lingual cortical plate and most notably the inferior cortex. inferior bowing of the lower border of the mandible is almost a constant feature in larger lesions. pathologic examination of the central cof shows a proliferation of irregularly shaped calcifications within a hypercellular fibrous connective tissue stroma. the calcifications are extremely variable in appearance and represent various stages of bone and cementum deposition. most of the calcified fragments are immature cementum, with basophilic coloration on hematoxylin and eosin - stained sections. the calcified fragments are osteoid, with typical eosinophilic coloration on hematoxylin and eosin - stained sections. central ossifying fibromas are slow - growing, ovoid or round - shaped, and well - demarcated bone tumors. they may appear at any age, especially in white adult female patients, usually in the mandible. expansion of the inferior border of the mandible and well - defined radiographic borders seem to be the most common manifestations of central ossifying fibroma. regarding the histology, trabecular structures are more common than cementicle - like masses. sometimes, a fibrous capsule is noted. the differential diagnosis includes benign and malignant fibro - osseous lesions and odontogenic cysts and tumors. | cemento - ossifying fibroma (cof) is considered a benign osseous tumor. herewith, we present a case of multiple central ossifying fibroma in a 35-yeaold woman. intraorally, there was swelling in the left upper posterior teeth region and another diffused swelling in the fourth quadrant. radiographs revealed the presence of well - defined mixed radiolucent radiopaque area having thin radiolucent rim followed by thick sclerotic margin. no genetic correlation could be established. as bilateral cof is a rare entity, we present such a case with different radiographic appearance, using various radiographic techniques. |
radiography is generally used in determining dental maturation and two - dimensional panoramic radiographs are mainly used in radiography as seen in the dental and forensic literature. however, different dental age estimation methods have been used to measure tooth development, which include diagrams, charts, and definition of the formative stages at present. cone beam computed tomography (cbct) allows obtaining three - dimensional images of the tooth. yang. mentioned that the clinical use of cbct has shown new techniques for acquiring three - dimensional dental radiographs, resulting in good image quality with low radiation dose. cameriere. emphasized that volumetric study with cbct would be the best method for dental age estimation. first describe an approach to dental age estimation by cbct using the ratio of pulp / tooth volume that could be calculated for living individuals. indicated that to improve age estimation goal, including the volume of dental tissues and their ratio, further investigation should aim : to acquire larger sample sizes in order to reduce standard errors of age estimation, study the effect of several factors on model parameters, and investigate the use of all types of teeth together. limited number of studies is available on the issue of ratio of pulp / tooth volume. dental age estimation is usually done on the mandibular incisor, canine, or premolar that is, always the monoradicular teeth in the afore - mentioned studies. nevertheless, no study has investigated the use of tooth germ volume for dental age estimation in multiradicular teeth. in addition to this, it is a well - known fact that dental development is a multi - factorial phenomenon. garn. in their study have reported in 1965 that they thought genes, harmonies, and even calories have an important role in tooth development. however, environmental factors are now better known to have a very important role in the dental development. orthodontic malocclusions on the sagittal, vertical and even horizontal dimensions have been reported to have an effect on dental development. posterior crossbite is characterized as any abnormal buccopalatinal relationship between mandibular and maxillary molars, premolars, or both in centric occlusion. uysal. indicated that the crossbite patient presented with a tendency to delayed dental maturation. in addition to this, there is no study investigating the development of 3ms in the crossbite and normal sides. in light of this fact, the aim of this study was to investigate mandibular 3ms maturation and volume in the crossbite side and to compare them with the 3ms in the normal sides by two - dimensional and three - dimensional analyses using cbct data. a retrospective study was performed using cbct data of 25 patients (16 females and 9 males ; aged 15 - 23 years), selected randomly from the archive of faculty of dentistry in erciyes university. cbct scans of these patients were previously obtained as a part of the diagnostic records for orthognathic surgery, temporomandibular dysfunction, and impacted canine ; and for this reason they were not subjected to additional radiation. ethics committee approval was not required due to the archival nature of the research. however, as a usual protocol, all the patients (or parents) signed an informed consent agreeing to the use of the patients data (age, gender, medical history, etc.) for scientific studies. all images were obtained in the supine position by using cbct (newtom 5 g, qr verona, italy). the scanning time was 18 s, collimation height 13 cm, exposure time 3.6 s, and voxel size 0.3 mm. patients in the study group met the following inclusion criteria : no history of trauma, developmental and acquired craniofacial disorders or maxillofacial surgery prior to orthodontic treatment.no missing tooth (excluding maxillary 3ms) and no systemic disease.skeletal class i (determined by anb ; 0 - 4) and normodivergent facial pattern (determined by sn - gogn angle ; 32 5).unilateral crossbite of at least two teeth determined by plaster models (11 patients had all teeth in crossbite, 8 patients had three teeth in crossbite and 6 patients had two teeth in crossbite). no history of trauma, developmental and acquired craniofacial disorders or maxillofacial surgery prior to orthodontic treatment. skeletal class i (determined by anb ; 0 - 4) and normodivergent facial pattern (determined by sn - gogn angle ; 32 5). unilateral crossbite of at least two teeth determined by plaster models (11 patients had all teeth in crossbite, 8 patients had three teeth in crossbite and 6 patients had two teeth in crossbite). all records were examined in axial slices, and patients who had two or more unilateral posterior crossbite teeth were determined using the same device (newtom 5 g, qr verona, italy) [figure 1 ]. primary reconstructions of the data were performed with the mimics software (materialise hq, leuven, belgium). second, the exported digital imaging and communication in medicine (dicom) files were viewed, and segmentations of the mandibular 3ms were carried out using software. observations were made regarding the patient 's sex, chronological age, anb, and sn - gogn angles. unilateral crossbite shown in a cone beam computed tomography image panoramic radiograms were obtained by the mimics software (materialise hq, leuven, belgium), and dental development of the 3ms was evaluated according to the method of demirjian. the formation stages of the tooth germs were evaluated based on the formative condition of the crowns and roots of the mandibular 3ms. this method divides the formation of the crowns and roots of permanent teeth into eight stages. the formation stage of the mandibular 3 m on the side of the crossbite was evaluated. the formative condition of the 3 m on the other side of the same jaw was used as the control [figure 3 ]. dicom files were imported in a cbct diagnostic and treatment planning software allowing for tooth volume calculations. the program automatically calculated the volume of the obtained three - dimensional images of the tooth [figure 4 ]. all measurements were carried out by a dentomaxillofacial radiologist without knowing the crossbite side and thus a blinding was performed. the eight stages of tooth development according to demirjian method the formative condition of the third molar on the other side of the same jaw was used as the control volumetric measurement of third molar on the cone beam computed tomography images to test the reproducibility of the assessments of dental developmental stages and volume of mandibular 3ms, the same investigator re - evaluated 15 randomly selected cbcts 2 weeks after the first evaluation. intra - class correlation coefficients were performed to assess the reliability of volumetric measurements as described by houston. the coefficients of reliability according to the houston method for volumetric measurements were 0.92 (mandibular right molar volume) and 0.98 (mandibular left molar volume), confirming measurement reliability. kappa coefficients were used to evaluate agreement between the first and second dental maturity assessments and kappa values were found to be above 0.90. student 's t - test was used to assess gender differences in the patient 's chronological age, anb, and sn - gogn angles. a paired t - test was used to compare the differences in the developmental stages and volume of the 3ms between the crossbite and control sides. since no gender difference was present for mandibular 3msdevelopment stages and volume, the data from both sexes were pooled. a linear regression analysis was performed to evaluate the effect of the number of teeth in crossbite. all data were analyzed with spss for windows (version 15.0, spss, chicago, illinois). panoramic radiograms were obtained by the mimics software (materialise hq, leuven, belgium), and dental development of the 3ms was evaluated according to the method of demirjian. the formation stages of the tooth germs were evaluated based on the formative condition of the crowns and roots of the mandibular 3ms. this method divides the formation of the crowns and roots of permanent teeth into eight stages. the formation stage of the mandibular 3 m on the side of the crossbite was evaluated. the formative condition of the 3 m on the other side of the same jaw was used as the control [figure 3 ]. dicom files were imported in a cbct diagnostic and treatment planning software allowing for tooth volume calculations. the program automatically calculated the volume of the obtained three - dimensional images of the tooth [figure 4 ]. all measurements were carried out by a dentomaxillofacial radiologist without knowing the crossbite side and thus a blinding was performed. the eight stages of tooth development according to demirjian method the formative condition of the third molar on the other side of the same jaw was used as the control volumetric measurement of third molar on the cone beam computed tomography images to test the reproducibility of the assessments of dental developmental stages and volume of mandibular 3ms, the same investigator re - evaluated 15 randomly selected cbcts 2 weeks after the first evaluation. intra - class correlation coefficients were performed to assess the reliability of volumetric measurements as described by houston. the coefficients of reliability according to the houston method for volumetric measurements were 0.92 (mandibular right molar volume) and 0.98 (mandibular left molar volume), confirming measurement reliability. kappa coefficients were used to evaluate agreement between the first and second dental maturity assessments and kappa values were found to be above 0.90. student 's t - test was used to assess gender differences in the patient 's chronological age, anb, and sn - gogn angles. a paired t - test was used to compare the differences in the developmental stages and volume of the 3ms between the crossbite and control sides. since no gender difference was present for mandibular 3msdevelopment stages and volume, the data from both sexes were pooled. a linear regression analysis was performed to evaluate the effect of the number of teeth in crossbite. all data were analyzed with spss for windows (version 15.0, spss, chicago, illinois). student 's t - test showed no significant sex differences for skeletal facial patterns and chronological ages and thus both genders presented similar characteristic features. demographic features of the subjects included to the study third molars development stages and volume in relation to crossbite presence are shown in table 2. paired t - test showed that no statistically significant differences were found in the development of the mandibular 3ms between the crossbite and control sides (p = 0.714). in addition, when we evaluated patients one by one, we detected differences in seven patients according to the developmental stages of the 3ms. on the other hand, 18 of 25 patients development of the 3ms were the same. although three patients had one developmental stage delay of the mandibular 3 m in the crossbite side, the other four patients had one developmental stage accelerated. third molar development stage in relation to cross bite presence because student 's t - test showed no significant sex differences for the volume parameters of the 3ms, the data for both genders were pooled. in contrast to developmental stage similarities, paired t - test showed statistically significant differences for the volume of the mandibular 3ms between the crossbite and control sides (p = 0.021). the volume of mandibular 3 m in the crossbite side was less than the volume of 3 m in the control side (529.81 199.01 mm and 578.52 217.43 mm, respectively). results of the regression analysis showed that the number of the teeth in crossbite had no affect on the volume of the mandibular 3ms (r = 0.004 ; p = 0.714). dental age determination is important in both medical jurisprudence and clinical dentistry. the most common method by which dental age assessment is done is the method of demirjian. this classification method distinguishes the first four stages of crown development (a - d) and the last four stages of root development (e - g) so it is easy to use. have reported that this method performed intra- and inter - examination best for radiographic stage assessment of 3ms. malocclusions in the sagittal, vertical, and horizontal dimensions were previously shown to affect dental development. therefore, patients who were included in this study were compatible with the horizontal and vertical dimensions as shown in table 1. lately, it was shown that children with hypodontia showed a significant delay in dental development when compared with case controls. to eliminate those factors, patients with hypodontia since above factors affecting dental development was eliminated, the effects of unilateral crossbite might be better understood. panoramic radiographs are noninvasive methods that are used in the formative condition of the teeth. dental maturation has been successfully determined from two - dimensional panoramic radiographs. however, these radiographic images were made from accumulated two - dimensional images of the horizontal or parallel aspects of the tooth and therefore, would be difficult to accurately evaluate the development of the teeth, especially 3ms that have generally different eruption and formation anomalies. furthermore, the entire three - dimensional morphological assessment of 3ms is not possible with panoramic radiographs. however, the clinical use of cbct has created new opportunities for obtaining three - dimensional tooth radiographs, resulting in reasonable fair image quality at a low radiation dose. in a recent paper, ahlowalia. in their study have reported that cbct was an accurate means of measuring the volume of artificially created bone cavities. it was stated that deviation of tooth maturation on the two sides of the same jaw is frequent and few months of tooth maturation difference can be observed. in this study, no statistically significant difference for the mandibular 3 m developmental stage between the crossbite and the normal sides was present. we detected differences only in seven patients in relation to the developmental stages of the 3ms. although there was no difference in dental maturation as assessed by two - dimensional analyses method, there were statistically significant volume differences between the crossbite and control sides (p = 0.021). it was found that mean volume (529.81 199.01 mm) of mandibular 3ms in the crossbite side was less than the mean volume (578.52 217.43 mm) of mandibular 3ms in the normal side. this result suggests that the presence of mandibular 3ms has a different growth potential even if a similar development stage was observed in two - dimensional radiographs. this difference might be due to the difference in crown volume in the erupted teeth. however, we did not differ crown and root volume due to the difficulties in performing this. furthermore, we investigated the effects of tooth number in the crossbite on volume difference and no effect was found according to the results of the linear regression analysis (r = 0.004 ; p = 0.714). the strength of this study is that it is the first study using cbct for the evaluation of mandibular 3 m maturation in unilateral crossbite patients. however, limitation of this study might be the number of the patients (25 patients) included to the study. cbct data from the archives were used in the present study because it is not ethically acceptable to expose patients to radiation. according to the findings of celikoglu., dental age of patients with sagittal skeletal malocclusions was approximately twice more advanced when compared with patients without sagittal skeletal anomaly patterns. a recently published study have shown that the dental age has been estimated to be more advanced than chronological age in all skeletal malocclusions and dental maturation advanced in cases with a tendency to develop class ii malocclusions. therefore, the inclusion criteria such as no hypodontia, no sagittal and vertical skeletal anomaly reduced the study sample of the present study. future studies including larger study samples and performed on cbct archive could be welcome to discuss and confirm our findings. as a result, although two - dimensional data showed that no statistically significant difference was present for the development of mandibular 3ms in the normal and crossbite sides, the volume of 3 m that was calculated by means of cbct was found to be less in the crossbite side than in the normal side. | objectives : the aim was to investigate mandibular third molar (3 m) 's maturation in the crossbite and normal sides by two- and three - dimensional analyses using cone beam computed tomography (cbct).materials and methods : a retrospective study was performed using cbct of 25 patients (16 females and 9 males ; mean age : 16.8 2.9 years) with unilateral posterior crossbite. the formation stages and the volume of the mandibular 3ms were evaluated by means of cbct data of the patients without knowing the crossbite side of the patients.results:statistically no significant differences were found in the development of the 3ms between the crossbite and the control sides, whereas the volume of 3 m was found to be less in the crossbite side than in the normal side (p = 0.021).conclusions : a volume of 3 m was found to be less in the crossbite side than in the normal side. |
anemia represents a frequent complication in cancer patients as well as in chronic inflammatory diseases. it is an important cause of cancer - related fatigue, which considerably affects quality of life. anemia is in fact considered as a bad prognostic factor for survival regardless of tumor type. up to 40% of cancer patients are anemic at diagnosis [3, 4 ] and the frequency even increases following chemotherapy. this incidence varies according to the stage and the tumor type as well as patient age. moreover, tumor responsiveness to radiotherapy seems to be weakened in the case of anemia. in substitution to blood transfusion as anti - anemia therapy, some erythroid stimulating agents have been developed including human recombinant erythropoietin (hrepo). despite this treatment improves quality of life by alleviating anemia, the use of hrepo as a treatment for cancer related anemia could be inappropriate for cancer patients. indeed, based on clinical trials [7, 8 ] hrepo was suspected to trigger tumor progression leading to decreased survival. the essential role of circulating erythrocytes is the transport of oxygen to the tissues. oxygen is bound to hemoglobin within erythrocytes that makes them highly prone to oxidative damage. for this reason, erythroid cells contain numerous antioxidant enzymes to protect them against oxygen radicals and deficient protection from reactive oxygen species (ros) results in disease of red blood cells including anemia. in fact, there are several causes of cancer - associated anemia including mechanical influence of the tumor on blood flow, and mainly the immune system activation with autoantibody formation and pro - inflammatory cytokines production. indeed, in vivo and in vitro studies have demonstrated the implication of interferon (ifn)-, tumour - necrosis factor (tnf)-, tnf - related apoptosis - inducing ligand (trail) and interleukin (il)-1 [1318 ] in the inhibition of the proliferation, and differentiation of erythroid progenitor cells. in fact, pro - inflammatory cytokines were shown to trigger the suppression of renal erythropoietin production and therefore erythropoiesis. inhibition of epo production was shown in vitro and in vivo to potentially involve ifn, il-1 and -6, and tnf [2022 ]. however, according to spivak, the suppression of erythropoietin production in inflammatory conditions such as cancers, can not be the solely explanation for anemia since the level of plasma erythropoietin is not affected in a sufficient amount. in this respect, hematopoietic stem / progenitor cells (hspc) express receptors for pro - inflammatory cytokines and several studies demonstrated that a direct action of the cytokines on hematopoietic cell lines in vitro could impair erythroid development and the number of erythroid progenitor cells [2326 ]. moreover, cytokines act in a microenvironment where they are produced and supposed to be concentrated, rather than in circulating blood. indeed, poor correlation has been reported between circulating cytokine levels and the high cellular cytokine production. furthermore, marrow - adherent cells from patients with the anemia of chronic disease suppressed erythroid progenitors. the molecular mechanisms involved in pro - inflammatory cytokine - mediated anemia, apart from epo down - regulation and iron metabolism deficiency, are poorly described. for that reason, we aim to review the current knowledge concerning the direct effect of pro - inflammatory cytokines on erythroid cell differentiation, especially on signal transduction pathways and the regulation of erythrospecific genes expression in the pro - inflammatory - mediated inhibition of erythroid differentiation. we will then focus on molecular regulation of erythroid differentiation rather than on iron or erythropoietin involvement in anemia. expansion and differentiation of erythroid progenitor cells are dependent on growth factors and hormones network, acting in a thinly regulated chronology. epo is the main erythropoietic hormone, acting by interaction with its specific membrane receptor epor. stimulation of epor triggers the activation of signaling pathways required for survival, proliferation, and differentiation of erythroblasts. another important cytokine involved in erythropoiesis is the stem cell factor (scf), a ligand of the membrane receptor c - kit. signal transduction pathways activated by scf have been reported to delay differentiation and to enhance progenitors proliferation in cooperation with epo [29, 30 ]. the activation of jak2 results from the ligand binding - induced conformational change of the epor dimer [31, 32 ]. activated jak2 induces phosphorylation of the tyrosine kinase ron that activates pi3k via the docking molecule grb2-associated binder (gab)1 also reported as phosphorylated after stimulation of epor. activation of the pi3k substrate akt / pkb induces downregulation of the cell cycle inhibitor p27/kip1 expression via inhibition of the transcription factor forkhead box 03a (foxo3a), a downstream target of epor / pi3k / akt signaling pathway. moreover, the transcription factor foxo3a has been recently reported as one of the main regulators of oxidative stress in erythropoiesis. in fact, foxo3a is inactivated by epo signaling pathway and its expression as well as its transcriptional activity is enhanced during the maturation of erythroid precursor cells when epor expression decreases. in the presence of epo and in the case of the loss of foxo3a, ros mediate the decrease in lifespan of circulating erythrocytes as well as the rate of erythroid cell maturation. this suggested that foxo3 is required for the regulation of oxidative stress in erythropoiesis. the cell - signaling cascade initiated by epo - dependent jak2 activation, leads to erythroblast expansion. moreover, pi3k activation mediates the mitogen - activated protein kinase (mapk, erk1/2) path in correlation with the expansion of erythroblasts. another epor - mediated pathway leading to cell proliferation involves the ras - raf - mek - erk pathway [3943 ] upon recruitment of the grb2-sos adapter molecules to the epor [44, 45 ]. phosphorylation of the kinase raf1 has been shown to delay erythroblast differentiation by restraining the caspase-3 activation. moreover, epo and scf activate jun - n terminal kinase (jnk) promoting proliferation and survival of hematopoietic cells [47, 48 ]. on the other hand, epo - induced differentiation of erythroid cells is also dependent on pi3k / akt signaling pathway that was suggested to act in concert with protein kinase c (pkc)-. pkc- isoform has a role in mediating epo - induced erythroid differentiation of the cd34 + progenitor cells from human bone marrow. furthermore, during epo - dependent phase of erythroid differentiation, epo and scf suppressed activity of p38 whereas during the epo - independent terminal - phase of differentiation, p38 and - phosphorylation was increased. this demonstrated both isoforms of p38 function to promote the late - stage differentiation of primary erythroid progenitors. this confirmed previous report, showing that activation of p38 as well as jnks was required for epo - induced erythroid differentiation in skt6 cells. also involved in erythropoiesis regulation is the jak / stat5 signaling pathway which is rapidly activated after epo binding to epor, on erythroid progenitors. in mice models, it was shown that early erythroblasts survival as well as normal erythropoiesis was controlled by stat5. indeed, silenced stat5 expression in mice led to an increase in early erythroblast numbers which nevertheless failed to progress in differentiation giving rise to anemia. silenced stat5-mediated anemia was correlated to down - regulation of the antiapoptotic bcl - xl gene and to increased apoptosis. this supported the jak / stat5 pathway implication in the regulation of differentiation by preventing pro - erythroblasts apoptosis. on the other hand, bcl - xl - mediated inhibition of apoptosis in erythroid cells was shown to be a response to epo / epor - induced inhibition of the caspase cascade amplification. indeed, caspase-3 activation led to degradation of the transcription factors scl / tal-1 as well as gata-1, which regulate bcl - xl gene expression [54, 55 ]. in fact, tal-1 protein was shown phosphorylated in response to epo stimulation by pi3k - activated mapk signaling pathway. stimulation of signaling pathways by epo / epor, scf / kit or other stress conditions results in the activation / repression of many transcription factors specifically involved in erythropoiesis regulation. the zinc finger protein gata-1 is considered as one of the most critical transcription factors in erythropoiesis as well as megakaryopoiesis. besides gata-1 that belongs to the gata - family of transcription factors, gata-2 is also involved in erythropoiesis and megakaryopoiesis regulation [58, 59 ]. both gata-1 and gata-2 transactivation activities require interaction with friend of gata (fog)-1 cofactor [60, 61 ]. in addition, both transcription factors have gata binding sites in their cis - acting elements allowing a cross - regulatory mechanism in which gata-1 can control the expression of gata-2 and vice versa. gata-2 is overexpressed in early immature hematopoietic progenitors to ensure their maintenance and proliferation whereas gata-1 is essential for the survival of erythroid progenitors as well as the terminal differentiation of erythroid cells [59, 62 ]. in fact, increased expression of gata-2 determines megakaryocytic differentiation whereas its down - regulation is required for erythroid differentiation. recently, a role for gata-2 in the regulation of quiescence in human hematopoietic stem and progenitor cells has been reported. epo - induced phosphorylation of gata-1 is important for maturation of fetal liver erythroid progenitor cells, specifically on serine 310 by pi3k / akt that enhances gata-1 transcriptional activity in vitro and in erythroid cells. however, gata-1 acetylation by cbp / p300 is also described as crucial for the binding to its dna target gata sequence possibly involving phosphorylation [6567 ]. moreover, phosphorylation of gata-1 could be mediated by mapk pathway, as an ubiquitination signal for its proteasomal degradation. on the other hand, besides fog1, gata-1 activity is highly dependent on interaction with many cofactors including eklf, sp1, cbp / p300, lmo2, ldb1, runx1, fli1 and pu.1, which represent a part of the best - described interacting proteins. these cofactors can constitute a very complex network regulating erythropoiesis and megakaryopoiesis, by promoting or repressing gata-1 activity [6973 ]. particularly, pu.1 is a strong inhibitor of gata-1 dna - binding activity and erythroid differentiation [74, 75 ]. evidences for tnf inhibiting effect on erythroid differentiation have been described 30 years ago. in 1987 blick. observed a decrease in hemoblobin synthesis in cancer patients treated with tnf (phase 1) while in vitro study showed that tnf inhibited the formation of bfu - e cells. later, xiao. reported that tnf inhibited the glycophorin a+ cells in correlation with an inhibition of erythropoiesis. moreover, an increasing hemoglobin level has been observed in patients suffering from anemia of chronic disease after an anti - tnf treatment. the reduction of epo production in the kidney partially explained the effect of the pro - inflammatory cytokins including tnf. reported that tnf-mediated inhibition of epo production in hepg2 cells was a consequence of gata-2 and nf-b over - expression. this was to some extent completed by imagawa. who showed that tnf-mediated inhibition of epo gene expression could be rescued by the k-7174, a gata - specific inhibitor, in hepg3 cells. however, tnf inhibiting effect on erythroid differentiation also occurs by a direct action on cells, including hematopoietic progenitors and cell lineages. recently, tsopra. published a study on disease - related anemia in chronic lymphocytic leukemia (cll) patients. they showed that cll - related anemia might result from the direct suppressive effect of tnf on the erythroid development in early stages of erythropoiesis instead of an intrinsic defect of erythroid precursors to differentiate or to respond to epo stimulation. in fact, the results from rusten and jacobsen in 1995 suggested for the first time that tnf--induced inhibition of erythroid colony formation could be directly mediated on the progenitor cells. by using bfu - e colony stimulated by various cytokine combinations (scf, il-3, il-9) with epo, they showed that tnf inhibiting effect was mediated predominantly through p55-tnf receptor (tnfr1). this result was correlated to the implication of nf-b transcription factor, a tnf/tnfr1 activated product, in the inhibition of erythroid specific genes. especially, transfection assays in k562 cells showed the suppression of human -like globin promoters by the nf-b pathway. on the other hand, our group recently showed that tnf inhibited hemoglobin production in aclacinomycin - induced k562 cells. aclacinomycin is an anthracyclin that was reported to induce over - expression of the key transcription factors for erythropoiesis, gata-1 and nf - e2 in this cell line [82, 83 ]. interestingly, the cytokine inhibiting effect was correlated to the down - regulation of gata-1 and nf - e2. these results were confirmed in the erythroleukemia cell lines hel and tf-1 [23, 24 ]. furthermore, studies in k562 and hel cells strongly suggested gata-1 as a key target of tnf inhibiting effect achievement (figure 1). indeed, the cytokine induced a decrease in the expression of fog-1, an essential cofactor of gata-1, a down - regulation of gata-1 by proteasomal degradation and a reduced acetylation level of gata-1 while the transcription factor gata-2 was over - expressed. in addition, an inhibition of epor, - and -globin, erythroid - associated factor (eraf), hydroxymethylbilane synthetase (hmbs), and glycophorin a (gpa) erythro - specific genes, was found in the epo - dependent tf-1 cell line. these results were concomitant with a reduction of gata-1/fog-1 complex formation and a significant and rapid increase in p38mapk phosphorylation. the inhibition of p38 abrogated the inhibitory effect of tnf on gata-1 as well as -globin expression in epo - induced tf-1 cells. thus, data related to tnf-mediated inhibition of erythropoiesis show the indirect but also the direct involvement of this cytokine in anemia development. however despite few publications describe the molecular mechanisms implicated, they clearly show the role of tnfr1 and nf-b, as well as other specific transcription factors, namely gata-1 and fog1, nf - e2, and gata-2. notably, the down - regulation of gata-1 at different levels by tnf might affect erythroblasts programmed cell death besides differentiation, by triggering early apoptosis via down - expression of the anti - apoptic bcl - xl gene whose transcription is regulated by gata-1. further studies on molecular mechanisms using hematopoietic stem / progenitor cells should allow bettering understanding of how tnf-mediated inhibition of erythropoiesis occurs. tnf - related apoptosis - inducing ligand (trail), also known as apo2l, is a member of the tnf - related proteins initially identified and characterized by wiley. in 1995. it is a type ii membrane protein that is also expressed as a soluble protein. both forms are able to induce apoptosis in a wide variety of transformed cell lines of diverse origins including several hematopoietic lineages. trail exhibits structural and functional similarities with fas ligand (fasl / cd95l), including the use of fadd as adaptor molecule [8690 ]. it interacts with four high - affinity membrane receptors (r) trail - r1 (dr4), trail - r2 (dr5), trail - r3 (dcr1) and trail - r4 (dcr2) that belong to the apoptosis inducing tnf - receptor family. a study on trail implication in the homeostatic control of hematopoiesis showed its negative effect on normal erythropoiesis, in a differentiation - stage specific manner. moreover, studies on trail activity and expression in myelodysplastic syndromes (mds) patients have been reported. mds are characterized by impaired erythropoiesis leading to anemia, the major clinical feature in this syndrome. by analyzing mds marrow, zang. showed that trail induced extensive apoptosis, including in the blast cell population while no increase in apoptosis was observed in normal marrow. they correlated this observation with high levels of surface expression of agonistic receptors trail - r1 and -r2 in mds marrow, which could explain the selective killing of tumor cells by trail. on the other hand, the authors suggested that apoptotic response could be modified by the variations in the anti - apoptotic protein flip expression in addition to the cell - surface - receptor expression in mds. indeed, they found that flip was expressed in marrow from healthy donors whereas the protein was not detectable in most of the mds marrows. together, the results suggested that trail might play a role in the regulation of hematopoiesis in mds marrow. in the same way, another study showed that trail expression was increased in the bone marrow mononuclear cells from mds patients and released at the bone marrow level probably contributing to the degree of anemia. in fact, mds bone marrow - conditionned media with released soluble trail, added to a normal cd34-derived erythroblasts culture, led to impairment of erythroid maturation, as assessed by the levels of gpa. this demonstrated the role of soluble trail in affecting the maturation of erythroid precursors in mds patients. a comparative study in multiple myeloma (mm) patients with or without anemia, showed an inverse correlation between the expression of trail (and fas - l) in malignant plasma cells and the relative erythroblast numbers, with a higher percentage of immature erythroblasts in enriched erythroblast populations from anemic mm patients. a downexpression of gata-1 transcription factor has been detected in immature erythroblasts from mm patients with severe anemia. they ascribed this decrease to fas - l and/or trail - mediated cleavage of gata-1 native form. indeed, gata-1 can be cleaved by several caspases in cd34 + cell - derived erythroblasts, following their treatment with trail leading to maturative arrest. found that trail - r3 and -r4 were never expressed neither on the surface of freshly purified cd34 + hematopoietic progenitor cells nor on gpa+ erythroblasts at early - intermediate and late culture times. on the contrary, they suggested that trail - r2 probably played a role in erythroid development since this receptor was found expressed at early phases of erythroid differentiation with a progressive increase along the erythroid maturation. the authors reported that trail induced map kinase erk1/2 activation in primary normal erythroblasts, but not p38 or jnk. considering that erk1/2 pathway was shown as involved in the early proliferative phases of erythropoiesis and in the inhibition of terminal erythroid differentiation [52, 9698 ], they suggested that trail - mediated activation of erk1/2 was responsible for the inhibiting effect of trail on normal erythroid development. on the other hand, the activation of erk1/2 by trail at all the stages of erythroid development correlated with the expression of trail - r2. together these studies clearly indicate that trail plays a role in erythropoiesis inhibition and anemia development via its receptor r2 and probably r1. interferon (ifn)- has been reported as inhibiting growth and differentiation of erythroid precursor cells [99, 100 ] as well as a potential mediator of hematopoietic failure in aplastic anemia (aa). aa belongs to the bone marrow failure syndromes, characterized by a breakdown in the stem- and progenitor - cell compartments. in fact, several authors had suspected the role of ifn- in erythropoiesis inhibition according to an elevated production of the cytokine by lymphocytes in aa patients while it was absent in normal bone marrow [100105 ]. a microarray analysis of rna - expression profile in cd34 + and bone marrow stroma cells showed that ifn- induced an increase in the expression of fas and trail genes, known to be involved in anemia promotion. furthermore, felli. reported that the tnf family members trail and tweak (apo-3 ligand) as well as its receptor fn-14 were involved in ifn--mediated suppression of erythropoiesis in purified human erythroblasts. moreover, they could restore erythroid cell survival, proliferation and maturation, inhibited by ifn-, performing combined neutralization of tweak, trail and fasl / cd95l. this pointed out the role of the three proteins as effectors of ifn- in erythropoiesis suppression. on the other hand, the modulation of adhesion molecule genes expression (icam1 and vcam1, integrin-5 and integrin-3) in stromal cells treated with ifn- correlated with previous works demonstrating the altering effect of ifn- (and tnf-) on the adhesive mechanism. therefore it was assumed that the cytokine may affect the function of bone hematopoietic stem cells by disturbing their adhesion to the marrow microenvironment in aa patients. until now, in opposition to tnf and trail, the direct effect of ifn- on erythroblastic cells at the molecular level has not been demonstrated. however, ifn- and also il-6 are known to activate the phosphorylation of the transcription factor stat3. it was recently reported that stat3 activation silenced -globin gene expression in primary erythroid cells and foetal hemoglobin production in k562 cells. in these experiments one of the major proteins of red blood cells is the acidic peroxidoxin, which plays a role in the response against oxidative stress. the expression of this protein is induced early in erythropoiesis, prior to hemoglobin production suggesting the importance of the protection of erythroid progenitors against oxidative stress. erythropoiesis regulation is indeed very sensitive to oxidative stress and the release of proinflammatory cytokines in neoplastic patients as well as chronic inflammatory diseases are often associated with increased production of reactive oxygen species (ros) (h2o2 and ho) [112, 113 ]. moreover, tnf links inflammation to carcinogenesis through ros and it was reported that tnf elevates ros production in glioma cells. however, it appears that the role of oxidative stress is complex since reactive oxygen species may either trigger or prevent [117, 118 ] hematopoietic differentiation and proliferation. it was demonstrated that the increase in oxidative stress and free radicals are associated with disorders implicated in anemia of chronic disease while anthracycline- and butyrate - mediated differentiation of k562 cells was prevented by anti - oxidant compounds. this last study suggested that butyrate- or anthracycline - generated oxidative stress was involved as the first step in the irreversible erythroid differentiation process. on the other hand, dallalio. reported that inflammatory cytokine - mediated anemia of chronic disease may occur through modulation of oxidative stress. elorza. also demonstrated the importance of the ros modulation during erythropoiesis, by studying the uncoupling protein (ucp)2 role. its expression leads to a decrease in mitochondrial superoxide and it was previously shown that ucp2 was related to erythroid differentiation since it was induced by gata-1 activation in the proerythroblast g1e - er cell line. ucp2 deficiency in progenitor cells at the epo - dependent phase of erythropoiesis led to a decrease in cell proliferation in correlation with erk reduced phosphorylation, which is known as a ros - dependent cytosolic regulator of cell proliferation. a relationship between ucp2 and the mapk / erk pathway has been reported in the case of elevated inflammatory response and inhibition of ucp2 could lead to the development of anemia. obviously pro - inflammatory cytokines have a negative effect on erythropoiesis development leading to anemia in multiple diseases including chronic infections, chronic inflammatory diseases, myelodysplastic syndromes, and malignancy. the pathophysiological effects of cytokine over - expressions have been well described but the mechanisms surrounding cytokine - mediated induction of anemia remain largely unknown. many studies show that erythroid colony formation in response to epo is impaired in the presence of pro - inflammatory cytokines [125127 ] without providing molecular mechanisms. in this paper we review data available on the molecular mechanisms involved in the defect of erythropoiesis due to three pro - inflammatory cytokines tnf, trail, and ifn. nonetheless, other pro - inflammatory cytokines have been involved in erythropoiesis defect, such as the interleukin (il)-6. impaired erythropoiesis is most likely due to apoptosis induction, cell growth inhibition and epor downregulation as a result of a local increased production of the cytokines, but also iron metabolism damage. in all cases pro - inflammatory cytokines affect epo either by inducing inhibition of its production by kidney or by preventing its physiological functions at the cellular level. indeed, cytokines activate signaling pathways that can have common feature with epor - triggered signaling pathways leading to cell proliferation, differentiation, or survival. regulation of erythropoiesis occurs in a tightly time dependent manner and changes in the timing of one specific signaling intermediate activation can disturb cell differentiation process. we could recently observe that this kinase was very early activated by tnf (10 minutes) while epo - mediated activation occurred much later in tf-1 cells (48 hours). this result was correlated to the inhibiting effect of tnf on epo - induced erythroid differentiation in tf-1 cells. thus, how specific signal transduction pathways of pro - inflammatory cytokines can interact with epor ones to prevent erythroid differentiation is an interesting issue. on the other hand, pro - inflammatory cytokines activate diverse transcription factors that can have contradictory effects in regard to the cell intention. for example, nf-b, which is typically activated by cytokines, was reported involved in the inhibition of epo production and the repression of globin genes expression. moreover, gata-1/gata-2 balance, which is a key element for erythropoiesis regulation was shown affected by tnf. on the other hand, besides erythroid genes expression, gata-1 and tal-1/scl are implicated in apoptosis regulation by controlling bcl - xl gene expression. common pro - apoptotic properties of pro - anemia cytokines seem involved in erythropoiesis delay or prevention. therefore, cytokine - mediated activation of caspases and epo - induced gata-1 expression might represent a crossing point in which gata-1 cleavage would lead to down - regulation of bcl - xl expression and then apoptosis activation. studying the pro - inflammatory cytokines effect at the transcriptional level should allow understanding how the erythroid specific genes are repressed and how the pro - apoptotic genes are activated. in addition to transcription factors network analysis, especially gata-1, micro - rnas implication could be taken in consideration according to the expected role of these molecules in erythropoiesis [128135 ]. in conclusion, the modulation of epo - mediated signaling pathways as well as transcription factors and cofactors by pro - inflammatory cytokines are required to achieve inhibition of erythroid differentiation. several in vitro and in vivo studies demonstrated that high levels of proinflammatory cytokines and increased oxidative stress contribute both to the development of anemia and to the resistance to recombinant human hrepo. the complexity of this phenomenon provides multiple targets for potential drugs in order to inhibit cytokines effect and/or to promote erythroid differentiation, mainly in cancer - related anemia. indeed, despite hrepo is clinically efficient for anemia treatment it is strongly suspected to induce tumor cell proliferation. therefore studies of the mechanisms involved in the inhibiting effect of cytokines on erythropoiesis are essential to intend future anti - anemic treatment, at least for cancer patients. | anemia of cancer and chronic inflammatory diseases is a frequent complication affecting quality of life. for cancer patients it represents a particularly bad prognostic. low level of erythropoietin is considered as one of the causes of anemia in these pathologies. the deficiency in erythropoietin production results from pro - inflammatory cytokines effect. however, few data is available concerning molecular mechanisms involved in cytokine - mediated anemia. some recent publications have demonstrated the direct effect of pro - inflammatory cytokines on cell differentiation towards erythroid pathway, without erythropoietin defect. this suggested that pro - inflammatory cytokine - mediated signaling pathways affect erythropoietin activity. they could interfere with erythropoietin - mediated signaling pathways, inducing early apoptosis and perturbing the expression and regulation of specific transcription factors involved in the control of erythroid differentiation. in this review we summarize the effect of tumor necrosis factor (tnf), tnf - related apoptosis - inducing ligand (trail), and interferon (ifn)- on erythropoiesis with a particular interest for molecular feature. |
acidithiobacillus ferrooxidans (a. ferrooxidans) is a gram - negative, extremely acidophilic, mesophilic, chemolithotrophic bacterium and the most well - studied acidophilic organism which is usually found in acid environments such as acid mine drainage,. due to its bioleaching capabilities, it is an important member of microbial consortia involved in the industrial recovery of metal under mesophilic conditions (bioleaching or biomining). recently, a. ferrooxidans has played important roles in bioleaching and harnesse environmental contamination,. like in other acidophilic iron - oxidizing bacterium, it grows optimally at about 35 c in 9k inorganic medium at extremely low ph (ph 1.02.0) and fixes both carbon and nitrogen from the atmosphere. a. ferrooxidans derives energy from oxidizing reduced sulfur compounds and fe ions to form sulfate and fe, respectively. dna was isolated from 1.01.5 g of cell paste using qiagen genomic 500 dna kit (qiagen, hil - den, germany) with a modified protocol, st / ft, for cell lysis, as described in valdes.. draft genome sequence of a. ferrooxidans type strain dlc-5 was obtained in illumina hiseq2000 sequencing technology by shanghai majorbio bio - pharm technology co., ltd. (shanghai, china), using the short oligonucleotides alignment program (soap) denovo alignment tool (http://soap.genomics.org.cn/) processes reads assemble. a library containing 300-bp inserts was constructed. altogether, 6,372,268 paired reads ; 398,580 single reads ; total 1,079,535,272 bp bases with average coverage of 221.1. reads were filtered to remove adapter sequences, low - quality bases (phred score, 1000 bp ; contig n50, 102 bp ; contig n90, 569 bp) and 573 scaffolds (> 1000 bp ; scaffold n50, 71 bp ; scaffold n90, 333 bp). until now, two genome sequences of a. ferrooxidans strains atcc 23270 and atcc 53993 are available in the public databases,. these genomic data are useful for the experimental identification of unique proteins or estimation of the phylogenetic relationship among the related strains. strain dlc-5 (cctcc - m 2014362) is the type strain of a. ferrooxidans, isolated from wudalianchi in heihe of heilongjiang province, and the type species of the genus acidithiobacillus, which currently contains five species. the draft genome sequences of strain dlc-5 have great importance to provide more explicit information for the physiology and metabolic potential of a. ferrooxidans. further analysis of the genome sequence via gene engineering might improve the oxidation of fe efficiency by strain dlc-5. the genome includes two plasmids, for a total size of 3,142,890 bp, with one circular chromosome of 1,832,305 bp (58.3% gc content). for the main chromosome, 4299 bp genes 3250 bp of protein coding genes were assigned to a putative function with the remaining annotated as hypothetical proteins. the properties and the statistics of the genome are summarized in table 1. the distribution of genes into cogs functional categories is presented in table 2. extremely acidophilic bacteria and archaea with special emphasis on bioleaching microorganisms are widely distributed in the extreme acidic environment. in this study, we analyzed the genome sequence of a. ferrooxidans dlc-5, which was isolated from acid mine drainage in northeast china. it may contribute to further studies on important process for bioleaching and acid mine drainage production, such as biofilm formation, energy resources utilization and quorum sensing that could play a role in a possible interrelationship of bioleaching heaps and other acidic environments. in addition, combining with genomes of other members in acidithiobacillus, will make an important advance in understanding of the ecological roles that acidithiobacillus species play in those acidic environments and their relationships with other extremely acidophilic microorganisms. the sequence of a. ferrooxidans dlc-5 under this whole genome shotgun project has been deposited at ddbj / embl / genbank under the accession jnnh00000000.1. | acidithiobacillus ferrooxidans type strain dlc-5, isolated from wudalianchi in heihe of heilongjiang province, china. here, we present the draft genome of strain dlc-5 which contains 4,232,149 bp in 2745 contigs with 57.628% gc content and includes 32,719 protein - coding genes and 64 trna - encoding genes. the genome sequence can be accessed at ddbj / embl / genbank under the accession no. jnnh00000000.1. |
protooncogenes expressed in murine embryonal carcinoma (ec) cells or their differentiated daughter cells include more or less ubiquitously expressed protooncogenes such as c - myc, c - k - ras, and c - abl, as well as c - onc genes with a very restricted expression pattern. examples of the latter are n - myc, c - mos, and int-2. these c - onc genes are transcriptionally active in ec cells, as well as in germ cells and/or early embryonic cells. when ec cells are induced to differentiate some protooncogenes or oncogene - related products undergo changes in expression. thus, ec cell differentiation has been associated with increased expression of c - src, c - fos, int-1, int-2, and the epidermal growth factor (egf) receptor, whereas decreased expression has been observed for c - mos, c - k - ras, c - myc, n - myc, and platelet - derived growth factor. the relationships between these changes in expression and ec cell differentiation are not understood. they may be important for the differentiation process or for expression of a differentiated phenotype. they may, however, also be secondary events with no functional significance to ec cell differentiation.imagesfigure 2.figure 2.figure 4.figure 4.figure 5.figure 5. |
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hiv / aids is increasingly affecting the health and welfare of children and undermining hard - won gains of child survival in highly affected countries. recent estimates from the joint united nations programs on hiv / aids (unaids) suggest that globally about 2.5 million children younger than 15 years of age are infected with hiv : 90% living in sub - saharan africa and about 64,813 living in ethiopia. without treatment 75% of hiv - infected children as highly active antiretroviral therapy (haart) becomes increasingly available in low resource settings, infected children are living longer. with increased survival, one of the greatest psychosocial challenges that parents and caregivers of hiv - infected children face is the disclosure of hiv - positive status to their infected children. one of the most difficult issues that families with hiv - infected children face is when and how to talk about hiv to their children. hiv - positive status disclosure to infected children and adolescents should take place in a supportive environment with collaboration and cooperation among caregivers and health care providers. disclosure is contingent on the caregiver 's acknowledgement of the illness, the readiness to disclose, and child 's cognitive skills and emotional maturity. despite emerging evidence of the benefit of disclosure, when and how to disclose the diagnosis of hiv to children remain a clinical dilemma. clinicians and other members of multidisciplinary teams should collaborate with caregivers of hiv - infected children to disclose hiv diagnosis to the child in a developmentally appropriate manner. children react to hiv disclosure in different ways and it is not uncommon for relatives to disagree about disclosing hiv - related information to children. disclosure has to be individualized taking into consideration the particular child, parent (s), family, household, and community. hiv diagnosis disclosure entails communication about a potentially life - threatening, stigmatized, and transmissible illness, and many caregivers fear that such communications may create distress for the child. the american academy of pediatrics strongly encourages disclosure of hiv - positive status to school - age children. but in ethiopia, no such recommendations and guidelines are available concerning disclosure of pediatric hiv, and disclosing the diagnosis of hiv or aids to a child is controversial and challenging among health care providers, parents, and caregivers. thus this study assessed the magnitude of hiv - positive status disclosure and the associated factors among hiv - infected children in northwest ethiopia. an institution - based cross sectional study design was carried out from march to april, 2012 at the three hospitals of north gondar zone. north gondar zone is one of the 11 zones in the amhara national regional state. all hiv - positive children aged 515 years who were on care and support followup at the pediatric art clinics of the three hospitals (gondar, metema, and dabark) in north gondar zone. all caregivers of the children enrolled in the chronic hiv care at pediatric art units of the three hospitals were included. children who came by themselves or with no caregiver or parent disclosure refers to when the caregiver said that the child knows his / her hiv / aids diagnosis regardless of who told the child. data were collected by an interview technique using a structured questionnaire which was first prepared in english then translated to the local language amharic. a clinical nurse working at the pediatric art clinic of each hospital and supervised by a supervisor collected the data. the prepared questionnaire was pretested and structured accordingly in a logical manner into sociodemographic, clinical characteristics and hiv - positive disclosure parts. the data were entered in to epi info version 3.5.1 statistical software and analyzed by spss version 20.0. variables having p value 0.2 in the bivariate analysis were entered into a multiple logistic regression model to control the confounding effect. odds ratios with their 95% confidence intervals were calculated to measure associations, and statistical significance was set at p 0.05 is considered as a good fit model. permission was obtained from the hospitals administration and the art focal persons at each hospital. after the purpose of the study was explained, verbal consent was obtained from each caregiver. participants also were informed that participation was on voluntary basis and that they can withdraw at any time if they are not comfortable about the questionnaire. names or personal identifiers were not included in the written questionnaires to ensure participants ' confidentiality. a total of 428 caregivers were interviewed. of these, 343 (80.1%) were from gondar university referral hospital. three hundred thirty - one (77.3%) of the caregivers were females, 368 (86%) were orthodox christians, and the majority (89.5%) were urban residents. about half (51.4%) of the caregivers had a monthly income of 300999 ethiopian birr per month. nearly two thirds (65.4%) of the caregivers were biological parents of the children and one third were daily labourers. almost half (49.3%) of children were males and the mean age of children was 9.96 3.0 sd years. the median age at diagnosis of hiv was 6.0 years (iqr = 5 years). three hundred four (71%) of the children attended their primary school and nearly two third of them were living with their biological parents (table 1). nearly two third (61.9%) of the caregivers were hiv - positive of whom 92.5% were on art and 86.4% had disclosed their hiv - positive status to someone else. majority (81.3%) of the children had a who clinical stage i disease. majority, that is, 344 (80.4%) children, had history of opportunistic infections (ois) and 42.5% were hospitalized. three hundred forty - eight (81.3%) children were on art at the date of interview (table 2). of the 428 children, 169 (39.5%, 95% ci : 34.8, 43.7) of the children living with hiv / aids were disclosed their hiv - positive status. the mean age at disclosure was 10.7 years (2.3 years). sixty - nine (40.8%) children were disclosed by their biological parents while 38.5% of children were disclosed by health care providers. the prominent reasons for disclosure as mentioned by caregivers were child thought to be matured (44.4%) and repeated questionings of what happened to me (27.2%) by the child (figure 1). participants mentioned reasons for not disclosing the child about his / her hiv - positive status. more than half still believe that the child is too young (57.1%) and another one fifth fear the negative emotional and health consequence (20.1%) of disclosure (figure 2). two hundred twenty - one (81.1%) of the caregivers believed that disclosing the hiv - positive status to the child is advantageous and three quarters (76.8%) had the intension to disclose in the near future. as clearly depicted on the multivariate logistic regression, caregiver 's relation with the child, age of the child and loss of a family member were independently and significantly associated with disclosure of hiv - positive status to hiv - infected children. however, factors related to the caregiver such as sex, religion, hiv - positive status, and educational status, as well as sex of the child, history of ois, and art status of children were not significantly associated with disclosure of hiv - positive status to hiv - infected children. accordingly, nonbiological parents were 4.14 (aor = 4.14, 95% ci : 1.22, 14.04) times more likely to disclose hiv - positive status to hiv - infected children as compared to biological ones. age of the child was one of the factors significantly associated with disclosure of hiv - positive status in which children older than 10 years of age were 8.54 (aor = 8.54, 95% ci : 4.5, 15.53) times more likely to be disclosed as compared their counterparts. those children who lost any of their family members were two (aor = 2.04, 95% ci : 1.16, 3.6) times more likely to be disclosed their hiv - positive status as compared to their counterparts (table 3). in ethiopia, due to the recent improvements in access to antiretroviral therapy, dramatic decline of mortality and morbidity of hiv - infected children has been observed. as children survived for longer periods of time, disclosure issues emerge related to pubertal development and sexuality, fear of transmission, and the need to promote adherence to complex and often toxic regimens. promotion of trust, improved adherence, open family communication, and better long - term health and emotional well - being in children are some of the advantages. in this study, 39.5% of hiv - positive children this finding is similar to studies conducted in usa which reported a disclosure rate of 3543% [1113 ]. but it is very low as compared to studies done in high - income countries in which the disclosure rate ranges from 57 to 100% [10, 14, 15 ]. the lower prevalence of disclosure in our study might be due to fear of stigma and discrimination by the family members. since the majority of hiv - infected children acquired the virus from their mothers, disclosure of a child 's hiv - positive diagnosis often leads to disclosure of other family secrets that leads to stigma and discrimination. caregiver 's perceived lack of emotional preparedness of children and [16, 17 ] and the absence of recommendations and guidelines for disclosure of hiv - positive children in ethiopia might have also contributed for the lower rate of disclosure. this finding was somewhat higher as compared to studies conducted in poland (16.2%), thailand (30.1%), ghana (21%), and nigeria (13.5%). it is also higher as compared to a study conducted in addis ababa, ethiopia (17.4%). the possible justification can be difference in time period and there might be also increased awareness on the benefit of disclosure by caregivers. additionally, this study assessed disclosure status among children 515 years of age, but the study conducted in addis ababa includes all pediatric age groups. age was identified as a factor for disclosure in this study and in another study conducted in ethiopia. this could be due to the caregivers ' belief that at early age, the child is lacking the emotional and cognitive maturity needed to understand the disease and its implications [12, 13, 21, 22 ]. in this study, the mean age at disclosure was 10.7 years which was high as compared to studies done in new york (7 years) and nigeria (8.7 years) but somewhat comparable with a study conducted in ghana (11.72 years) [7, 17, 20 ]. reasons cited by the caregivers were consistent with that of studies in resource - limited countries ; namely, child is too young, fear of emotional and health consequences, fear of stigma and discrimination, and fear that the child would not keep diagnosis to themselves. caregivers believed their children were too young to know their status. in our study, the factors that were independently and significantly associated with disclosure were the age of the child, nonbiological parent relation with the child, and loss of family member. consistent with previous studies done in ghana and london, children were more likely to be disclosed if they were orphaned [11, 23 ]. the results of our study supported previous studies done in nigeria, thailand, london, and massachusetts [11, 19, 20, 23 ] that showed older age of infected children as a determinant factor for hiv - positive status disclosure. children older than 10 years were more likely to be disclosed than those younger than 10 years. the child 's theory of cognitive understanding of illness is also in favour of this finding. accordingly, the age from 9 to 10 years and older is considered to be the best time for hiv - infected children to know about their sickness as at this age children can understand about the complex causes of illness and its consequences. in this study, nonbiological caregivers were more likely to disclose the child 's hiv - positive status than biological caregivers. this finding is in agreement with studies done in philadelphia and thailand [19, 21 ] where most children who knew their diagnosis were living with caregivers who were not related to them, whereas the majority of children who did not know the diagnosis were living with biological parents. as argued by these studies biological parents might not be willing to confront the fact of their own responsibilities in passing the infection onto their children. the sample size is relatively larger than other studies done in sub - saharan africa, and generalization can be made to children on chronic hiv / aids care in ethiopia. but as a cross - sectional study, the associations observed may not be causal. because of lack of data on adherence to treatment, we could not include it in the analysis. furthermore, the study did not explore the benefits of disclosure on adherence and clinical improvement in hiv / aids. the rate of disclosure of hiv - positive status to hiv - infected children is low in this study. non biological parent caregivers, children older than 10 years of age, and loss of family member were independently and significantly associated with disclosure of hiv - positive status to hiv - infected children. hence, it is important to target young children living with their biological parents and those having young parents. guideline for disclosure of children with hiv / aids has to be established in ethiopian context. we recommend further studies to be undertaken to explore the benefits of disclosure of hiv - positive status to hiv - infected children. | introduction. clinical reports have indicated positive outcomes associated with disclosure of hiv - positive status in children. this study assessed the level and associated factors of hiv - positive status disclosure to hiv - infected children in northwest ethiopia. methods. institution - based cross - sectional study was conducted among hiv - positive children from march to april 2012. data were collected using a structured questionnaire by face - to - face interview technique. bivariate and multivariate analyses were performed. results. of the 428 children, 169 (39.5%) were disclosed their hiv - positive status. the mean age of hiv - positive status disclosure was at 10.7 (2.3) years. having a nonbiological parent (aor = 4.14, 95% ci : 1.22, 14.04), child 's age older than 10 years (aor = 8.54, 95% ci : 4.5, 15.53), and death of a family member (aor = 2.04, 95% ci : 1.16, 3.6) were significantly and independently associated with disclosure of hiv - positive status to infected children. conclusions. the rate of disclosure of hiv - positive status to infected children still remains low in north gondar. hence, it is important to target children living with their biological parents and having young parents and children younger than 10 years. the guideline for disclosure of children with hiv / aids should be established in an ethiopian context. |
decreasing healthcare costs while maintaining optimal patient outcomes has been a recent focus of many institutions given changes in united states healthcare. cost reduction with optimal outcomes has been shown by switching from intravenous (iv) to oral (po) antibiotics for the treatment of pneumonia in the non - intensive care unit (icu) population. this practice has potential for significant economic impact if expanded to the icu population, as pneumonia affects 1.1 million people annually and accounts for more than 50,000 mortalities (16.5 deaths per 100,000) in the united states alone. while oral antibiotic therapy is routinely used to treat community - acquired pneumonia (cap), particularly in less severe infections that are treated on an outpatient basis, patients admitted to the icu are customarily treated with iv antibiotics for the duration of therapy. acquisition cost for the majority of iv antibiotics is significantly more expensive than their oral equivalents. in addition, iv antibiotics can have additional indirect costs, specifically involving preparation, nursing administration, and waste. the effectiveness of switching from iv to oral antibiotics has been demonstrated in cap. a multicenter, randomized trial of clinically stable patients transitioned to oral antibiotics after 3 days of iv therapy for a 10-day total course found no difference in rates of clinical cure, mortality, or clinical deterioration compared to those receiving iv treatment for duration of therapy. however, those transitioned to oral therapy had significantly shorter hospital length of stay. similarly, a prospective, observational study of patients with cap who were transitioned from iv to oral therapy within 24 h of clinical improvement demonstrated clinical cure rate of 97%. guidelines for cap developed by the infectious disease society of america recommend the switch from iv to oral therapy once patients are clinically improving, hemodynamically stable, and able to adequately absorb and take oral therapy. the american thoracic society and infectious disease society of america guidelines for hospital - acquired pneumonia (hap), ventilator - associated pneumonia (vap), and healthcare - associated pneumonia recommend all patients be initiated on iv therapy, but state conversion to enteral may be appropriate in certain patients, citing the proven effectiveness and bioavailability of certain antibiotics. dosing recommendations in the guidelines, however, are solely for iv administration. in vitro studies are available for many enteral antibiotic options for treatment of pneumonia, including those caused by multidrug resistant bacteria. multiple studies have shown that oral linezolid has approximately 100% bioavailability, with equivalent half - life and volume of distribution between enteral and iv administration, even in patients receiving enteral nutrition. pharmacokinetic studies of ciprofloxacin and moxifloxacin have demonstrated that both enteral and iv administration achieve pharmacokinetic and pharmacodynamic targets when administered in comparable doses. at present time there are no published data assessing the efficacy of enteral antibiotic therapy for critically - ill patients with bacterial pneumonia. the primary objective of this study was to assess rates of clinical improvement in patients who are initiated on or transitioned to enteral antibiotics compared to those who solely receive iv antibiotic therapy for treatment of bacterial pneumonia in a surgical icu (sicu). a single - center, retrospective, cohort study was performed after obtaining expedited approval from our institution 's institutional review board. patients treated for bacterial pneumonia in a 44-bed sicu at an academic medical center between 1/1/09 and 3/31/11 were identified electronically. patients were eligible if they had a positive quantitative bronchoalveolar lavage (bal) and were treated for bacterial pneumonia for organisms susceptible to antibiotics available in an enteral preparation. pneumonia was diagnosed with at least one the following : (i) temperature > 100.4f ; (ii) white blood count 11,000 per microliter or 50% bands ; or(iii) new or worsening altered mental status plus two at least of the following : (a) new or worsening dyspnea, cough, or tachypnea ; (b) new - onset purulent sputum or change in sputum character ; (c) worsening gas exchange or increased oxygen requirements on ventilator support ; (d) rales in combination of > 10,000 colony forming units of bacteria per milliliter from protective - catheter bal quantitative cultures. patients excluded from data analysis include those 48 h following endotracheal intubation. the primary study outcome was clinical improvement of bacterial pneumonia on day of antibiotic discontinuation. clinical improvement was defined as improvement in at least two of the following signs and symptoms of pneumonia : temperature, sputum production, cxr, white blood cell (wbc), hemodynamic stability, o2 saturation, or cpis. cpis were calculated based on temperature (36.538.4 = 0, 38.538.9 = 1, and 39.0 or 36.5 = 2), leukocytosis (411,000 = 0, 11,000 = 1, and 11,000 and bands 50% or > 17,000 = 2), tracheal secretions (none / scant = 0, non - purulent = 1, and purulent = 2), oxygenation (pao2/fio2 100.4f ; (ii) white blood count 11,000 per microliter or 50% bands ; or(iii) new or worsening altered mental status plus two at least of the following : (a) new or worsening dyspnea, cough, or tachypnea ; (b) new - onset purulent sputum or change in sputum character ; (c) worsening gas exchange or increased oxygen requirements on ventilator support ; (d) rales in combination of > 10,000 colony forming units of bacteria per milliliter from protective - catheter bal quantitative cultures. patients excluded from data analysis include those 48 h following endotracheal intubation. the primary study outcome was clinical improvement of bacterial pneumonia on day of antibiotic discontinuation. clinical improvement was defined as improvement in at least two of the following signs and symptoms of pneumonia : temperature, sputum production, cxr, white blood cell (wbc), hemodynamic stability, o2 saturation, or cpis. cpis were calculated based on temperature (36.538.4 = 0, 38.538.9 = 1, and 39.0 or 36.5 = 2), leukocytosis (411,000 = 0, 11,000 = 1, and 11,000 and bands 50% or > 17,000 = 2), tracheal secretions (none / scant = 0, non - purulent = 1, and purulent = 2), oxygenation (pao2/fio2 0.99). the majority of patients were started on empiric therapy for nosocomial infections in the sicu, consisting of linezolid, piperacillin / tazobactam, and either tobramycin or amikacin for double coverage of gram - negative organisms. thirteen patients (43.3%) in the po group and 24 (26%) in the iv group had multi - organism growth (p = 0.07). causative organism was similar between groups ; multiple organisms were reported in the setting of polymicrobial growth [table 3 ]. there was significantly more methicillin - susceptible staphylococcus aureus (mssa) and serratiaspecies in the po group, but significantly less enterobacterspecies compared to the iv group [table 3 ]. as expected, de - escalated antibiotics were quite different between groups [table 2 ]. there was significantly more linezolid, amoxicillin / clavulanic acid, and cephalexin in the po group compared to the iv group. median duration of antibiotics was similar between groups, with 8 (79) days in the po group and 9 (810) days for patients on iv therapy (p = 0.17). twenty - six patients (86.7%) in the po group exhibited clinical improvement, with 71 patients (75.5%) on iv antibiotics achieving this outcome (p = 0.31). median cpis on day of antibiotic discontinuation was decreased to 3 in both groups, no longer correlating with presence of pneumonia. secondary outcomes, including duration of mechanical ventilation, sicu and hospital length of stay, and all - cause and infection - related mortality were similar between groups [table 4 ]. microbiological data, assessed independently of the primary outcome, was not different between groups [table 4 ]. recurrence rates were low, with one patient (3.3%) in the po group and seven patients (7.4%) in the iv group exhibiting recurrence with the initial causative organism (p > 0.99). while there was no significant difference in median total healthcare cost ($ 55,787 (42,41084,248) po group versus $ 67,690 (41,200105,632) iv group, p = 0.39) those in the po group had a significant lower infection - related cost ($ 17,381 (14,77920,310) versus $ 20,776 (16,26731,497), p = 0.012) and antibiotic cost ($ 697 (5131,176) versus $ 1,042 (8251,303), p = 0.04). empirical and de - escalated antibiotic therapy for enteral verses intravenous antibiotics isolated organism from bronchoalveolar lavage for enteral verses intravenous antibiotics a total of 647 patients with bal cultures were screened for inclusion based on treatment of bacterial pneumonia. there was no difference in surgical services between the groups and the majority of patients in each group were admitted to the trauma / acute care surgery service. greater than 90% of all patients were mechanically ventilated, although administration of vasopressors on day of antibiotic initiation was low in both groups [table 1 ]. most patients were admitted to the icu on hospital day 1, however time to bal collection was approximately 1 week for all patients. on day of antibiotic initiation, vap accounted for the majority of infections, followed by hap and cap [table 1 ]. causative organism was susceptible to initial antibiotic therapy in 29 patients (96.7%) in the po group and 90 patients (95.7%) in the iv group (> 0.99). the majority of patients were started on empiric therapy for nosocomial infections in the sicu, consisting of linezolid, piperacillin / tazobactam, and either tobramycin or amikacin for double coverage of gram - negative organisms. thirteen patients (43.3%) in the po group and 24 (26%) in the iv group had multi - organism growth (p = 0.07). causative organism was similar between groups ; multiple organisms were reported in the setting of polymicrobial growth [table 3 ]. there was significantly more methicillin - susceptible staphylococcus aureus (mssa) and serratiaspecies in the po group, but significantly less enterobacterspecies compared to the iv group [table 3 ]. as expected, de - escalated antibiotics were quite different between groups [table 2 ]. there was significantly more linezolid, amoxicillin / clavulanic acid, and cephalexin in the po group compared to the iv group. median duration of antibiotics was similar between groups, with 8 (79) days in the po group and 9 (810) days for patients on iv therapy (p = 0.17). twenty - six patients (86.7%) in the po group exhibited clinical improvement, with 71 patients (75.5%) on iv antibiotics achieving this outcome (p = 0.31). median cpis on day of antibiotic discontinuation was decreased to 3 in both groups, no longer correlating with presence of pneumonia. secondary outcomes, including duration of mechanical ventilation, sicu and hospital length of stay, and all - cause and infection - related mortality were similar between groups [table 4 ]. microbiological data, assessed independently of the primary outcome, was not different between groups [table 4 ]. recurrence rates were low, with one patient (3.3%) in the po group and seven patients (7.4%) in the iv group exhibiting recurrence with the initial causative organism (p > 0.99). while there was no significant difference in median total healthcare cost ($ 55,787 (42,41084,248) po group versus $ 67,690 (41,200105,632) iv group, p = 0.39) those in the po group had a significant lower infection - related cost ($ 17,381 (14,77920,310) versus $ 20,776 (16,26731,497), p = 0.012) and antibiotic cost ($ 697 (5131,176) versus $ 1,042 (8251,303), p = 0.04). empirical and de - escalated antibiotic therapy for enteral verses intravenous antibiotics isolated organism from bronchoalveolar lavage for enteral verses intravenous antibiotics this retrospective analysis suggests that initiation of or early transition to enteral antibiotics produces comparable clinical outcomes to iv therapy in a sicu population, as evidenced by no difference in rates of clinical improvement between groups. patients in the po group had a significant reduction in antibiotic cost and overall infection - related costs compared to those patients receiving iv antibiotics for the duration of therapy. while a reduction in antibiotic cost was predicted, the decrease in infection - related costs may be attributed to indirect costs such as fewer procedures for line placement and no need for additional radiographs for placement confirmation. on the day of bal collection, initial antibiotic therapy and patient characteristics were similar between groups. however, there was significantly more mssa and serratia pneumonia in the po group, likely due to the fact that these organisms are often less resistant and generally thought to be easier to eradicate. thus, clinicians may be more inclined to initiate or transition to enteral antibiotics for treatment. on the other hand, enterobacterspecies were more commonly isolated in the iv group, which may be a result of practitioner 's concern for antimicrobial resistance with these organisms. while many of the antibiotics, specifically those for gram - negative coverage, were not transitioned to an enteral preparation until causative organism was identified, it is common practice in our sicu for linezolid to be initiated enterally for empiric coverage. this was the first study assessing clinical outcomes for surgical critically - ill patients treated with enteral antibiotics for bacterial pneumonia. benefits of utilizing enteral antibiotics are substantial, including decreased medication and labor costs, ease of administration for nurse and patient, and reduction in length of stay. with changes in healthcare in the united states, decreasing while we expected the antibiotics cost to be significantly less in the po group, we were surprised that the infection related cost was significantly lower. patients may avoid additional line placement for administration of iv antibiotics (e.g., central or peripherally inserted central catheters), additional radiographs to confirm line placement, and use of these lines may put patients at risk for catheter - related bloodstream infections. however, there remain concerns regarding enteral administration in the critically - ill population. due to acute physiologic changes, including changes in volume of distribution, organ function, and medication clearance, there is concern that bioavailability may be compromised. this is particularly of concern in the surgical population, when impaired gastrointestinal motility and adequacy of enteral absorption are potential issues. the majority of these patients are also receiving enteral feeding, which may compromise exposure of certain antibiotics if nutrition is not appropriately held surrounding enteral antibiotic administration. it was a single - center, retrospective study that relied on documentation in the electronic medical record. sample size was also limited, particularly for those meeting criteria for inclusion into the enteral group. patients included in this study belong to a specific subset of the critically - ill population, the sicu ; thus extrapolation to other critically - ill populations should be done with caution. bacterial pneumonia caused by multidrug resistant organisms was also excluded, which may make these results less applicable to certain institutions based on local antibiograms. while not statistically significant, there was a trend toward increased clinical improvement in patients treated with enteral antibiotics. this may be partially attributed to the fact that clinicians are less likely to initiate or transition to enteral therapy in unstable or severely ill patients, thus artificially inflating the clinical improvement in the enteral group. although cpis and apache ii scores were similar between groups ; differences in these patient populations or clinical status may not have been captured when utilized for analysis due to inherent limitations with such scoring systems. critically - ill surgical patients initiated or transitioned to enteral antibiotics for treatment of pneumonia had similar outcomes as those treated solely with iv antibiotics. the results of this study show potential for significant cost savings via utilization of enteral antibiotics for the treatment of bacterial pneumonia. decreased infection - related cost and antibiotic cost savings, as well as advantages regarding administration, exist while producing comparable clinical outcomes in a critically - ill surgical population. however, prospective studies are necessary to confirm the appropriateness of enteral therapy for bacterial pneumonia in the critically - ill surgical patients. | background : controlling healthcare costs without compromising patient care is a focus given recent healthcare changes in the united states. the purpose of this study was to assess clinical improvement in surgical intensive care unit (sicu) patients initiated on or transitioned to enteral antibiotics compared to those who solely receive intravenous (iv) antibiotic therapy for treatment of bacterial pneumonia.materials and methods : this retrospective cohort study included patients with a positive quantitative respiratory culture being treated for bacterial pneumonia in a sicu from 1/1/09 to 3/31/11. two distinct patient groups were identified : those treated with iv antibiotics exclusively (iv) and those either initiated on or transitioned to enteral antibiotics within 4 days of antibiotic initiation (po). the primary endpoint of clinical improvement was assessed on day of antibiotic discontinuation.results:a total of 647 patients were evaluated ; 124 met inclusion criteria (30 patients po group and 94 iv group). there was no difference in clinical improvement (86.7 po vs 72.3% iv, p = 0.14) or recurrence (10 po vs. 12.8% iv, p > 0.99) between groups. secondary outcomes of duration of mechanical ventilation, icu and hospital length of stay, and all - cause mortality were also similar. antibiotic and infection - related costs were significantly decreased in the po group ($ 1,042 vs $ 697, p = 0.04 ; $ 20,776 vs $ 17,381, p = 0.012, respectively).conclusions : sicu patients initiated on or transitioned to po antibiotics for pneumonia had similar clinical outcomes, but significantly less infection - related and antibiotic costs compared to those receiving iv therapy. further, prospective studies are warranted. |
ganciclovir, a drug against cytomegalovirus (cmv) infection, is generally well tolerated, but can cause neurotoxicity such as encephalopathy. although ganciclovir - induced encephalopathy has been described in several reports, a literature search revealed that ganciclovir concentrations in the blood or cerebrospinal fluid were previously measured in only 3 patients with encephalopathy. prompt and accurate diagnosis is thus sometimes difficult, and is derived solely from accumulated clinical information of definite cases, since ganciclovir concentrations, not routinely measured, become available after several days or a few weeks. here, we summarize clinical information of all patients with definite ganciclovir - induced encephalopathy including our own patient, who had severe symptoms, with the highest reported trough concentration of ganciclovir in the blood, and underwent therapeutic dialysis with complete recovery. encephalopathy can be caused by neurotoxicity with prophylactically or therapeutically administered drugs such as acyclovir, ganciclovir, and their prodrugs, valacyclovir and valganciclovir. the drugs are structurally similar nucleoside analogues, but their effects on neurons are poorly understood. acyclovir - induced encephalopathy is more widely known than ganciclovir - induced encephalopathy, possibly because acyclovir is used against herpes virus infection, which is more common than cytomegalovirus (cmv) infection, for which ganciclovir is used. ganciclovir was also reported to be effective against hepatitis b infection, but has rarely been used for this indication recently. although ganciclovir - induced encephalopathy has been documented previously, a literature search revealed that ganciclovir concentrations in the blood or cerebrospinal fluid (csf) have been reported in only 3 patients [1, 2, 3 ]. previous reports on patients with such definite ganciclovir - induced encephalopathy have suggested that the trough concentration of ganciclovir in the blood is important. here, we summarize clinical information of patients with definite ganciclovir - induced encephalopathy including our own patient, who had severe symptoms, with the highest reported trough concentration of ganciclovir in the blood, and underwent therapeutic dialysis. a 55-year - old man started to receive hemodialysis because of diabetic renal failure 2 years previously. he underwent renal transplantation 1.5 years previously, and had been receiving immunosuppressants since then. eight months after transplantation, the serum creatinine level increased to 4.4 mg / dl. he had cmv enteritis with occult blood in the stool and an elevated cmv pp65 (c7-hrp) antigen level in blood mononuclear cells. intravenous ganciclovir (150 mg / day) was administered for 11 days, followed by valganciclovir (450 mg / day). because the enteritis was very severe, ganciclovir and valganciclovir two days after starting valganciclovir, he had unsteady gait, but could walk unaided. on the next day, the patient needed assistance with walking. his consciousness was mildly disturbed (e3, v5, and m6 on the glasgow coma scale). two days later, he was found on the floor after falling, without major injuries. nine days after starting valganciclovir, his level of consciousness worsened (e3, v3, and m5), and he could not receive oral drugs, including valganciclovir. encephalitis was unlikely, since no meningeal signs or fever was noted ; the cell count was normal (0.33 cells/l) in the csf, and the protein concentration marginally elevated (54 mg / dl). cmv, herpes simplex virus, varicella - zoster virus, and epstein - barr virus dna was later found to be negative in the csf. because of the risk of further falls, hemodialysis using a vps-15 ha membrane, a vitamin e - coated polysulfone membrane (asahi kasei kuraray medical, japan) was performed twice in 2 days. his consciousness improved considerably after the first session of dialysis (e3, v4, and m6) and was completely restored on the next morning after the second session (e4, v5, and m6). the trough levels of ganciclovir in the serum and csf were retrospectively measured and are shown in table 1. generally, ganciclovir is well tolerated, but caution is required in patients with renal impairment. additionally, the severe symptoms in our patient may have been attributed to high doses of ganciclovir or valganciclovir for treatment of severe cmv enteritis. although the trough blood ganciclovir concentration in our patient was the highest reported to date, his csf ganciclovir concentration was the second highest among the 4 patients reported to date in the literature. such discrepancies between the csf data and blood data may be explained by other factors, including penetration rates (how much ganciclovir passes through the blood - brain barrier), which are reported to vary among individuals. however, csf concentrations may not necessarily reflect the severity of encephalopathy. as an extreme example, 1 previous patient with an undetectable level of ganciclovir in the csf (patient 2 in table 1) still had disturbed consciousness. we speculate that the severe symptoms in our patient might have been attributed to peripheral nervous system involvement, which may be more sensitive to blood ganciclovir concentrations. although definitive conclusions must await further studies, blood trough concentrations of ganciclovir may be more closely related to symptom severity than csf concentrations. however, a fall, as observed in our patient, can cause devastating complications, an extended hospital stay, or both. in such patients dialysis also has a positive effect on the function of the transplanted kidney. in summary, our experience suggests that therapeutic dialysis is a safe and effective treatment for encephalopathy as well as for a damaged transplanted kidney. the measurement of ganciclovir concentrations in the csf may not be feasible for the management of encephalopathy in individual patients because it is not routinely performed, and because several days to several weeks are required for the results. nonetheless, measurement of ganciclovir concentrations can play an important role in confirming the diagnosis and evaluating disease severity. we believe that accumulated knowledge on such patients with confirmed encephalopathy will lead to prompter and more accurate diagnoses. | backgroundganciclovir, a drug against cytomegalovirus (cmv) infection, is generally well tolerated, but can cause neurotoxicity such as encephalopathy. although ganciclovir - induced encephalopathy has been described in several reports, a literature search revealed that ganciclovir concentrations in the blood or cerebrospinal fluid were previously measured in only 3 patients with encephalopathy. symptoms usually include confusion and disturbed consciousness, which mimic cmv encephalitis. prompt and accurate diagnosis is thus sometimes difficult, and is derived solely from accumulated clinical information of definite cases, since ganciclovir concentrations, not routinely measured, become available after several days or a few weeks.case presentationhere, we summarize clinical information of all patients with definite ganciclovir - induced encephalopathy including our own patient, who had severe symptoms, with the highest reported trough concentration of ganciclovir in the blood, and underwent therapeutic dialysis with complete recovery.conclusionour summary of patients with definite encephalopathy could lead to prompt and accurate diagnoses. |
plasma proteins are the primary targets of glycation following elevated levels of glucose in diabetes. amongst plasma proteins, albumin is one of the heavily glycated proteins because of its abundance, comparatively longer half - life and a higher number of free lysine and arginine residues. glycation accelerates albumin degradation via increasing catabolic rate and decreasing protein half - life, thus decreasing the albumin levels in diabetes. it has been mechanistically shown that albumin competes with other proteins for glycation and low albumin level was associated with increased plasma protein glycation in diabetes. this study was corroborated in a recent finding where low albumin levels were associated with increased fibrinogen glycation. it has also been suggested that low plasma albumin predicts the glycated hemoglobin (hba1c) in type 2 diabetes, thus, strongly implicating albumin in regulation of plasma protein glycation and hba1c. glycated hemoglobin is an important marker of glycemic control as it estimates average blood glucose of the previous 3 months. recent guidelines by the american diabetes association also recommended hba1c as a diagnostic tool for diabetes, in addition to its well - known use to define control. studies showed that its level correlates with average plasma glucose and the progression of diabetes complication. however, several biological, ethnic and therapeutic factors are known to affect hba1c values, one of being albumin levels. despite a significant negative correlation between plasma albumin levels and hba1c in type 2 diabetes, levels of albumin are not routinely monitored in diabetes. only in diabetic nephropathy, therefore, we hypothesized that low albumin levels may be associated with higher hba1c levels and vice versa. thus we pursued this hypothesis by analyzing the association between albumin and hba1c in clinical setting. we analyzed clinical, anthropometric and biochemical data of subjects who attended outpatient 's clinic of chellaram diabetes institute, pune during year 2012 - 2014 and who have had a simultaneous measurement of fasting plasma glucose (fpg), hba1c and albumin levels via the same blood collection. we screened data of 929 subjects and excluded 319 cases with anemia (hb 2 mg / dl), pregnancy, chronic liver disease (serum total bilirubin > 3 mg / dl ; serum direct bilirubin > 0.6 mg / dl ; serum indirect bilirubin > 3 mg / dl ; serum aspartate aminotransferase > 120 iu / l ; serum alkaline phosphatase > 387 iu / l), hypertriglyceridemia (triglycerides > 500 mg / dl), iron or vitamin b12 deficiency and also those who were on drugs that can induce variability in hba1c estimation. all biochemistry was done using commercially available kits. hba1c estimation was based on principles of ion - exchange high - performance liquid chromatography using fully automated d-10 hemoglobin a1c analyzer (bio - rad laboratories, inc. albumin was estimated by colorimetric assay with endpoint method using cobas integra 400 plus (roche, switzerland). albumin binds with bromcresol green, an anionic dye, at ph 4.1 to form a blue - green complex. the intensity of the blue - green color was measured at 583 nnm which is directly proportional to the albumin concentration in the sample. subjects were classified according to their albumin concentrations in tertiles - q1 4.44 g / l, q2 = 4.44 - 4.73 g / l and q3 4.73 the mean (hba1c) and fpg of these albumin - level groups were compared using anova with post - hoc tukey 's correction. step - wise multivariate regression analysis was done to find out the independent predictors of hba1c. based on fpg, subjects were grouped into group 1 (fpg 0.05) between q2 and q3 of serum albumin in gp2 of fpg [table 3 ]. the distribution of average hba1c according to the tertiles of albumin concentration and three levels of fpg the results of our study showed a statistically significant negative correlation between hba1c and serum albumin levels. this persisted despite adjusting for confounding factors like fpg age, bmi, hb, serum creatinine, serum globulin, total protein. notably, common clinical conditions like anemia and drugs interfering with hba1c estimations like aspirin were excluded. while the magnitude of hba1c change with serum albumin variations was admittedly small (0.3%) compared to a previous study we believe that beyond altering hba1c, this phenomenon may have other important physiological effects too. for example, if serum albumin was to compete with hemoglobin for glycation and lower the hba1c negatively, it is possible that other proteins could be designed that may become progressively glycated, and therefore, prevent tissue glycation, and alter the prevalence of complications. indeed this has been tested in vitro and in vivo, with some suggestions of benefit. interestingly, our results also suggest that the hba1c - albumin association existed only at non - hyperglycemic fpg ranges, but not at hyperglycemic ranges. this leads us to speculate whether this means that higher glucose levels were able to glycate both hemoglobin and albumin - thus warding off any correlation between the two at higher levels. it is well known that hba1c may falsely overestimate pre - diabetes in indians, and this has been attributed to iron deficiency anemia, among other factors. in the study by hardikar., it has been shown that among 116 subjects (hba1c and ogtt measured on the same day), hba1c overestimated prediabetes (23.3%) compared with ogtt (7.8%), but did not overestimated diabetes (2.3% by both hba1c and ogtt) and this has been attributable to iron deficiency. it is possible that additional factors like low albumin level could also, play a role in overestimating hba1c in indians with pre - diabetes. the results of our study are consistent with this finding as the increased hba1c correlated with low albumin in those with fpg between 100 to < 126 mg / dl, but not with fpg 126 mg / dl. whether this increase in hba1c attributable to a lower albumin our study in indian subjects suggests that higher serum albumin levels may decrease hba1c levels and that lower serum albumin levels may raise hba1c levels as reported previously from western studies. as we did not measure glycated albumin levels, we can only cautiously speculate that this could be due to higher albumin levels competing with hb to get excessively glycated. further, we caution that our study may be interpreted as hypothesis - generating, rather than hypothesis proving results, as this study has several limitations - importantly, it was a retrospective study. also, we classified subjects into hyperglycemia and non hypergylcemia and did not group them into diabetes and non - diabetes. hence, those without hyperglycemia could have been nondiabetic or could have been well - controlled diabetes. first, the group of both prediabetes and well - controlled diabetes under a single group of fpg between 100 to < 126 mg / dl mean that our data could be generalizable to both diagnostic and therapeutic settings. second, the finding of an association of statistically increasing hba1c with low albumin tertiles (in the subgroup of fpg 100 to < 126 mg / dl) suggest that albumin could be one more factor that alters hba1c in prediabetes subjects. this further strengthens the current understanding that hba1c may not be as reliable in diagnosing prediabetes among indian subjects. we believe that the results of this study, which showed statistically significant negative correlations between hba1c and albumin in the indian population, could lead to new approaches in studying the ways in which glucose and proteins (albumin and hb are examples of such proteins) might interact with one another and such studies could have an impact on understanding hyperglycemia and it 's estimation. | background : protein glycation plays a significant role in diabetic complications. glycated hemoglobin (hba1c) is a known predictor of diabetes and its complications. albumin, found to be profoundly glycated in diabetes, and its level could regulate plasma protein as well as hemoglobin glycation.aim:we aimed to evaluate the association between variations in albumin level with hba1c in the asian indian population.materials and methods : we screened data of 929 subjects who have had a simultaneous measurement of fasting plasma glucose (fpg), hba1c and albumin levels via the same blood collection. data were analyzed by spss for 610 subjects who met the study criteria.results:there was a significant negative correlation between hba1c and albumin concentration (r = 0.284 ; p < 0.001). univariate analysis showed the statistically significant decrease of average hba1c but not for fasting plasma glucose (fpg) across increasing tertiles of albumin. stepwise multiple regression model showed a significant correlation between hba1c and serum albumin (p < 0.05), fpg (p < 0.001), hemoglobin (hb) (p < 0.001) and serum globulin (p < 0.05). fpg was the strongest predictor (63.4%) of variation of hba1c. the albumin concentration (r = 0.114) accounted for 0.3% (p < 0.05) of the total variance in hba1c independent of age, body mass index, fpg, hb, creatinine, total protein and globulin. it was also observed that hba1c decreases with increasing albumin concentration in those having fpg between 100 to < 126 mg / dl.conclusion : serum albumin negatively correlates with hba1c in asian indians independent of other variables. this study suggests that predicting diabetes and its complication based on the hba1c needs to be further investigated in indian subjects. |
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the relation between spatial and numerical cognition was first assumed by galton (1880) at the end of the nineteenth century. taking into account introspective reports he proposed that magnitude information might be analogically arranged through the location of numbers along a spatial axis oriented from left to right. the concept of a mental number line (mnl), where smaller numbers occupy leftward locations and larger numbers rightward locations, later found consistent evidence in the spatial (1993), in a seminal study where participants were asked to decide if a centrally presented number was even or odd by pressing one of two lateralized keys. they reported that large numbers were responded to faster with the right than with the left key and small numbers were responded to faster with the left than with the right key. such preferential mapping effect (see, e.g., kornblum., 1990) between the magnitude of a target number and the location of a correct response in external space would thus corroborate the idea of the existence of a mental representation linking numbers to space. even if magnitude information is irrelevant to the task of parity judgment, the display and subsequent processing of an arabic number was thus assumed to obligatorily activate its numerical magnitude code (i.e., cardinality ; see, e.g., santens and gevers, 2008 ; fitousi., 2009 for more recent proposals with a different emphasis). the snarc effect is nowadays an established finding (see fischer, 2006, for reservations) and it has been consistently found across different tasks, materials, response modalities, and populations (fias and fischer, 2005 ; wood., 2008). 1993) found that the snarc effect does not reverse in left handed individuals or when participants are asked to respond with their hands crossed (but see wood., 2006a). they found weaker snarc effects in subjects who were originally educated in a right to left writing system, such as iranian immigrants ; and the longer their iranian participants had dealt with a left - to - right writing system (i.e., the longer they had been living in france), the more likely they were to show the typical western snarc effect. later on zebian (2005) provided more direct evidence, by showing a significant reverse snarc effect in monoliterate arabic readers. these findings are interesting as they highlight the possibility that the association between number and space is the byproduct of educational factors rather than some biologically determined connection (see also nez, 2011). other sources have pointed to finger counting habits, in alternative or in addition to reading direction, as a crucial component of the mental representation of number from which spatial attributes could originate (e.g., butterworth, 1999 ; fias and fischer, 2005 ; rusconi., 2005 ; fischer and brugger, 2011). in many cultures the use of fingers develops spontaneously in childhood, and tends to precede the use of more abstract numerical codes (butterworth, 1999). accordingly, influences from finger representations and counting habits have been recently shown both in children and in adult numerical cognition (see, e.g., nol, 2005 ; di luca., 2006, 2010 ; di luca and pesenti, 2010 ; domahs., 2008, 2010 ; finger counting habits appear to influence also the snarc effect as measured in a parity judgment task with bimanual responses (fischer, 2008). fischer (2008) suggests that a systematic relation exists between the hand one starts counting with and the strength of the preferential mapping of numbers on bimanual lateralized responses. more precisely, the snarc effect is weaker in right - starters compared to left - starters because their counting routine consistently associates smaller numbers to their right hand and larger numbers to their left hand, in contrast with the mnl - based correspondence effect (fischer, 2008). predominance of a counting- over a mnl - based representation was reported by di luca. they asked participants to respond to arabic digits by pressing 1 of 10 keys with all 10 fingers and with their hands in prone and in supine posture. consistent with their participants being right - starters, performance was better when small numbers were associated to the right hand and large numbers to the left hand (with modulations). such advantage was present in either postures and top performance was achieved when the specific number - to - finger mapping was also congruent with the prototypical direction of counting within a hand, which therefore can be said to influence the way numerical information is projected into physical space via hand motor outputs (see also sato., 2007 for neurophysiological evidence). in conclusion, part of the available evidence suggests that finger counting habits modulate the association between numbers and space as measured via manual responses. on the other hand, dehaene. (1993) obtained a significant snarc effect also with an incongruent hand - to - response key mapping, that is having participants respond with their hands crossed. they thus concluded that the snarc effect is not driven by the association between number magnitude and any lateralized effectors but it rather depends on response location. (2006a) later failed to replicate dehaene.s (1993) result, as the snarc effect disappeared when their participants responded with their hands crossed. fischer (2006) suggested that, although it is true that several spatial frames of reference may exist that either conflict with or boost each other, it is also possible that one single number to space association (which does not necessarily reflect any long - term representation) is strategically instantiated by working memory, depending on contingent task requirements and settings. in agreement with fischer s (2006) proposal, (1998) have shown that the classical (and supposedly mnl - related) snarc effect can be easily overwritten by a reverse snarc effect when asking participants to perform simple tasks with numbers while imagining them as hours on a clock face (whereby small numbers are on the right hand side, large numbers on the left hand side). thus different long - term associated frames of reference and/or working memory strategic representations can contribute to the resulting behavioral snarc effect. finally, wood. (2006b) convincingly argued that the presence (absence) of a snarc effect in their study may not only reflect the activation (or lack of activation) of the mnl but it may also represent the end result of an interaction between different, and at times conflicting, spatial frames of reference evoked by numbers. (2010) have also advanced the proposal that different mechanisms (categorical vs. coordinate spatial reference frames) may be at the origin of endogenous snarc effects as detected in parity vs. magnitude judgment tasks. the proposal is especially interesting, considered that it would see these mechanisms naturally mapped on different macro - anatomical substrates (e.g., left vs. right hemisphere ; kosslyn, 2006 ; gevers., 2010) and thus predict a specific role for the language dominant hemisphere in the snarc effect from parity judgment and for the non - dominant hemisphere in the snarc effect from magnitude judgment tasks (gevers., 2010 ; see, e.g., rusconi., 2011a, for consistent neuro - functional evidence) data from left - sided visuo - spatial neglect patients and studies with transcranial magnetic stimulation (tms) applied on the right (non - dominant) hemisphere of healthy participants (e.g., zorzi., 2002 ; oliveri., 2004 ; doricchi., 2005 ; gbel., 2006 ; see sandrini and rusconi, 2009 ; umilt., 2009 ; sandrini., 2011 for related reviews) reported a systematic bias toward larger numbers in numerical bisection tasks analogous to the bias that is produced by actual or virtual lesions to the right hemisphere in physical space processing. neglect patients have also been reported to show a rightward bias in binary - choice magnitude judgment tasks on arabic digits (vuilleumier., 2004) but an intact snarc effect in parity judgments (priftis., 2006), and tms on the right anterior hemisphere eliminates the snarc effect in magnitude judgments but not in parity judgments (rusconi. while the right hemisphere is generally considered dominant for space processing, the left hemisphere has been historically recognized as dominant for the skilled use of hands and their coordination (liepmann, 1905 ; binkofski., 1999). it has also been indicated as the site of body - related schemas (kinsbourne and warrington, 1962 ; sirigu., 1991 ; guariglia., 2002), in addition to hosting a language - related categorical space reference system (kosslyn, 2006). furthermore, left hemisphere lesions often produce spurious (i.e., either incomplete or with additional deficits) and sometimes pure gerstmann s syndrome, a cluster of neuropsychological symptoms characterized by left right confusion, agraphia, acalculia, and finger agnosia (gerstmann, 1940 ; see rusconi. likewise, tms studies have identified contiguous neural substrates with causal effects on numerical processing, finger gnosis, and categorical left if there is any cross - talk between a supposed embodied spatial reference frame and the snarc effect, it thus appears more likely to occur by virtue of left hemisphere fronto - parietal networks. building on neuropsychological insights (e.g., gerstmann, 1940 ; kinsbourne and warrington, 1962) and on current knowledge of somatosensory stimulus processing we have recently identified an abstract structural representation of the hand and fingers that is posture - invariant (rusconi., 2009). such body structural representation would constitute a very basic form of self - awareness, and is thought to embed long - term information about the identity and the relative position of fingers rather than their current position in egocentric space (which would instead be continuously updated via proprioceptive input and be functional to action systems). as counting consists of an overlearnt sequence of movements that is essentially rooted in the invariant structure of the hand, the fixed order of fingers and their identity (e.g., butterworth, 1999) we hypothesize the existence of a long - term association between small digits and the internal structure of the hand (i.e., the relative position of fingers) that, in addition to the side of the starting hand (fischer, 2008), may influence the behavioral effects of number space associations in a predictable way. the issue of a relation between hands and number has been so far tackled from two complementary perspectives : an action - related and a representational perspective (sandrini and rusconi, 2009). as the possible mechanism linking counting routines to the mnl is still underspecified and far from definitively established (fischer, 2008), we propose that the posture - invariant body structural representation referred above may provide a relevant frame of reference (a within - hand directional vector) involved in the cross - talk between numbers, bodily representations, and the mnl. in the present study we thus address the relation between number, mental space, and finger representations by investigating whether the intrinsic directionality of the finger schema, which may lie behind the widespread use of anatomical counting routines (see lindemann., 2011), will exert any measurable effects in unimanual parity and magnitude judgment tasks that is simple numerical tasks that are known to reliably produce spatial stimulus response (s r) correspondence effects with bimanual response (umilt and nicoletti, 1990 ; wood., 2008) but to the best of our knowledge have never been systematically studied in unimanual version and with posture manipulation (one notable exception being leuthard., 2005, who thoroughly investigated clock - related snarc effects for different postures of the dominant hand, in a person s front and back space). in certain experimental and clinical settings, however, bimanual responses are best avoided, impractical, or impossible (e.g., some tms experiments, studies with hemineglect or hemiplegic patients), and the possibility to probe number space associations by measuring the snarc effect with unimanual responses should not be given for granted. in order to minimize potential carry over effects in the mapping of stimuli to responses from one posture to the other and mental rotation strategies (see, e.g., leuthard., 2005) we manipulated hand posture between rather than within participants. since the mechanisms of implicit and explicit access to number magnitude may be supported by different neuro - functional networks or even by different hemispheres (see, e.g., priftis., 2006 ; gevers., 2010 ; rusconi., 2011a), all of our participants engaged both in a number magnitude judgment and in a parity judgment task for exploratory reasons. in particular we were interested in detecting whether hand posture may affect the snarc effect in a different way, when probed in the context of a number parity or a number magnitude judgment task. we thus measured unimanual snarc effects from either hands in two different postures and with two classical numerical tasks. typically, the snarc effect emerges in settings requiring bimanual key - press responses, with response keys aligned along the horizontal dimension and therefore being defined one as left key and the other as right key (dehaene., although the right hand typically operates the right response key, and the left hand operates the left response key, dehaene. (1993) manipulated also the hand - to - key assignment in their seminal study and reported that the snarc effect follows the laterality of response keys rather than that of the response effectors (but see wood., 2006a). later studies adopted a unimanual response version of the same task, to produce an equivalent measure of the snarc effect for left hemispatial neglect patients who could only respond with their ipsilesional effector (i.e., the right hand only ; e.g., priftis., 2006). rather than operating a left and a right response key with their left and right hands, participants operated a left and a right response key with a left and a right finger of their right hand (see leuthard., 2005 for extensive background information and rationale of the unimanual variant). in the current study we will maintain the typical definition of the snarc effect, as a preferential association of small numbers to a left response key and large numbers to a right response key. when present, the snarc effect will be signaled by a significant interaction between number magnitude and response side (e.g., bchtold., 1998), and by a negative linear regression slope for the difference between right and left response latencies having number (19, 5 excluded) as a regressor (e.g., fias., 1996). unlike the usual snarc effect, unimanual snarc is characterized by the preferential mapping of numerical stimuli to lateralized responses operated by different fingers of the same hand rather than homologous fingers on different hands. our participants showed anatomical finger counting routines whereby, within each hand, counting starts from the thumb and ends with the little finger, thus invariably associating small numbers (in relative terms) to the thumb and large numbers to the little finger. we thus predicted instances of conflict between the direction of an active hand spatial framework and the mnl, while processing single - digit numbers. a responding right hand in prone posture will see the two frames of reference aligned, a responding right hand in supine posture will see the two frames run in opposite directions. a responding left hand in prone posture will have its intrinsic hand direction misaligned with the mnl, whereas its supination will have them aligned (see figure 1). if the mnl dominates over the within - hand reference frame in a unimanual context, the snarc effect when present should remain unaffected by posture manipulations. if the hand reference frame dominates over the mnl, the snarc effect should be significant in either aligned posture and of reverse sign in the posture with a misalignment between hand direction and mnl. if both frames of reference contribute about equally to the mapping of numbers onto response space, then it is possible that the snarc effect is significant when they are aligned and reduced or eliminated when they are misaligned. with this manipulation it is thus possible to investigate the influence of multiple competing spatial representations in numerical cognition. an alternative view could maintain that the absence of an effect in the misaligned condition indicates the absence of any spatial frames of reference (see, e.g., fischer, 2006 ; but see wood. this position however, based on a view of the snarc effect as byproduct of working memory strategies, would require the ad hoc assumptions that posture but not responding hand in one group (right hand in supine posture) and responding hand but not posture (left hand in prone posture) in the other group make the use of mnl too taxing or task - inefficient, while being instead useful when responding with the left hand in prone posture or with the right hand in supine posture., 2005 ; wood., 2006b), we will propose that the coexistence of two conflicting frames of reference may be indicated by the lack of an overall snarc effect in the misaligned condition due to increased variability in the leading frame of reference between participants rather than to the reciprocal neutralization of coexisting frames within individuals. if this was true, two comparable groups having significant but opposite snarc effects should be found in the misaligned condition. absence of both frames of reference in the misaligned condition would instead be signaled by the lack of an overall snarc effect in concomitance with low inter - individual variability in the snarc effect (expected to be close to null for most of the participants, with occasional deviations due to random error ; wood., 2006b). in the aligned condition, variability of the snarc effect would depend in any case on random error plus inter - individual differences in the overall strength of number space associations, with most of the participants showing a snarc effect in one direction. (a, b) show a right hand in prone and supine posture respectively. (c, d) show a left hand in prone and supine posture respectively. in (a, c), within - hand counting direction (white arrow) is aligned with the mental number line direction (mnl, black arrow) ; in (b, d), within - hand counting direction is misaligned with the direction of the mental number line. when unimanual responses are required with index and middle fingers, a regular snarc effect should be present in condition (a, c), whereas its presence in condition (b, d) may depend on the relative weight of the within - hand counting direction and the mental number line spatial frame. forty - eight healthy participants (26 females ; 45 right - handed) took part in the investigation, all of whom were nave to its purpose and were born and educated in a western country (left - to - right reading direction). they had a mean age of 26 (sd = 5) years. the study was approved by the ethical committee for experiments on humans at the university of trento and participants gave informed written consent before taking part in the experiment. one of the groups (11 females, 22 right - handed, mean age = 25, sd = 4) responded with either hands in prone posture, the other (14 females, 23 right - handed, mean age = 27, sd = 5) responded with either hands in supine posture. to avoid priming or carry - over effects in the experimental session, only at the end of the task participants were asked to show the experimenter how they count with their fingers when both their hands are free. most participants (44 out of 48, more precisely 22 in each group) reported using the conventional italian and french counting sequence starting from the right thumb, except for four participants who were reportedly left - starters. all of them, however, counted the smallest number on the thumb and the largest on the little finger of the opposite hand, and therefore switched from one hand to the other by following an anatomical (as opposed to spatial) sequence (see, e.g., lindemann., 2011). on each trial, participants fixated the center of a computer display where a white digit (range : 19, 5 excluded ; font and size : arial 48 bold) subtending horizontally about 1.2 and vertically about 1.9 of visual angle was shown on black background for 1,300 ms (see figure 2). in one of the two tasks, digits were to be classified as smaller / larger than 5, in the other digits were to be classified as even / odd. in the prone posture condition, participants kept their hands with their palms down throughout the experiment. while the responding hand was placed on the keyboard, the non - responding hand was resting comfortably on the ipsilateral knee. for half the trials participant responded with their right hand by pressing a left key with their index finger and a right key with their middle finger (see figure 3a), and for the other half with their left hand by pressing a left key with their middle finger and a right key with their index finger. response keys were aligned on participants vertical meridian, with the left key (corresponding to v on a qwerty keyboard) in left hemispace and the right key (corresponding to n) in right hemispace. in the supine posture condition, participants kept their hands with their palms up throughout the experiment. while the responding hand was placed on the keyboard, the non - responding hand was resting comfortably on the ipsilateral knee. for half the trials participants responded with their right hand by pressing a left key with their middle finger and a right key with their index finger (see figure 3b), and for the other half with their left hand by pressing a left key with their index finger and a right key with their middle finger. response keys were aligned on participants vertical meridian, with the left key (corresponding to n, as the keyboard was reversed) in left hemispace and the right key (corresponding to v, as the keyboard was reversed) in right hemispace. in either postures they were instructed to keep their non - responding hand comfortably resting on their ipsilateral knee in the same posture as their responding hand (e.g., see figure 3b). their compliance was visually monitored by the experimenter throughout the entire session. on each trial, participants fixated the centre of a display where a digit (19, 5 excluded) appeared for 1,300 ms. in half the blocks, digits were classified as smaller / larger than five in the other half as even / odd. participants responded with either their right index and middle finger or their left index and middle finger, and the experiment was divided in two main parts according to which hand was used to respond. since nine is large in the experimental range, the right - side key is compatible with a left - to - right representation of the numbers 19, and the left - side key is incompatible. (a) in the prone posture, participants fingers (index and middle of the same hand) were placed on the v and n keys of an upright qwerty keyboard. the response keys were centered on the participant s vertical meridian while the other hand rested comfortably on the ipsilateral knee in the same posture as the responding hand. (b) in the supine posture, participants fingers (index and middle of the same hand) were placed on the v and n keys of a reverse qwerty keyboard that was firmly attached to the table with its two bottom rows of keys (including v and n) protruding from the edge. as in the prone posture condition, the non - responding hand rested comfortably on the ipsilateral knee in the same posture as the responding hand. each main task (magnitude or parity judgment) included four blocks presented in abba order : two blocks with a s r mapping (block - type a, e.g., respond to small or odd with the left key, to large or even with the right key) and two with the alternative mapping (i.e., block - type b, respond to large or even with the left key, to small or odd with the right key). for this reason, subjects were instructed to carefully read the instructions preceding each block and containing precise indications about the required s r mapping. in order to avoid confounding the effects of interest with switching / remapping costs, the first eight trials of each block were considered as practice and excluded from subsequent analyses (see rusconi., 2011a, for a similar procedure). a 800-ms visual feedback (error in case of incorrect or too slow in case of missing response) or blank screen (in case of correct response) followed, and was then replaced by another 1,200 ms blank screen before the start of a new trial. since the experimental set comprised numbers ranging from 1 to 9, numbers from 1 to 4 were considered small and numbers from 6 to 9 were considered large in either task (dehaene., 1993). we therefore expected, in the baseline, to find an advantage for left key responses to 14 and for right key responses to 69. the experiment was divided in two parts : one in which participants responded with index and middle fingers of their left hand, and one in which they responded with index and middle fingers of their right hand. half participants responded with their hands in a prone posture, half with their hands in a supine posture. in total, the experiment comprised 384 experimental and 128 practice trials and was completed in a single session. each cell of the design response hand (left, right) task (magnitude, parity) magnitude (small, large) response key (left, right) contained 24 observations per each individual. response latency (mean rts) and accuracy (arcsin - transformed percentages of correct responses) were entered in an exploratory mixed design anova having one between participant factor (hand posture) with two levels and four within participant factors (responding hand, task, number magnitude, and response side) having two levels each (see below). follow - up f- or t - tests were then carried out to disambiguate interactions. whenever left unspecified, all of the reported follow - up tests remain significant with a family - wise bonferroni - corrected threshold equal to 0.05/(number of comparisons in a cluster). the presence of a significant snarc effect was then investigated more specifically by performing directional t - tests on individual weights, as obtained from linear regressions on the rt differences between right and left responses for each target number (lorch and myers, 1990), in the critical posture by response hand combination for either tasks. proportion of participants showing negative weights are also provided for conditions in which the snarc effect was significant, as well as the proportion of participants whose values were higher when mnl and hand - related frames of reference were misaligned. finally, proportions of participants having negative vs. positive s are reported for the aligned and the misaligned condition across experiments. relevant measures of effect size are provided throughout (rosenthal, 1991 ; field, 2007). forty - eight healthy participants (26 females ; 45 right - handed) took part in the investigation, all of whom were nave to its purpose and were born and educated in a western country (left - to - right reading direction). they had a mean age of 26 (sd = 5) years. the study was approved by the ethical committee for experiments on humans at the university of trento and participants gave informed written consent before taking part in the experiment. one of the groups (11 females, 22 right - handed, mean age = 25, sd = 4) responded with either hands in prone posture, the other (14 females, 23 right - handed, mean age = 27, sd = 5) responded with either hands in supine posture. to avoid priming or carry - over effects in the experimental session, only at the end of the task participants were asked to show the experimenter how they count with their fingers when both their hands are free. most participants (44 out of 48, more precisely 22 in each group) reported using the conventional italian and french counting sequence starting from the right thumb, except for four participants who were reportedly left - starters. all of them, however, counted the smallest number on the thumb and the largest on the little finger of the opposite hand, and therefore switched from one hand to the other by following an anatomical (as opposed to spatial) sequence (see, e.g., lindemann., 2011). on each trial, participants fixated the center of a computer display where a white digit (range : 19, 5 excluded ; font and size : arial 48 bold) subtending horizontally about 1.2 and vertically about 1.9 of visual angle was shown on black background for 1,300 ms (see figure 2). in one of the two tasks, digits were to be classified as smaller / larger than 5, in the other digits were to be classified as even / odd. in the prone posture condition, participants kept their hands with their palms down throughout the experiment. while the responding hand was placed on the keyboard, the non - responding hand was resting comfortably on the ipsilateral knee. for half the trials participant responded with their right hand by pressing a left key with their index finger and a right key with their middle finger (see figure 3a), and for the other half with their left hand by pressing a left key with their middle finger and a right key with their index finger. response keys were aligned on participants vertical meridian, with the left key (corresponding to v on a qwerty keyboard) in left hemispace and the right key (corresponding to n) in right hemispace. in the supine posture condition, participants kept their hands with their palms up throughout the experiment. while the responding hand was placed on the keyboard, the non - responding hand was resting comfortably on the ipsilateral knee. for half the trials participants responded with their right hand by pressing a left key with their middle finger and a right key with their index finger (see figure 3b), and for the other half with their left hand by pressing a left key with their index finger and a right key with their middle finger. response keys were aligned on participants vertical meridian, with the left key (corresponding to n, as the keyboard was reversed) in left hemispace and the right key (corresponding to v, as the keyboard was reversed) in right hemispace. in either postures they were instructed to keep their non - responding hand comfortably resting on their ipsilateral knee in the same posture as their responding hand (e.g., see figure 3b). their compliance was visually monitored by the experimenter throughout the entire session. on each trial, participants fixated the centre of a display where a digit (19, 5 excluded) appeared for 1,300 ms. in half the blocks, digits were classified as smaller / larger than five in the other half as even / odd. participants responded with either their right index and middle finger or their left index and middle finger, and the experiment was divided in two main parts according to which hand was used to respond. since nine is large in the experimental range, the right - side key is compatible with a left - to - right representation of the numbers 19, and the left - side key is incompatible. (a) in the prone posture, participants fingers (index and middle of the same hand) were placed on the v and n keys of an upright qwerty keyboard. the response keys were centered on the participant s vertical meridian while the other hand rested comfortably on the ipsilateral knee in the same posture as the responding hand. (b) in the supine posture, participants fingers (index and middle of the same hand) were placed on the v and n keys of a reverse qwerty keyboard that was firmly attached to the table with its two bottom rows of keys (including v and n) protruding from the edge. as in the prone posture condition, the non - responding hand rested comfortably on the ipsilateral knee in the same posture as the responding hand. each main task (magnitude or parity judgment) included four blocks presented in abba order : two blocks with a s r mapping (block - type a, e.g., respond to small or odd with the left key, to large or even with the right key) and two with the alternative mapping (i.e., block - type b, respond to large or even with the left key, to small or odd with the right key). for this reason, subjects were instructed to carefully read the instructions preceding each block and containing precise indications about the required s in order to avoid confounding the effects of interest with switching / remapping costs, the first eight trials of each block were considered as practice and excluded from subsequent analyses (see rusconi., 2011a, for a similar procedure). a 800-ms visual feedback (error in case of incorrect or too slow in case of missing response) or blank screen (in case of correct response) followed, and was then replaced by another 1,200 ms blank screen before the start of a new trial. since the experimental set comprised numbers ranging from 1 to 9, numbers from 1 to 4 were considered small and numbers from 6 to 9 were considered large in either task (dehaene., 1993). we therefore expected, in the baseline, to find an advantage for left key responses to 14 and for right key responses to 69. the experiment was divided in two parts : one in which participants responded with index and middle fingers of their left hand, and one in which they responded with index and middle fingers of their right hand. half participants responded with their hands in a prone posture, half with their hands in a supine posture. in total, the experiment comprised 384 experimental and 128 practice trials and was completed in a single session. each cell of the design response hand (left, right) task (magnitude, parity) magnitude (small, large) response key (left, right) contained 24 observations per each individual. response latency (mean rts) and accuracy (arcsin - transformed percentages of correct responses) were entered in an exploratory mixed design anova having one between participant factor (hand posture) with two levels and four within participant factors (responding hand, task, number magnitude, and response side) having two levels each (see below). follow - up f- or t - tests were then carried out to disambiguate interactions. whenever left unspecified, all of the reported follow - up tests remain significant with a family - wise bonferroni - corrected threshold equal to 0.05/(number of comparisons in a cluster). the presence of a significant snarc effect was then investigated more specifically by performing directional t - tests on individual weights, as obtained from linear regressions on the rt differences between right and left responses for each target number (lorch and myers, 1990), in the critical posture by response hand combination for either tasks. proportion of participants showing negative weights are also provided for conditions in which the snarc effect was significant, as well as the proportion of participants whose values were higher when mnl and hand - related frames of reference were misaligned. finally, proportions of participants having negative vs. positive s are reported for the aligned and the misaligned condition across experiments. relevant measures of effect size are provided throughout (rosenthal, 1991 ; field, 2007). total error rate averaged 3.9% and both latency and accuracy data were analyzed. a mixed design 2 2 2 2 2 anova having hand posture (prone, supine) as between subject factor and response hand (left, right), task (magnitude, parity), magnitude (small, large), and response key (left, right) as within subject factors was performed on mean reaction times (rts) for correct responses. significant main effects of task [f(1,46) = 126.11, mse = 4300 ; p 0.10 ]. the four - way interaction between posture, responding hand, magnitude, and response key on mean rts is depicted. (a, b) when participants kept their hands in a prone posture, the snarc effect was significant for the right hand only (and likely driven by the difference between left and right key for large numbers). (c, d) when participants kept their hands in a supine posture, the snarc effect was significant for the left hand only (and likely driven by the difference between left and right key for small numbers). the same design 2 2 2 2 2 anova performed on arcsin - transformed proportions of accurate responses detected a significant main effect of task [f(1,46) = 55.92, mse = 0.026 ; p < 0.001, = 0.55 ], magnitude comparison being more accurate than parity judgment (m = 1.48, se = 0.01 and m = 1.39, se = 0.01, respectively). moreover, a significant two - way interaction between magnitude and response key [f(1,46) = 10.42, mse = 0.021, p < 0.003 ; = 0.18 ] indicating a regular snarc effect was qualified by a four - way interaction between posture, hand, magnitude, and response key [f(1,46) = 6.77, p < 0.02 ; = 0.13 ]. consistently with the latency analysis, the snarc effect was present and significant when responses were given with the right hand [f(1,46) = 7.43, p < 0.009, r = 0.37 ] but not when they were given with the left hand in a prone posture (f < 1 ; see figure 4b). the snarc effect was present and significant when responses were given with the left hand [f(1,46) = 9.71, p < 0.004, r = 0.42 ] but not when they were given with the right hand in a supine posture (f < 1 ; see figure 4d). the significant interactions between magnitude and response, for participants responding with their right hand in prone posture and participants responding with their left hand in supine posture, signals the presence of a classical snarc effect. differential rts (or drts ; rts of right responses minus rts of left responses) were thus computed for all target numbers in each of the critical experimental conditions for every participant : if a classical snarc effect was present, it should be possible to fit drts with a line having negative slope (i.e., modeling faster left responses to smaller numbers and faster right responses for large numbers). directional single - sample t - tests on individual regression slopes (see lorch and myers, 1990 ; fias., 1996) showed that weights were significantly smaller than zero [prone posture, right hand : magnitude comparison, t(23) = 2.72, p = 0.006, r = 0.49 ; m = 0.25, se = 0.10 ; parity judgment : t(23) = 4.01, p = 0.006, r = 0.64, m = 0.30, se = 0.07 ; supine posture, left hand : magnitude comparison, t(23) = 1.80, p = 0.042, r = 0.35 ; m = 0.18, se = 0.10 ; parity comparison, t(23) = 4.54, p < 0.0001, r = 0.71 ; m = 0.35, se = 0.08 ]. finally, in the presence of a significant snarc effect, 17 out of 24 participants had negative weights in the magnitude task and 18/24 in the parity task for the prone posture condition. in the presence of a significant snarc effect, 15/24 had negative weights in the magnitude task, and 19/24 in the parity task, for the supine posture condition. overall, the experimental manipulation within participants (i.e., misalignment of the spatial frames of reference by changing the responding hand) caused a significant increase in the weights, by pushing them toward 0, of 0.21 units [t(47) = 3.27, p = 0.001, r = 0.43 ; m = 0.21, se = 0.06 ], with two - thirds of the participants (i.e., 32 out of 48) showing a change in the expected direction [(1)2 = 5.33, p = 0.021 ]. finally, two separate groups of participants could be identified based on the sign of individual weights in the misaligned condition across experiments, showing opposite snarc effects of large size in each group [negative : n = 27, m = 0.31, se = 0.04, t(26) = 8.29, p < 0.0001, r = 0.85 ; positive : n = 21, m = 0.26, se = 0.04, single - sample t(20) = 6.62, p < 0.0001, r = 0.83 ]. in the aligned condition, the proportion of participants having negative vs. positive s appeared much more unbalanced in favor of negative s ; effect sizes were in the large range for either group [negative : n = 39, m = 0.38, se = 0.04, single - sample t(38) = 10.05, p < 0.0001, r = 0.85 ; positive : n = 9, m = 0.19, se = 0.04, single - sample t(8) = 4.46, p < 0.01, r = 0.84 ]. a mcnemar s test for dichotomous variables in paired samples detected a significant difference between the misaligned and the aligned conditions (p = 0.017). in this study, we have used a unimanual version of the snarc effect to test for the possible presence of an hand - related allocentric frame of reference (see, e.g., kinsbourne and warrington, 1962 ; rusconi., 2009) that may be evoked by number processing. the directional vector of such representation was predicted to run from thumb - to - little based on our participants counting habits. by introducing conflict between the hand - related and mnl - related vectors, we predicted opposite modulations of the snarc effect for the two hands, depending on their posture. more precisely, when the right hand is pronated (see figure 1a) or the left hand is supinated (see figure 1c), the direction of either hand is aligned with the direction of the mnl as their thumb - to - little axis runs from left - to - right. when the right hand is supinated (as in figure 1b) or the left hand is pronated (as in figure 1d), the direction of either hand is opposite to the direction of the mnl because their thumb - to - little axis run from right to left. in the former cases, a regular snarc effect was found, in the latter cases no snarc effect was found. however, for each hand the snarc effect was found in just one of the tested postures. we showed that our manipulation acts at a group level by increasing inter - individual variability in the misaligned condition, rather than by neutralizing individual snarc effects in the misaligned condition. this is more compatible with the coexistence, in the misaligned condition, of two vectors having similar force but opposing direction, of which only one takes the lead and influence individual performance, rather than with the absence of any frames of reference. a much less clearcut, because found in the rts anova only and the smallest in size, finding was the different reliability of the unimanual snarc effect in parity judgment and the unimanual snarc effect in magnitude judgment. task, however, was not involved in any significant interactions with posture and response hand. the above results prompt interesting speculations about the cognitive mechanisms underlying interactions between numerical magnitude and representational space. first of all, they make it implausible that the unimanual snarc effect originates in a long - term mnl that is indiscriminately activated by number magnitude processing, because the snarc effect was involved in an interaction with response hand and hand posture. had it been the byproduct of mnl processing, the snarc effect might have interacted with task but in the opposite direction than the one we reported here (i.e., stronger snarc effect when number magnitude is relevant to the task). the fact that unimanual snarc effects, when present, were particularly strong in the parity judgment task, seem to corroborate the idea that a within - hand frame of reference, if present, may be more active in concomitance with the activation of categorical spatial representations from the dominant hemisphere (provided that gevers., 2010 perspective about the origin of the snarc effect in parity judgments is tenable ; see introduction). on the other hand, if the snarc was solely determined by finger identity, a reverse snarc effect would have been found for either hand in the misaligned condition (i.e., right hand in the supine posture, figure 1b, and left hand in prone posture, figure 1d) since the assignment of finger to response key was reversed. the fact that this systematic association (index - small and middle - large) was not present in the aligned conditions at the group level, suggests that a fully embodied model of the mental number - space is unsatisfactory as well. our data could be best accommodated by assuming that, in unimanual two - choice tasks involving numbers, at least two pre - existing frames of reference may simultaneously influence performance. in particular, with a supine posture, the thumb - to - little preferential mapping of the right hand could have competed with the left viceversa, with a prone posture the thumb - to - little preferential mapping of the left hand could have competed with the left since the non - responding hand was always kept in the same posture as the responding hand, it is unlikely that our results could be explained by conflict between active vs. inactive hand frames of reference, because this was kept constant across all conditions. alternatively, one should postulate that a group of participants was strategically evoking a number spatial representation when responding with their right hand but not with their left hand in a prone posture, and another group was strategically evoking a numerical spatial representation when responding with their left hand but not with their right hand in supine position, which would not be theoretically parsimonious. the concomitant presence of two frames of reference fits better than the absence of any frames of reference in the misaligned condition with the proportion of participants showing negative vs. positive weights and the detection of large and significant but reverse snarc effects (see also leuthard., 2005 ; wood.. a few other studies had previously introduced postural manipulations in simple numerical tasks (e.g., leuthard., 2005 ; di luca. (2008), for example, had their participants perform a tactile detection task by foot pedal responses. the tactile stimulus could be delivered either on their right thumb or on their right little finger following the appearance of a digit on a computer display. participants performed the test with their right hand both in supine and in prone posture, and results indicated faster detection whenever a stimulus was delivered to the left - side after the appearance of a small than a large digit and viceversa with a right - side stimulus, irrespective of hand posture. (2008) rightly concluded that arabic digits may evoke an extrapersonal spatial frame of reference that remains active and influences behavior even when attention is focused on the hand and on tactile stimuli to individual fingers. however, brozzoli.s set - up required no motor response selection stage as the spatial effects of number magnitude processing were measured in simple reaction times. no competition between mnl and hand - related frames of reference could be detected, if their interaction becomes manifest only when a response selection stage is involved. foot responses, moreover, may be relatively unaffected from correspondence effects arising from hand - structural representations (it would be probably different if responses required toes differentiation ; see, e.g., tucha., 1997). finally, the task did not require fine finger discrimination and only the most external fingers (thumb and little), which are usually told apart even in the presence of an acquired deficit in the structural representation of the hand (see, e.g., kinsbourne and warrington, 1962) received stimulation. in our study, on the contrary, participants were to continuously discriminate and select between two internal fingers (index and middle) and we employed a more demanding two - choice task. note that a study requiring discrimination between all the 10 fingers (di luca., 2006 ; see below) reported a striking predominance of hand - related counting associations over mnl - related associations, that is diametrically opposite to brozzoli.s conclusions. (2005), who investigated spatial s r compatibility effects in a unimanual two - choice task. their participants had to imagine numbers as they appear on a clock face, and were to answer whether a centrally presented number came earlier or later than six oclock. with similar instructions but bimanual responses, participants typically present a reverse snarc effect (i.e., a spatial s r compatibility effect consistent with the clock representation having smaller numbers on its right hand side, larger numbers on its left hand side ; bchtold., 1998). by originally adopting a unimanual response modality, with keys operated by the index and ring fingers of the dominant hand, leuthard. moreover, the typical reverse snarc effect was found to follow the relative position of response keys rather than finger identity (i.e., the preferential association between finger and side of the clock interacted with posture) when participants responded in peripersonal front space (i.e., in a condition very similar to ours, except they had the same participants doing both postures and with their dominant hand only). an opposite effect of posture was found instead when participants responded with their right hand in back space. in that condition, a reverse snarc effect was present for the supine posture only whereas it was absent for the prone posture. absence was due to increasing variability in mental imagery strategies between participants rather than elimination of any s the pattern of results that we found here for right hand responses look very similar to the pattern of results that leuthard. notably, that was also the condition in which participants were left free to choose their own frame of reference (i.e., they could choose to imagine a clock in front space or a clock in back space) and lack of a reliable s r correspondence effect in the group analysis was not taken as evidence for the absence of any spatial frames of reference. since leuthard.s participants were actively engaged in a mental imagery task, those claims could be verified against individual strategy self - reports. thus, unlike in our present study, a spatial frame of reference was intentionally used by participants throughout the experimental session. a task - relevant allocentric spatial representation might thus have been superimposed and given precedence over other pre - existing frames of reference (either mnl or hand - related), and consequently have overridden their potential effects our results appear consistent with the interplay between finger counting habits and mnl - related effects as reported by fischer (2008), who showed a reliable snarc effect for left - starters (associating small numbers with left space via counting routines, similarly to the mnl) and a weaker snarc effect for right - starters (associating small numbers with right space via counting routines, opposite to the mnl) with bimanual responses. whether the direction of counting routines may exert a causal influence on the direction of mnl, however, here we adopted a complementary approach by zooming in on the within - hand directional vector that may be identical for either hand, rather than focusing on the between hands counting sequence. we reported reliable unimanual snarc effects within either the dominant or the non - dominant hand of a group of participants that was mainly composed by right - starters (results did not change when the same analyses were performed without the four left - starters who participated in our study) and, based on fischer (2008), would therefore be expected to show relatively weak bimanual snarc effects. the interaction between snarc, hand and posture, and the pattern of inter - individual variability here described suggest that counting may affect number to space mappings at multiple levels and all possible frames of reference should be taken into account when attempting to model the possible effects counting routines on mnl representations. like fischer s (2008) study, our study is in partial agreement with di luca.s (2006), as for the supremacy of finger counting routines over mnl in numerical cognition. di luca.s (2006) participants were are asked to respond to arabic digits by pressing a key with one of their 10 fingers. performance was significantly faster when the mapping of digits to fingers matched individual finger counting habits rather than mnl. (2008), however, here the association between number and finger identity is largely unaffected by a change in the spatial position, and not viceversa. (2006) employed a bimanual response modality where discrimination between the 10 fingers was necessary to the task, the counting - based frame of reference as opposed to the mnl - based frame was highly emphasized by the task. our set - up, like leuthard.s, still required finger discrimination, however only two response alternatives were provided and two fingers (or their homologous on the other hand) were actively engaged throughout the session. emphasis was thus not so heavily posed on the finger series and other available mental frames of references may have been activated with equal strength. in conclusion, with the current study we provide novel evidence against a uni - dimensional model of number space associations. in particular we propose that a posture - invariant structural representation of the hand should be taken in consideration, in addition to the side of the hand where counting starts, when investigating the relation between individual counting routines and the mnl. in addition to the distinction of concepts such as bodily left and right, finger gnosis can reliably predict numerical abilities in developmental age (nol, 2005).. such combination of functions and their habitual use to manipulate and represent numerosities may be rooted in and facilitated by the contiguity of left parietal circuits in which they reside (see, e.g., rusconi., 2005, 2010). left - lateralized embodied representations, however, although important, may be only one of the cross - domain support systems that are available to the adult number processing system. visuo - spatial representations from a right - lateralized attentional system may also play an equally important role in number processing (see sandrini., 2011 for a review on relevant studies). clarifying how these separate but interacting systems can influence basic number processing will enable us to better understand both potentiality and limitations of human numerical cognition, as well as to identify new rehabilitative and educational paths toward facilitation and improvement in number skills. 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. | a structural representation of the hand embedding information about the identity and relative position of fingers is necessary to counting routines. it may also support associations between numbers and allocentric spatial codes that predictably interact with other known numerical spatial representations, such as the mental number line (mnl). in this study, 48 western participants whose typical counting routine proceeded from thumb - to - little on both hands performed magnitude and parity binary judgments. response keys were pressed either with the right index and middle fingers or with the left index and middle fingers in separate blocks. 24 participants responded with either hands in prone posture (i.e., palm down) and 24 participants responded with either hands in supine (i.e., palm up) posture. when hands were in prone posture, the counting direction of the left hand conflicted with the direction of the left right mnl, whereas the counting direction of the right hand was consistent with it. when hands were in supine posture, the opposite was true. if systematic associations existed between relative number magnitude and an allocentric spatial representation of the finger series within each hand, as predicted on the basis of counting habits, interactions would be expected between hand posture and a unimanual version of the spatial numerical association of response codes (snarc) effect. data revealed that with hands in prone posture a unimanual snarc effect was present for the right hand, and with hands in supine posture a unimanual snarc effect was present for the left hand. we propose that a posture - invariant body structural representation of the finger series provides a relevant frame of reference, a within - hand directional vector, that is associated to simple number processing. such frame of reference can significantly interact with stimulus response correspondence effects, like the snarc, that have been typically attributed to the mapping of numbers on a left - to - right mental line. |
ticks are blood - sucking ectoparasites which transmit serious diseases to animals and humans. they are considered as main vectors for transmission of many viral, bacterial, rickettsial and parasitical pathogens (garcia 2007). several tick species are important in veterinary medicine as vectors of theileriosis, babesiosis and anaplasmosis. in addition, lyme disease, ehrlishiosis, babesiosis, rocky mountain fever, colorado tick fever, tularemia, q fever, spotted fever, tick paralysis and tick encephalitis are the most common diseases which have been transmitted to human by ticks. they transmit diseases, produce paralysis, weight loss and cause economic damages to livestock (wall 2001). in view of the disease relationships of ticks, rhipicephalus species are important vectors of ovine babesiosis and ovine ehrlichiosis. annulata and crimean - congo hemorrhagic fever virus (jongejan and uilenberg 1994, chinikar. (2010) carried out a study in yazd province, and reported 7 species of ticks including : hy. numerous studies on the distribution of tick fauna in iran have been conducted (delpy 1936, abbasian 1961, mazlum 1971, razmi. 2004) reported different species of ixodidae (hard ticks) in rawalpindi and islamabad, pakistan. sulcata and hy. anatolicum was highly significant in this region. this article was conducted to collect and identify the tick species prevalent in domestic animals in zabol county. sistan and baluchestan is located in the southeast of iran and zabol is in the north of this province with hot and dry desert climate. the latitude and longitude gps coordinates of zabol (iran) is : lat : 31.0385, long : 61.4962. this survey was carried out to investigate the prevalence of hard tick species (acari : ixodidae) on cattle in zabol county during 2012. sampling was done during the activating seasons of ticks (i.e. summer and spring). the number of ticks was determined according to the cochran formula : (n = z2pqd2) where, n= sample size, z= 1.96, p= 0.56 prevalence estimated, q= 1p, d= 0.45. a total number of 469 hard ticks were collected from sheep, goats, cattle and camels. the ticks were collected from the body of infested animals and stored in 70% ethanol, then transported to the laboratory of zabol university of medical sciences. following examinations under stereomicroscope, ticks were identified using available taxonomic keys (kaiser. a total number of 469 adult ticks (381 males and 88 females) were collected from, sheep, goats, cattle, and camels. subspecies and numbers of male ticks collected in zabol, during 2012 (percentage of male ticks was calculated) table 2 shows the frequency of different tick species on various hosts. the maximum number of ticks was collected from sheep and goats 253(66.4%), followed by camels 73(19.16%) and cattle 55 (14.43%). the host of collected ticks in zabol, during 2012 rhipicephalus species were observed in sheep and goats. this article reports a study conducted to collect and identify the tick species prevalent in domestic animals in zabol, during year 2012. a similar study was also reported from meshkinshahr (a northwest area in iran) in 2009 by telmadarraiy, in which most of the ticks belonged to genus rhipicephalus and hyalomma (telmadarraiy. rhipicephalus sanguineus ticks are widely distributed around the world and one of the most common species in sheep herds in northeast of iran (razmi. 2007). in view of the importance of these species, hyalomma ticks are widespread in north africa, southern europe, middle east, central asia and china (durrani. 2009), including the species that are vectors of various diseases in humans and domestic animals. different species of ticks play an important role for the transmission of crimean - congo hemorrhagic fever (cchf) across the country. according to the findings, geographical distribution of cchf cases corresponds most closely with the distribution of hyalomma ticks, although, some species of dermacentor and rhipicephalus genera have the ability of transmission (chinikar. a total number of 469 adult ticks (381 males and 88 females) were collected. (2007) indicated that the number of ticks on each animal was low and male ticks were more than the females. (2010) collected ticks from sheep in abdanan township and showed that the frequency of male and female ticks was (77%) and (23%), respectively. (2004) reported different species of ixodidae (hard ticks) in rawalpindi and islamabad of pakistan. sulcata and hy. anatolicum was highly significant in this region. in the above mentioned studies, the researchers reported the genus of dermacentor and heamaphysalis which were not found in our article. as was found in this article, schulzei is the common species in the saravan area of sistan and baluchestan, iran. abbassian - lintzen (19601961) and mazlum (1971) found that these ticks usually occur on camels. in ethiopia, camels were infested with hy. (2011) reported hard ticks infestation of one - humped camels in qeshm island. abbasian - lintzen (1961) described it as the single species in south - eastern iran near the pakistan borders, but nabian. were collected from sheep and goats because they are the dominant livestock of the surveyed area. as the incidence of the tick - borne disease increases and the geographic areas in which they are found is expanding, it becomes increasingly important to distinguish tick species, which is essential to promote tick and tick - borne disease control. the results obtained from the present study serve as the starting point for future epidemiological studies and further investigations are needed to detect the vector role of ticks in this area. | background : ticks are important vectors of human and animal pathogens. they are considered as main vectors for transmission of many viral, bacterial, rickettsial and parasitical pathogens. the aim of the present study was to find out species diversity of ticks, which infested the domestic ruminants in zabol county, eastern iran in 2012.methods:ticks were selected randomly from sheep, goats, cattle and camels. the ticks were collected from the body of infested animals and stored in 70% ethanol, then transported to the laboratory of zabol university of medical sciences. following examinations under stereomicroscope, ticks were identified using available taxonomic keys.results:in this study, a total number of 469 adult ticks (381 males and 88 females) were collected. ticks were classified into 2 genera and 9 species including : hyalomma dromedarii (17.3%), hy. schulzei (1.8%), hy. marginatum (0.5%), hy. anatolicum excavatum (12.60%), hy. anatolicum anatolicum (11.2%), hy. asiaticum asiaticum (11.0%), rhipicephalus sanguineus (21.2%), rh. bursa (10.2%) and rh. turacunis (13.911%). the frequency of genus hyalomma (54.6%) was higher than rhipicephalus. rh. sanguineus was the predominant tick species and accounted for 21.26% of the ticks. the ratio of males was more than the female ticks.conclusion:hyalomma and rhipicephalus species are commonly distributed in the study area. further investigations are needed to identify the role of above tick species as vectors of pathogenic organisms. |
modest cognitive dysfunction is consistently reported in children and young adults with type 1 diabetes (t1d) (1). mental efficiency, psychomotor speed, executive functioning, and intelligence quotient appear to be most affected (2) ; studies report effect sizes between 0.2 and 0.5 (small to modest) in children and adolescents (3) and between 0.4 and 0.8 (modest to large) in adults (2). whether effect sizes continue to increase as those with t1d age a key issue not yet addressed is whether aging individuals with t1d have an increased risk of manifesting clinically relevant cognitive impairment, defined by comparing individual cognitive test scores to demographically appropriate normative means, as opposed to the more commonly investigated cognitive dysfunction, or between - group differences in cognitive test scores. unlike the extensive literature examining cognitive impairment in type 2 diabetes, we know of only one prior study examining cognitive impairment in t1d (4). this early study reported a higher rate of clinically relevant cognitive impairment among children (1018 years of age) diagnosed before compared with after age 6 years (24% vs. 6%, respectively) or a non - t1d cohort (6%). cognitive impairment is important to study in an aging t1d population because these individuals are concurrently exposed to the consequences of long - term t1d and the negative effects of advancing age on cognition. furthermore, several risk factors for age - related cognitive impairment, such as hypertension (5) and impaired renal function (6), are highly prevalent in t1d, yet the contribution of these factors to worsening cognitive function in an aging t1d cohort remains unknown. characterizing cognitive impairment in aging participants with t1d is warranted based on findings from cognitive studies of aging populations without t1d : cognitive impairment is related to poor self - care (7), high costs (8), and disability (9). this study tests the hypothesis that childhood - onset t1d is associated with an increased risk of developing clinically relevant cognitive impairment detectable by middle age. we compared cognitive test results between adults with and without t1d and used demographically appropriate published norms (1012) to determine whether participants met criteria for impairment for each test ; aging and dementia studies have selected a score 1.5 sd worse than the norm on that test, corresponding to performance at or below the seventh percentile (13). we also assessed relationships between t1d - specific variables and cognitive impairment ; based on the diabetes control and complications trial (dcct)/epidemiology of diabetes interventions and complications (edic) study s 18-year cognitive follow - up results, we hypothesized that cognitive impairment would be associated with smoking history, high blood pressure, prevalent microvascular complications, and poor metabolic control (14). middle - aged adults with t1d (n = 97 ; mean age and duration of diabetes 49 and 41 years, respectively) were recruited from the pittsburgh epidemiology of diabetes complications (edc) study, an ongoing prospective study of individuals diagnosed before age 18 years and listed in the children s hospital of pittsburgh s diabetes registry. the first edc clinical assessment occurred in 19861988 (n = 658 ; mean age and duration of diabetes 28 and 19 years, respectively). biennial physical exams and questionnaires occurred through 19961998, with an additional exam in 20042006 (for details see ref. all locally dwelling edc participants as of 1 january 2010 (n = 263) were invited to participate in an ancillary neuroimaging / neurocognitive study. of those, 81 refused, 26 never responded, and 2 were lost to follow - up. of the 154 interested, 37 were ineligible for mri (e.g., metallic implants, claustrophobic) and 5 had scheduling conflicts, leaving 112 eligible and scheduled for neuroimaging / neurocognitive testing. of these, three failed to show for their scheduled visit. on their scheduled testing date, another three refused the mri and nine refused the cognitive test battery, yielding an analytic sample of n = 97 (supplementary fig. adults without t1d who were participating in an observational study of the effects of prehypertension (blood pressure higher than normal but not high enough to qualify as hypertensive) on cerebral structure and function served as a comparison group. inclusion criteria were age 3560 years, local to pittsburgh, and blood pressure within the values of 120139/8089 mmhg. of the 414 who responded to the mailing / advertisement and were screened to participate, 110 were ineligible for mri, 60 changed their mind, and 14 withdrew, leaving 230 enrolled (mean age 46 years). to mirror the edc s primarily caucasian racial distribution, only caucasian participants with complete neuroimaging / neurocognitive data as of august 2013 (n = 138) were included. all study procedures received local institutional review board approval, and all participants provided informed consent before any research procedures were initiated. for all participants, blood pressure, bmi, and serum glucose participants with serum glucose 70 mg / dl were given a snack then retested after 15 min ; no cognitive testing was performed if serum glucose was 70 mg / dl. weekly caloric expenditure (kcal) metabolic control and blood pressure were assessed biennially from baseline through 19961998, again in 20042006, and at the time of neurocognitive testing (20102013) ; this allowed calculations of long - term average blood pressure and glycemic control (hba1c and a1c months, a calculated measure assessing the degree and duration of hyperglycemia [for details see ref. 16 ]). in 19901992, arterial health was assessed via the ankle - brachial index (abi) ; two readings were averaged, then dichotomized as 1.3/noncompressible. an abi > 1.3 is correlated with medial arterial calcification (17), which is associated with arterial stiffening (18). prevalence of t1d complications (nephropathy, coronary artery disease, distal symmetric polyneuropathy, cardiac autonomic neuropathy, retinopathy) was assessed biennially from baseline through 19961998, then again in 20042006, using highly standardized methods (for details see ref. cognitive measures included estimated verbal intelligence (north american adult reading test [naart ]) ; psychomotor efficiency (digit symbol substitution test [dsst ] and grooved pegboard [gp ]) ; executive function (trail making test, part b [tmtb ], verbal fluency [vf : animal naming and fas ], stroop color - word and letter / number sequencing [ln sequence ]) ; learning and working memory (rey auditory verbal learning tests, four - word short - term memory [4wstm ], and rey - osterrieth complex figure [rocf ] delayed task) ; and visuoconstruction skills (rocf copy task [rocf - copy ]). participant characteristics were compared using the t test, fisher exact test, and wilcoxon rank sum test, as applicable (table 1). comparison of characteristics of participants without t1d (no t1d) and with t1d (t1d ; edc) and by degree of cognitive impairment among participants with t1d all measures were collected at the time of neurocognitive assessment (20102013) unless otherwise noted. data are n (%), mean sd, or median (interquartile range). degree of cognitive impairment : 0 = no cognitive impairment, no test scores 1.5 sd worse than published norms ; 1 = mild, only one test score 1.5 sd worse than the published norm ; 2 = clinically relevant, two or more test scores 1.5 sd worse than published norms. for participants with t1d, systolic blood pressure (sbp) 140 mmhg or diastolic blood pressure (dbp) 90 mmhg or self - report of ever using antihypertensive medication at any edc physical exam from baseline (19861988) through the time of neurocognitive assessment (20102013) ; for participants without t1d, a history of high blood pressure or use of antihypertensive medication based on self - report at the time of neurocognitive assessment. raw cognitive test scores were compared between groups using ancova, adjusted for education (table 2). standardized effect sizes for between - group differences in test scores were computed using cohen d t(n1 + n2)/df(n1 n2) and were classified as small (d 0.1). to adjust for multiple comparisons education - adjusted p values were sorted from lowest to highest, then the benjamini - hochberg correction was applied to identify statistically significant variables, accepting that 20% may be false positives. this corresponded to a p value of 0.03 (supplementary table 2) when comparing factors between participants by t1d status (table 1, no t1d and t1d columns), of 0.06 (supplementary table 3) when comparing factors among participants with t1d by cognitive status (table 1, degree of cognitive impairment columns), and of 0.10 (supplementary table 4) when comparing cognitive test scores by t1d status (table 2). we chose this method over family - wise correction methods because of the paucity of prior studies ; we would rather falsely identify factors as deserving further investigation than reject factors that actually warrant additional study. in the ordinal logistic regression models (table 3, supplementary table 1), middle - aged adults with t1d (n = 97 ; mean age and duration of diabetes 49 and 41 years, respectively) were recruited from the pittsburgh epidemiology of diabetes complications (edc) study, an ongoing prospective study of individuals diagnosed before age 18 years and listed in the children s hospital of pittsburgh s diabetes registry. the first edc clinical assessment occurred in 19861988 (n = 658 ; mean age and duration of diabetes 28 and 19 years, respectively). biennial physical exams and questionnaires occurred through 19961998, with an additional exam in 20042006 (for details see ref. all locally dwelling edc participants as of 1 january 2010 (n = 263) were invited to participate in an ancillary neuroimaging / neurocognitive study. of those, 81 refused, 26 never responded, and 2 were lost to follow - up. of the 154 interested, 37 were ineligible for mri (e.g., metallic implants, claustrophobic) and 5 had scheduling conflicts, leaving 112 eligible and scheduled for neuroimaging / neurocognitive testing. of these, three failed to show for their scheduled visit. on their scheduled testing date, another three refused the mri and nine refused the cognitive test battery, yielding an analytic sample of n = 97 (supplementary fig. adults without t1d who were participating in an observational study of the effects of prehypertension (blood pressure higher than normal but not high enough to qualify as hypertensive) on cerebral structure and function served as a comparison group. inclusion criteria were age 3560 years, local to pittsburgh, and blood pressure within the values of 120139/8089 mmhg. of the 414 who responded to the mailing / advertisement and were screened to participate, 110 were ineligible for mri, 60 changed their mind, and 14 withdrew, leaving 230 enrolled (mean age 46 years). to mirror the edc s primarily caucasian racial distribution, only caucasian participants with complete neuroimaging / neurocognitive data as of august 2013 (n = 138) were included. all study procedures received local institutional review board approval, and all participants provided informed consent before any research procedures were initiated. for all participants, blood pressure, bmi, and serum glucose were measured using standardized techniques at the time of cognitive testing. participants with serum glucose 70 mg / dl were given a snack then retested after 15 min ; no cognitive testing was performed if serum glucose was 70 mg / dl. weekly caloric expenditure (kcal) was estimated using the self - report paffenbarger questionnaire. as part of edc, metabolic control and blood pressure were assessed biennially from baseline through 19961998, again in 20042006, and at the time of neurocognitive testing (20102013) ; this allowed calculations of long - term average blood pressure and glycemic control (hba1c and a1c months, a calculated measure assessing the degree and duration of hyperglycemia [for details see ref. 16 ]). in 19901992, arterial health was assessed via the ankle - brachial index (abi) ; two readings were averaged, then dichotomized as 1.3/noncompressible. an abi > 1.3 is correlated with medial arterial calcification (17), which is associated with arterial stiffening (18). prevalence of t1d complications (nephropathy, coronary artery disease, distal symmetric polyneuropathy, cardiac autonomic neuropathy, retinopathy) was assessed biennially from baseline through 19961998, then again in 20042006, using highly standardized methods (for details see ref. cognitive measures included estimated verbal intelligence (north american adult reading test [naart ]) ; psychomotor efficiency (digit symbol substitution test [dsst ] and grooved pegboard [gp ]) ; executive function (trail making test, part b [tmtb ], verbal fluency [vf : animal naming and fas ], stroop color - word and letter / number sequencing [ln sequence ]) ; learning and working memory (rey auditory verbal learning tests, four - word short - term memory [4wstm ], and rey - osterrieth complex figure [rocf ] delayed task) ; and visuoconstruction skills (rocf copy task [rocf - copy ]). cognitive measures included estimated verbal intelligence (north american adult reading test [naart ]) ; psychomotor efficiency (digit symbol substitution test [dsst ] and grooved pegboard [gp ]) ; executive function (trail making test, part b [tmtb ], verbal fluency [vf : animal naming and fas ], stroop color - word and letter / number sequencing [ln sequence ]) ; learning and working memory (rey auditory verbal learning tests, four - word short - term memory [4wstm ], and rey - osterrieth complex figure [rocf ] delayed task) ; and visuoconstruction skills (rocf copy task [rocf - copy ]). participant characteristics were compared using the t test, fisher exact test, and wilcoxon rank sum test, as applicable (table 1). comparison of characteristics of participants without t1d (no t1d) and with t1d (t1d ; edc) and by degree of cognitive impairment among participants with t1d all measures were collected at the time of neurocognitive assessment (20102013) unless otherwise noted. data are n (%), mean sd, or median (interquartile range). degree of cognitive impairment : 0 = no cognitive impairment, no test scores 1.5 sd worse than published norms ; 1 = mild, only one test score 1.5 sd worse than the published norm ; 2 = clinically relevant, two or more test scores 1.5 sd worse than published norms. statistically significant using an fdr of 0.20. for participants with t1d, systolic blood pressure (sbp) 140 mmhg or diastolic blood pressure (dbp) 90 mmhg or self - report of ever using antihypertensive medication at any edc physical exam from baseline (19861988) through the time of neurocognitive assessment (20102013) ; for participants without t1d, a history of high blood pressure or use of antihypertensive medication based on self - report at the time of neurocognitive assessment. raw cognitive test scores were compared between groups using ancova, adjusted for education (table 2). standardized effect sizes for between - group differences in test scores were computed using cohen d t(n1 + n2)/df(n1 n2) and were classified as small (d 0.1). to adjust for multiple comparisons education - adjusted p values were sorted from lowest to highest, then the benjamini - hochberg correction was applied to identify statistically significant variables, accepting that 20% may be false positives. this corresponded to a p value of 0.03 (supplementary table 2) when comparing factors between participants by t1d status (table 1, no t1d and t1d columns), of 0.06 (supplementary table 3) when comparing factors among participants with t1d by cognitive status (table 1, degree of cognitive impairment columns), and of 0.10 (supplementary table 4) when comparing cognitive test scores by t1d status (table 2). we chose this method over family - wise correction methods because of the paucity of prior studies ; we would rather falsely identify factors as deserving further investigation than reject factors that actually warrant additional study. in the ordinal logistic regression models (table 3, supplementary table 1), both cohorts were comparable in age, male - to - female distribution, systolic blood pressure, bmi, and apolipoprotein e4 (apoe4) status (table 1). compared with participants without t1d, participants with t1d were significantly more likely to have a history of high blood pressure (38% vs. 8% ; p 1.3 ; and were more likely to have prevalent distal symmetric polyneuropathy or proliferative retinopathy measured 5 years earlier (table 1) relationships remained similar when controlling for serum glucose or a1c at the time of neurocognitive testing. among participants with t1d, ordinal logistic regression models with level of cognitive impairment as the outcome showed that having a 14-year (19962013) average hba1c 7.5% (58 mmol / mol) tripled the odds of cognitive impairment (supplementary table 1, model 1). results were similar for prevalent proliferative retinopathy or distal symmetric polyneuropathy (supplementary table 1, models 2 and 3). each unit increase in bmi was associated with a 10% increased odds of cognitive impairment (supplementary table 1, model 4). an average abi > 1.3/noncompressible quadrupled the odds of cognitive impairment (supplementary table 1, model 5). these relationships remained statistically significant after adjusting for years of education and the interval of time from the most recent edc exam (20042006) to the time of neurocognitive testing after bonferroni correction. unlike previous reports of mild / modest cognitive dysfunction in young adults with t1d (1,2), we detected clinically relevant cognitive impairment in 28% of our middle - aged participants with t1d. this prevalence rate in our t1d cohort is comparable to the prevalence of mild cognitive impairment typically reported among community - dwelling adults aged 85 years and older (29%) (20). we know of only one prior study that investigated clinically relevant cognitive impairment in t1d, and this was done in a pediatric population (4). overall, 12.8% of the pediatric participants with t1d met this early study s definition of clinically relevant cognitive impairment, a rate less than half that observed in our middle - aged population with t1d (28%). interestingly, rates observed in the participants who did not have t1d were similar (56%) in both studies. this suggests that, for people without t1d, the rate of cognitive impairment remains basically unchanged from childhood into middle age, whereas the rate more than doubles over the same time among people with childhood - onset t1d. unlike the pediatric study, we found no effect of early versus late age at t1d diagnosis (age 6 years) on cognitive impairment. it is possible that during youth an earlier t1d onset contributes to worse brain function, but this effect becomes secondary to other factors as people with t1d age, prolonging their exposure to the deleterious effects of this metabolic disorder. our findings contradict results from two previous studies of older participants with t1d, which reported mild disturbances in cognitive function (21) and no between - group differences in the rate of cognitive decline (22). it is important to note differences between our study and these prior studies that may contribute to the disparate findings. only 33% of the participants in the earlier studies were diagnosed during childhood, whereas 100% of our participants with t1d were diagnosed during childhood. consequently, although younger, with a mean age of 49 years compared with 61 (21) and 65 years (22), our participants with t1d had a longer t1d duration (mean 41, 34, and 38 years, respectively). we postulate that the developing brain (i.e., during childhood) may be especially vulnerable to insults of glycemic dysregulation and fluctuating insulin concentrations compared with the adult brain. while mild cognitive differences can be detected early, more severe effects may become especially apparent later in life as these individuals grow older and experience the effects of normal age - related changes in brain function and structure in combination with the effects of long - term t1d and its comorbidities (e.g., hypertension, microvascular complications). while the dcct / edic study found no evidence of substantial long - term declines in cognitive function over 18 years (23), many of the dcct participants were diagnosed during adulthood (at dcct baseline, mean age 27.0 years and mean duration of diabetes 5.7 years), unlike our cohort that includes only individuals diagnosed during childhood. in addition, the dcct participants were young adults at their baseline neurocognitive exam and displayed relatively good metabolic control. having t1d was the only factor significantly associated with the between - group difference in clinically relevant cognitive impairment in our sample. traditional risk factors for age - related cognitive impairment, in particular older age and high blood pressure (24), were not related to the between - group difference we observed. the design of our study does not allow us to determine whether these traditional risk factors contributed to the development and/or progression of cognitive impairment in these participants, only that these factors were not related to the degree of cognitive impairment already present in these participants at the time of cognitive testing. among participants with t1d, a 14-year average hba1c 7.5% (58 mmol / mol), prevalent distal symmetric polyneuropathy, and proliferative retinopathy assessed 5 years earlier were related to cognitive impairment, with effect sizes larger than those reported in prior studies (21,25). two other measures of glycemic control, hba1c > 7.5% (58 mmol / mol) at the time of cognitive testing and 25-year a1c months, also were related to higher odds of having cognitive impairment. we chose to study the relationship between cognitive impairment and 14-year average a1c rather than because the latter is not widely understood ; we chose 14-year average a1c rather than a1c at a single assessment concurrent with cognitive testing to assess the chronicity of hyperglycemia. all analyses were repeated, controlling separately for serum glucose then for a1c at the time of cognitive testing, to ensure glucose concentrations were not confounding study results. similar to previous studies of younger adults with t1d (14,26), we found no relationship between the number of severe hypoglycemic episodes and cognitive impairment. rather, we found that chronic hyperglycemia, via its associated vascular and metabolic changes, may have triggered structural changes in the brain that disrupt normal cognitive function. like others (14), we found no statistically significant relationships between cognitive impairment and apoe4 status. whereas earlier studies found relationships between cognitive dysfunction and age at t1d diagnosis (4,2729), diabetes duration (29,30), and high blood pressure (21,29,30), this could be a result of our outcome of cognitive impairment compared with previous studies outcome of cognitive dysfunction. in addition, participants in those earlier studies were younger, on average, than our participants and may therefore have not yet developed cognitive impairment. we also identified two less - studied factors related to cognitive impairment in our sample. first, we found that higher bmi was related with higher odds of cognitive impairment (or 1.10 ; table 3), similar to results reported by brismar. (29) showing relationships between higher bmi and deficits in psychomotor speed as well as general intelligence in adults with t1d. while the exact mechanism relating bmi and cognitive function remains unclear, one plausible route is via an inflammatory pathway (31). in animal models of t1d, higher levels of circulating glucocorticoids and higher susceptibility to stress were associated with high bmi, increased brain inflammation, and reduced neurogenesis (32). higher bmi could also indicate insulin resistance, which also is related to worse cognitive outcomes in older participants who are healthy or have type 2 diabetes (33). future studies should further investigate the influence of bmi on cognitive function in young participants with t1d to determine whether this could be a risk factor suitable for early intervention. second, we found that participants with an average abi > 1.3/noncompressible had quadrupled odds of clinically relevant cognitive impairment. high abi is a marker of subclinical vascular conditions including atherosclerosis, vessel stiffness, and calcification, all of which are known risk factors for cognitive impairment in older adults without t1d (34,35). peripheral vascular stiffness, as a result of endothelial dysfunction, may serve as an indicator of early changes to brain vasculature and structure (36). as an example, hepatic hypoperfusion resulting from endothelial dysfunction leads to increased adhesion of circulating inflammatory cells to the endothelium, activation of the coagulation cascade and constriction of the microvasculature (37). cerebral hypoperfusion likely exerts similar effects on the brain s vasculature ; diffuse subclinical ischemia and resultant damage to oligodendrocytes and focal necrosis in gray and white matter offer a mechanistic explanation for the relationship with cognitive impairment. while the applicability of this finding is limited because of the small sample size, this novel potential risk factor deserves further investigation in participants with t1d. performance on several neurocognitive tests used in this study (e.g., dsst, gp, rocf - copy) depend on visual acuity and motor capabilities, skills that are compromised in participants with t1d with proliferative retinopathy and distal symmetric polyneuropathy. while these complications may contribute to poor performance on these tasks, psychomotor speed and executive function appear to be affected even in pediatric populations with t1d, well before such microvascular complications occur (3,4). furthermore, performance on several tasks requiring the same degree of visual acuity and fine motor skills (e.g., the rocf delayed task) did not differ by cognitive status among participants with t1d, suggesting that prevalent retinopathy and/or polyneuropathy alone do not explain the cognitive impairment observed in these individuals. rather, it suggests that long - term t1d causes microvascular damage in the brain similar to that known to affect the eyes and nerves, thereby explaining why participants with proliferative retinopathy and distal symmetric polyneuropathy were more likely to meet the study definition of clinically relevant cognitive impairment. the number of participants with t1d completing each neurocognitive task varied (see supplementary fig. however, the completers did not significantly differ from the noncompleters by retinopathy, polyneuropathy, 14-year average a1c > 7.5%, overall, incompleteness was due to time constraints, that is, insufficient time to complete all tests within the 90 min allotted : 12 participants arrived at the cognitive testing center 10 min late, and another 6 were hypoglycemic, requiring at least 1530 min for serum glucose concentrations to normalize before initiating cognitive testing. lack of time affected completion of the final tasks (fas, vf animals, stroop, gp) ; we do not believe this was dependent on cognitive status. first, we included only participants with childhood - onset diabetes ; the cognitive effects of t1d on participants diagnosed during adulthood may differ from our participants who were exposed to chronic hyperglycemia in childhood, during crucial stages of brain and cognitive functioning development (38). in addition, 99% of our participants are caucasian, whereas 93% of people diagnosed with t1d in allegheny county, pennsylvania, from 19651979 were caucasian (39). a survivor bias may be present in that these participants have survived with t1d for a substantial period of time. a selection bias is also possible ; those participating in this cognitive study are, in general, healthier than the parent edc cohort, especially since the eligibility criteria for participation in the cognitive study included being able to undergo brain imaging. many causes of mortality and morbidity that prevented participation in this study are related to cognitive impairment, potentially underestimating its true prevalence in t1d. because of the study design, we can not determine direction of the relationships between cognitive impairment and factors assessed concurrently, such as a1c on the day of cognitive testing ; it could be that those with worse cognitive impairment are less able to manage their diabetes, leading to higher a1c concentrations. the study design also prohibits determination of the life stage at which clinically relevant cognitive impairment first developed in our participants. strengths of this study include the use of a well - defined cohort with over 20 years of data, thus allowing computation of long - term trajectories of risk factor profiles, and the use of an extensive neuropsychological test battery to assess multiple cognitive domains. the cognitive impairment algorithm has been used in previous studies (13), is easily replicable, and could be adapted by others to improve comparability across studies. in summation, results of this study show that a large percentage of middle - aged adults with childhood - onset t1d are currently living with clinically relevant cognitive impairment. the effects of cognitive impairment on these individuals diabetes self - management, health service utilization, disability, and quality - of - life issues deserve further investigation. future studies with larger sample sizes and a wider range of ages at diagnosis are needed to improve our understanding of the development and progression of t1d - related cognitive impairment. such studies should use repeated neurocognitive testing, beginning shortly after the time of diagnosis. furthermore, incorporating repeated neuroimaging with extensive measures of metabolic control and vascular health would help to clarify the mechanisms contributing to the development of cognitive impairment in these individuals. | objectivethe aim of this study was to investigate the presence and correlates of clinically relevant cognitive impairment in middle - aged adults with childhood - onset type 1 diabetes (t1d).research design and methodsduring 20102013, 97 adults diagnosed with t1d and aged 7.5% (58 mmol / mol) (odds ratio [or ] 3.0 ; p = 0.009), proliferative retinopathy (or 2.8 ; p = 0.01), and distal symmetric polyneuropathy (or 2.6 ; p = 0.03) measured 5 years earlier ; higher bmi (or 1.1 ; p = 0.03) ; and ankle - brachial index 1.3 (or 4.2 ; p = 0.01) measured 20 years earlier, independent of education.conclusionsclinically relevant cognitive impairment is highly prevalent among these middle - aged adults with childhood - onset t1d. in this aging cohort, chronic hyperglycemia and prevalent microvascular disease were associated with cognitive impairment, relationships shown previously in younger populations with t1d. two additional potentially modifiable risk factors for t1d - related cognitive impairment, vascular health and bmi, deserve further study. |
a 57-year - old woman developed multiple (> 20) melanoma in - transit metastatic nodules on her right thigh (figure 1a), as confirmed by histological examination, after removal, six months earlier, of a primary 2.2 mm thick ulcerated nodular melanoma on her right leg for which the initial sentinel lymph node biopsy was negative. after completion of fdg pet - ct to exclude distant metastasis and assessment of the extent of limb metastasis, the patient was referred to a specialized center to receive, isolated limb perfusion (ilp) of melphalan under hyperthermia, tnf- and interferon. superficial and deep pelvic (ilioinguinal) lymphadenectomy was simultaneously performed and revealed two additional deep pelvic lymph node metastases. one month after the end of this procedure, most in - transit lesions had undergone necrosis. four months later, all lesions had healed and persisted as non - changing pigmented macules (figure 1b) with no clinical or radiological (including fdg pet - ct) signs of relapse. in order to confirm that none of the residual pigmented macules was an active lesion, two of them were excised after being previously examined by rcm. histological examination showed a persisting melanophage granuloma (nodular melanosis) within areas of fibrosis in the upper dermis with no sign of tumor infiltration (figure 2a and 2b). rcm imaging, performed using the vivascope 1500 (vivascope systems, munich, germany) showed ill - defined aggregates of dermal bright cells, typical of melanophages with almost no visible nuclei. sometimes a characteristic ring - shaped deposit was seen within the network of bright collagen fibers and bundles (figure 3). such deposits could be seen in many areas of the excised lesions and correlated with the histological images. we then performed rcm imaging on 12 other main persisting pigmented lesions (figure 1b) in which a similar characteristic image was consistently seen. the patient has now been followed for more than a year without any clinical or radiological sign of relapse and most lesions have slowly resolved (figure 4). around 510% of patients with malignant melanoma develop lymphatic dissemination and in - transit metastasis. even in the era of effective systemic therapies, isolated limb perfusion (ilp) with hyperthermia performed using various agents (melphalan, with addition of chemokines, i.e., tnf- and/or other chemokines) is still a valuable therapeutic approach and is being used for patients who develop numerous limb lesions simultaneously and are therefore considered inoperable. according to the literature nevertheless complete response can be difficult to assess if post - necrotic persisting pigmented lesions occur, especially if numerous and unresectable. these residual lesions occur mainly in the more superficial and pigmented types of metastatic disease. as it was the case with our patient, these lesions can often correspond to the residual melanophage granuloma (nodular melanosis) that persists after tumor tissues undergo complete necrosis and can be difficult to distinguish from partial response and persisting in - transit metastasis. rcm is a non - invasive technique that allows the exploration of the superficial dermis. confocal - histopathological correlation has been shown to be possible for the visualization of some histologic features in the superficial dermis up to 300um deep [36 ] where, in the case of superficial in - transit metastasis, post - necrotic residual melanophage granuloma (nodular melanosis) might be localized. morphologic features of melanophages under in vivo rcm have been well characterized, allowing their distinction from melanocytes. here, using combined rcm and histological examination of two sample lesions, numerous unresectable post - ilp limb residual pigmented lesions were considered by rcm to be non - active as confirmed by the subsequent dermoscopy follow - up. although in the case presented here there was not any doubt at follow - up regarding the absence of residual disease, considering that the deeper reticular dermis can not be explored with rcm, biopsy remains mandatory in case of doubt during the subsequent needed close follow - up. | complete response can be difficult to assess after isolated limb perfusion (ilp) for metastatic in - transit melanoma, especially when numerous and unresectable post - necrotic persisting pigmented lesions occur. these residual lesions are mainly seen in the more superficial and pigmented types of metastatic disease and correspond to the residual melanophage granuloma that persists after tumor tissues undergo complete necrosis. reflectance confocal microscopy (rcm) is a non - invasive technique that allows the exploration of the superficial dermis. here, we present the case of a patient in whom numerous post - ilp limb residual pigmented lesions were explored using combined rcm and histological examination of sample lesions and could be characterized as non - active. this approach allowed us to avoid additional excisions. |
prior to np[xl184 ] synthesis, the biodegradable copolymer plga 50:50 (17 kda ; 0.18 dlg ; lake shore biomaterials) was modified to incorporate a peg moiety (cooh - peg - nh2 ; 3.5 kda ; jenkem technology), which enhances both nanoparticle stability and circulation time. nanoparticles were synthesized with various xl184 (> 99.0% purity ; selleck chemicals) and peg - plga drug / polymer ratios ranging from 110% (w / w). of the tested ratios, the xl184 molar loading efficiency was maximal at a drug / polymer (w / w) ratio of 1% (supplemental figure 2). in addition, a range of solvent : water ratios (1:21:10) were tested and the optimal ratio for np[xl184 ] formation was 1:3 acetone : water. for the optimized synthesis protocol, xl184 was co - solubilized in 1 ml of acetone with plga - peg at a 1% (w / w) drug to polymer concentration. nanoprecipitation was achieved by adding this mixture drop - wise using a 27-gauge needle to 3 ml of h2o stirred magnetically at 400 rpm. the reaction mixture was then stirred uncovered for 6 h to allow acetone evaporation, passed through a 0.2 m filter and purified by ultrafiltration (amicon - millipore ; 30 kda cut - off) at 2500 rpm for 10 min with intermittent washing (4 cycles ; 4 ml phosphate - buffered saline per wash). the xl184plga nanoparticle loading efficiency was determined by optical absorption measurements following solvation of the nanoparticles in dimethyl sulfoxide, using the formula : 100(no. all np[xl184 ] size and charge measurements were made by dynamic light scattering (malvern, zetasizer nano zs). pmils co - encapsulating bpd (verteporfin ; vwr international) and np[xl184 ] were prepared by modification of existing synthesis methods. the lipids (dppc, dotap, cholesterol, and dspe - peg ; avanti polar lipids) were each dissolved separately in chloroform, and then mixed together in a molar ratio of 2:0.2:1:0.2 (dppc : dotap : cholesterol : dspe - peg) with 100 nmoles of bpd. this lipid composition was selected based on the previously reported pharmacological success of similar compositions. inclusion of the cationic lipid, dotap, resulted in a zeta potential (surface charge) of + 3 mv. this slightly cationic surface charge promotes cellular uptake without significant cytotoxicity to maintain biocompatibility. to form thin lipid films containing bpd, chloroform was removed by roto - evaporation and by placing the sample under vacuum overnight. next, lipid film hydration was achieved by adding np[xl184 ] (50 nmoles of xl184) in 1 ml of phosphate - buffered saline. to ensure adequate encapsulation of np[xl184 ], the thin film was subjected to 10 freeze - thaw cycles (6 min per cycle) at 0c and 45c, below and above the highest transition temperature of the lipid mixture (dppc ; tm = 41c). the resulting dispersion of multilamellar liposomes was extruded through a 200-nm - diameter polycarbonate membrane using a mini - extruder system (avanti polar lipids) to form unilamellar liposomes. bpd and xl184 not loaded into the pmil were removed by dialysis (spectra / por ; 300 kda cut - off ; 1 ml sample in 4 l of phosphate - buffered saline at 4c for 18 h). during the dialysis period initially, these measurements indicated a bimodal distribution with peaks at 80 nm (np[xl184 ]) and 150 nm (pmil), which gradually became a single monodispersive peak (pdi 99.0% purity ; selleck chemicals) and peg - plga drug / polymer ratios ranging from 110% (w / w). of the tested ratios, the xl184 molar loading efficiency was maximal at a drug / polymer (w / w) ratio of 1% (supplemental figure 2). in addition, a range of solvent : water ratios (1:21:10) were tested and the optimal ratio for np[xl184 ] formation was 1:3 acetone : water. for the optimized synthesis protocol, xl184 was co - solubilized in 1 ml of acetone with plga - peg at a 1% (w / w) drug to polymer concentration. nanoprecipitation was achieved by adding this mixture drop - wise using a 27-gauge needle to 3 ml of h2o stirred magnetically at 400 rpm. the reaction mixture was then stirred uncovered for 6 h to allow acetone evaporation, passed through a 0.2 m filter and purified by ultrafiltration (amicon - millipore ; 30 kda cut - off) at 2500 rpm for 10 min with intermittent washing (4 cycles ; 4 ml phosphate - buffered saline per wash). the xl184plga nanoparticle loading efficiency was determined by optical absorption measurements following solvation of the nanoparticles in dimethyl sulfoxide, using the formula : 100(no. all np[xl184 ] size and charge measurements were made by dynamic light scattering (malvern, zetasizer nano zs). pmils co - encapsulating bpd (verteporfin ; vwr international) and np[xl184 ] were prepared by modification of existing synthesis methods. the lipids (dppc, dotap, cholesterol, and dspe - peg ; avanti polar lipids) were each dissolved separately in chloroform, and then mixed together in a molar ratio of 2:0.2:1:0.2 (dppc : dotap : cholesterol : dspe - peg) with 100 nmoles of bpd. this lipid composition was selected based on the previously reported pharmacological success of similar compositions. inclusion of the cationic lipid, dotap, resulted in a zeta potential (surface charge) of + 3 mv. this slightly cationic surface charge promotes cellular uptake without significant cytotoxicity to maintain biocompatibility. to form thin lipid films containing bpd, chloroform was removed by roto - evaporation and by placing the sample under vacuum overnight. next, lipid film hydration was achieved by adding np[xl184 ] (50 nmoles of xl184) in 1 ml of phosphate - buffered saline. to ensure adequate encapsulation of np[xl184 ], the thin film was subjected to 10 freeze - thaw cycles (6 min per cycle) at 0c and 45c, below and above the highest transition temperature of the lipid mixture (dppc ; tm = 41c). the resulting dispersion of multilamellar liposomes was extruded through a 200-nm - diameter polycarbonate membrane using a mini - extruder system (avanti polar lipids) to form unilamellar liposomes. bpd and xl184 not loaded into the pmil were removed by dialysis (spectra / por ; 300 kda cut - off ; 1 ml sample in 4 l of phosphate - buffered saline at 4c for 18 h). during the dialysis period initially, these measurements indicated a bimodal distribution with peaks at 80 nm (np[xl184 ]) and 150 nm (pmil), which gradually became a single monodispersive peak (pdi < 0.2) as purification of the pmils completed. however, electron microscopy revealed the presence of residual np[xl184 ] not incorporated within liposomes within the pmil samples (supplementary note 3). the pmil bpd and xl184 concentrations and loading efficiencies were determined by fluorescence and absorbance spectroscopy or by high - performance liquid chromatography (hydrosil c18 ods ; 2.0 cm 14.0 cm ; 50% acetonitrile in h2o 100% acetonitrile ; 0.5 h), respectively, following solvation of the pmils in dimethyl sulfoxide. all pmil size and charge measurements pmil and np[xl184 ] size stability during storage at 4c was investigated by repeated dynamic light scattering measurements over a period of 40 d (supplementary fig. release and photoinduced drug release were measured using dialysis membranes in phosphate - buffered saline at 37c with 10% fetal bovine serum added to each dialysis tube (spectra / por ; np[xl184 ], 100 kda cut - off ; pmils, 300 kda cutoff ; fig. 2f). a 690 nm diode laser (high power devices, inc.) was used for all nir irradiation experiments. during dialysis samples were collected periodically and placed immediately in acetonitrile containing 1% of the internal standard n-(1-naphthyl)ethylenediamine. separation and quantification of drug components was achieved by liquid chromatography - tandem mass spectrometry (lc - ms / ms) using standard curves for each drug normalized to the internal standard. briefly, 1.0 l of dialysis sample was injected into a zorbaxc18 (2.1 50 mm) column eluted at 0.400 ml / min with acetonitrile and 10 mm ammonium formate (80% 20% over 4 min). detection of drug components was made using triple quadrupole ms / ms detection with an ion source esi+ in mrm scan mode to identify the product ions for bpd (ret. time= 2.68 min, 232 m / z). quantitative analysis of chromatograms allowed for area under curve integrations of each product ion normalized to the internal standard (ret. total moles were determined using standard curves and percent loss calculated for each time point after correcting for sample volume changes. the resulting bpd and xl184 release profiles were fit individually to a simple one- or two - phase exponential model : a0 + a1 e + a2 e, where a0 is an offset, a1 and a2 are the maxima release plateaus (at equilibrium) of phases 1 and 2, k1 and k2 are the release rate constants of phases 1 and 2, and t is time from placing the sample in serum media within the dialysis tube. note that xl184 release from np[xl184 ] is sufficiently described by a single - phase model (p=0.067, two - phase alternative hypothesis), whereas xl184 release from the pmil with or without laser irradiation is best described by the multi - phase model (p = 0.00030.0079). dynamic light scattering and transmission electron microscopy were also performed before and after photoirradiation (fig. 3 and supplementary fig. cryo - em was performed using a fei technai g2 polara microscope equipped with an energy filter (gatan) and a k2 summit direct detection device (gatan). briefly, 5 l of nanomaterial sample (~60 m bpd for the pmil or l[bpd ] ; ~30 or 125 m xl184 for the pmil or np[xl184 ], respectively) mixed with 2 l of bsa gold tracer (em - grade 6 nm ; electron microscopy sciences, 25484) re - suspended in phosphate - buffered saline were deposited onto glow - discharged holey carbon grids (quantifoil r 2/1 200 mesh, copper ; electron microscopy sciences), blotted and rapidly vitrified in a liquid ethane and propane mixture (50:50) using a custom - built plunger (max planck institute of biochemistry, germany). imaging was performed at 300 kv under low - dose conditions with 4.98 or 6.12 sampling and a defocus of 3 or 6 m for 2d or 3d images, respectively. 2d images were obtained using the dose - fractionation mode of the detector (~2040 e / cumulative dose). tilt series (60) for tomography were collected around a single axis with a 2 sampling increment using serialem software (~100 e / cumulative dose). renders of 3d pmil and np[xl184 ] objects were created by manual segmentation in imod and rendered using vmd. 5. only unambiguous np[xl184 ] objects were counted (~20 nm in diameter or greater). mean lamellarity was calculated as : l(nl l)/lnl, where nl is the number of objects with lamellarity l (e.g., l = 1 for unilamellar liposomes). tem (philips cm10) was performed using negative staining either on untreated (200 mesh nickel pelco support film with a formvar / carbon coating, ted pella inc.) or ionized carbon coated grids (to promote sample adhesion). briefly, 10 l of sample was added to the grid, air - dried and stained (2 l, 1.0% phosphotungstic acid). monolayer cultures of aspc1 cells (american type culture collection, crl-1682 ; low passage number, < 20)recently tested (july 2015) and found to be negative for mycoplasma contamination (mycoalert mycoplasma detection kit, lonza)were maintained in media (rpmi 1640, mediatech) supplemented with 10% fetal bovine serum (invitrogen), 100 units / ml penicillin and 100 g / ml streptomycin. the aspc1 cell line has not been listed in the database of cross - contaminated or misidentified cell lines maintained by the international cell line authentication committee. cellular uptake of pmils and l[bpd ] was tested in multi - well plates with coverslip bottoms (greiner bio - one) plated with aspc1 cells allowed to attach and grow overnight. nanoconstructs were added to the wells at staggered time points to reach a concentration of 100 nm bpd and to achieve varying incubation times at 37c (1590 min). imaging was performed with an olympus fv1000 confocal microscope with a 20 0.75 na (numerical aperture) objective. bpd excitation was performed using a 405 nm diode laser with an emission spectrograph centred on the 696 nm bpd fluorescence emission peak. the laser, photomultiplier tube detector and pinhole settings, as well as brightness - contrast adjustment settings for display, were kept constant for all images. in addition, phase contrast images were collected during microscopy in order to focus on a high - density field of cells (not shown). images were also collected for untreated cells (0 min) to quantify the autofluorescence background and to define a fluorescence intensity threshold that rejects 99.5% of the background signal. this intensity threshold was applied to all images to select pixels above the autofluorescence background (true bpd signal) for analysis. the resulting cellular uptake data was fit to a simple biexponential pharmacokinetic model : a (e e), where k and j are the elimination and absorption rate constants, a is a coefficient dependent on the administered bpd dose as well as its bioavailability and t is time post - administration. for in vitro pdt, 0.2510 aspc1 cells were grown on a 35-mm culture dish for 24 h and incubated with nanoconstructs containing bpd (250 nm equivalent) and/or xl184 (100125 nm equivalent) in 1 ml complete medium for 1 h. the incubation media was then replaced with 2 ml of fresh, complete media prior to photoirradiation. this removal of nanoconstructs not uptaken by cells prior to irradiation limits the release of xl184 and the generation of photocytotoxic species to intracellular and cell - associated nanoconstructs. singlet oxygen sensor green (sosg ; molecular probes) and d - mannitol (sigma - aldrich) were used to probe reactive oxygen species involvement in bpd - pdt - induce met activation. tyrphostin ag1478 (sigma - aldrich) and bacterial toxin b (toxin b, clostridium difficile - calbiochem, millipore) were used to test for involvement of enzymes known to participate in met transactivation. sosg and mannitol were added to cells immediately prior to laser irradiation in fresh media and then removed immediately after pdt by a second media replacement step. ag1478 and toxin b were incubated with cells in fresh media for 30 min and 2 h, respectively, prior to laser irradiation and then removed immediately after pdt by a second media replacement. all animal experiments were conducted with approval and according to guidelines established by the massachusetts general hospital institutional animal care and use committee. experiments were carried out on 6-week - old male swiss nude mice weighing 2025 grams (cox breeding laboratories). for tumour implantations and photoirradiation, tumours were implanted by injection of a 50 l volume containing 10 aspc1 cells in a 1:1 mixture of matrigel (bd biosciences) and culture media. subcutaneous tumours were implanted above the hind leg and tumour volumes were estimated longitudinally by measuring the three tumour dimensions using a calliper and the hemi - ellipsoid formula : volume = l w h/6, where l, w and h, are the tumour length, width and height. note that here, h, represents the measured height of the hemi - elliptical tumour, which is half the height of a full ellipsoid. eighteen days following cancer cell implantation, subcutaneous tumours reached volumes of ~50 mm prior to the start of treatment. for orthotopic tumour implantation, animals were laid supine, a small left abdominal flank incision was made to exteriorize the pancreas and the cell suspension was injected into the pancreas. a small amount of 10% povidone / iodine was applied topically to the injection site. then the incision was closed with 4 - 0 sutures and 10% povidone / iodine was then applied to the incision site to prevent infection. ten days after cancer cell implantation, orthotopic pancreatic tumours reached volumes of ~25 mm prior to the start of treatment. all injections for treatment were done intravenously (tail vein) in 200 l sterile phosphate - buffered saline. mice were randomized into the various treatment groups, and the tumours of mice receiving bpd were irradiated with nir light (using the 690 nm diode laser listed above) 1 h post - injection, delivered at an irradiance of 100 mwcm. subcutaneous tumours were irradiated transcutaneously while orthotopic tumours were surgically exposed as for tumour implantation and irradiated. fourteen days after treatment, orthotopic tumours were excised to estimate their volumes using callipers and the ellipsoid formula : volume = l w h/6. briefly, orthotopic pancreatic tumours were excised 2 weeks post - treatment, embedded in optimal cutting temperature compound and frozen at 80c. sections were (1) fixed in 1:1 acetone : methanol for 15 minutes at 20c, (2) air dried for 30 minutes, and (3) washed three times in phosphate - buffered saline. a blocking solution (dako protein block reagent) was applied for 30 minutes, followed by application of the immunostains, at ~5 g / ml monoclonal antibody (mab) each diluted in background reducing dako antibody diluent for 2 h at room temperature in a humidifying chamber. finally, the slides were washed again three times, mounted (invitrogen slowfade gold with 4,6-diamidino-2-phenylindole, dapi) with a coverslip and sealed with nail polish. confocal fluorescence imaging was performed using an olympus fluoview 1000 confocal microscope with a 10 0.4 numerical aperture (na) or a 20 0.75 na objective. excitation of dapi, anti - mouse pecam-1 (cd31 ; clone 390 ; cbl1337, millipore) mab - alexa fluor 568 conjugates and anti - human cytokeratin 8 (clone lp3k ; mab3156, r&d systems) mab - alexa fluor 647 conjugates was carried out using 405-, 559- and 635-nm lasers, respectively. mosaic images of entire tumour cross - sections were collected and stitched together using the olympus fluoview software. the anti - human cytokeratin 8 stain (a cytoskeletal protein highly expressed by aspc1 cells) has dual selectivity for the epithelial cancer cells because it does not react with mouse proteins. all analyses were performed using custom matlab (mathworks) routines for batch image processing. microvessel density values were calculated from whole tumour sections, within viable tumour tissue only, and averaged over slices from the entire tumour rather than a more complex hot spot identification and calculation, which is difficult to define objectively. intratumoural microvessel volume is calculated by multiplying microvessel density with the viable tumour volume in each slice and then summing over the whole tumour by interpolation. here, we used the minimum tumour subsampling necessary based on a mathematical model to resolve a statistically significant change in intratumoural microvessel volume, as validated previously using the orthotopic aspc1 tumour model (supplementary note 9). a quantitative reverse transcription polymerase chain reaction (qrt - pcr) assay was performed on excised liver and iliac lymph nodes to estimate the number of human cancer cells in excised organs as described and validated previously. briefly, qrt - pcr is used to measure the total number of human cancer cells from the level of human and mouse glyceraldehyde 3-phosphate dehydrogenase (gapdh) housekeeping genes. at least 300 mg of freshly excised liver and retroperitoneal lymph nodes were collected at the treatment endpoint and snap frozen in liquid nitrogen. the frozen samples were then pulverized and homogenized, followed by rna extraction (rnaeasy plus mini kit ; qiagen). human and mouse gapdh gene were measured using custom synthesized primers (invitrogen). for each specimen, the cycle threshold (ct) from human gapdh gene was normalized by ct from mouse gapdh gene. the normalized ct was quantified into number of cancer cells using a standard curve generated with a set of organ lysates from no - tumour control mice mixed with different numbers of human cancer cells. specific statistical tests are indicated in the figure captions and were carried out using graphpad prism (graphpad software). all reported p values are two - tailed. parametric tests (one - way anova with tukey s post - test) were used for in vitro drug release (fig. 6) studies ; and, the dagostino & pearson omnibus normality test (= 0.05 ; requires n 8 replicates per group) did not identify significant deviations from normality within these data sets (testing could only performed for groups with n 8 replicates). note that all groups within the drug release data were analysed together (some groups appear only in supplementary fig. 1) were analysed using nonparametric tests (the mann - whitney u test or kruskal wallis one - way anova). the brown forsythe test (= 0.05) was applied to all data sets with n 3 replicates to test for equal variance (regardless of whether parametric or nonparametric analysis was used) and identified significant deviations from equal variance (figs. in these cases, the data were analysed following a logarithmic transform of the data to pass the brown forsythe test. 5d) following a natural logarithm transform of the data to pass the dagostino & pearson omnibus normality test. no exclusion criteria were used, and no data points or animals were excluded from analysis. animal sample sizes were selected to ensure adequate power (80%) to detect a 20% difference using a maximum of 16 animals per group assuming a standard deviation of 15%. for the subcutaneous model, significance | nanoscale drug delivery vehicles can facilitate multimodal therapies of cancer by promoting tumour - selective drug release. however, few are effective because cancer cells develop ways to resist and evade treatment. here, we introduce a photoactivatable multi - inhibitor nanoliposome (pmil) that imparts light - induced cytotoxicity in synchrony with photo - initiated and sustained release of inhibitors that suppress tumour regrowth and treatment escape signalling pathways. the pmil consists of a nanoliposome doped with a photoactivatable chromophore (benzoporphyrin derivative, bpd) in the lipid bilayer, and a nanoparticle containing cabozantinib (xl184)a multikinase inhibitor encapsulated inside. near infrared tumour irradiation, following intravenous pmil administration, triggers photodynamic damage of tumour cells and microvessels, and simultaneously initiates release of xl184 inside the tumour. a single pmil treatment achieves prolonged tumour reduction in two mouse models and suppresses metastatic escape in an orthotopic pancreatic tumour model. the pmil offers new prospects for cancer therapy by enabling spatiotemporal control of drug release whilst reducing systemic drug exposure and associated toxicities. |
a 66-year - old woman with sjgren syndrome presented with a 5-year history of bilateral shoulder pain that was significantly worse on the right than the left side. the pain was reported to be constant in nature but worse at night, and completely prohibited activities above shoulder height. examination of the right shoulder revealed a globally restricted range of movement at the glenohumeral joint. treatment with copeland resurfacing hemiarthroplasty was performed in june 2007 through an anterosuperior (mackenzie) approach.. 1 interrupted pds (polydioxanone) sutures were used for reattachment of the anterior border of the osteo - periosteal sleeve to the acromion. the sutures were placed transosseously through the acromium on one side and through the deltoid fascia, incorporating an osteo - periosteal sleeve, on the other [figure 2 ]. preoperative radiograph demonstrating severe osteoarthritis radiograph taken in the immediate postoperative period showing a copeland resurfacing hemiarthroplasty passive elevation past 90 could be achieved on the first postoperative day. a shoulder immobilizer was worn for 3 weeks and the patient was instructed to avoid active elevation. the patient had returned to aerobics and table tennis by the 4 postoperative month, at which point increased pain in the shoulder was reported. at the end of the 1 postoperative year, significant impingement symptoms were reported and x - rays demonstrated a new bony spur on the anteroinferior edge of the acromion [figure 3 ]. an ultrasound scan showed a bony spur indenting the supraspinatus, but there was no accompanying cuff tear. the pain continued to worsen and diagnostic arthroscopy was performed in july 2009, which confirmed the presence of the spur at the anteroinferior edge of the acromion and excluded the presence of significant glenoid arthrosis or adhesions as contributing factors to the patient 's symptoms. radiograph at 1 year showing new bony spur on the inferior surface of the acromion appearance following arthroscopic resection of the spur the mackenzie anterosuperior approach has been popularized for several types of shoulder arthroplasties. in this approach the anterior deltoid is commonly reflected with an osteo - priosteal sleeve. we postulate that the bony spur responsible for the impingement symptoms in our patient was formed by malunion of the osteo - periosteal sleeve. at the time of arthroscopy, a sharp spur of bone of the same size and shape as the osteo - periosteal sleeve was found to be united in the correct position on the anterior acromion but, from its shape, it had apparently rotated 90 from the original orientation. the onset of symptomatic impingement along with new x - ray findings at 4 months postoperatively is consistent with rotation of the fragment during healing following the patient 's return to sporting activity. full resolution of the patient 's symptoms following resection of the spur also supports this conclusion. intra - articular factors are the most common reasons for postoperative pain following copeland resurfacing hemiarthroplasty and are due to the nonanatomical shape of the prosthesis, which does not accurately recreate the original shape of the humeral head. as our patient was initially pain free with good shoulder function, we carried out other investigations, including diagnostic arthroscopy, to look for other causes for her pain preoperative investigation to establish the diagnosis of subacromial impingement could include an injection of local anesthetic under aseptic technique into the subacromial space. immediate pain relief would be convincing evidence of the existence of subacromial impingement. to the best of our knowledge, this is the first report of formation of an acromial spur following the mackenzie approach. complications of the copeland resurfacing hemiarthroplasty have been reported as infection, aseptic loosening, periprosthetic humeral fracture, and osteolysis. subacromial impingement has been reported in patients after this procedure and the symptoms have necessitated subacromial decompression, but this has not previously been reported to be associated with a malunited osteo - periosteal sleeve. nonunion (or fibrous union) has been reported by previous authors following surgical access using the mackenzie approach but they did not report symptomatic subacromial impingement. in our patient, simple arthroscopic excision of the bony spur proved curative. this case has altered our practice, as we have now abandoned the osteoperiosteal sleeve technique and instead opt for subperiosteal dissection of the anterior deltoid. we conclude that the cause for the delayed, severe impingement symptoms in this patient following successful copeland resurfacing hemiarthroplasty was malunion of the osteo - periosteal sleeve that was made as part of the mackenzie approach. all symptoms resolved following arthroscopic resection of the spur. | we report the case of a patient with end - stage osteoarthritis who received a successful copeland resurfacing hemiarthroplasty through a mackenzie anterosuperior approach, which involves taking the anterior portion of the deltoid attachment from the acromion along with an osteo - periosteal sleeve. the patient went on to develop severe subacromial impingement symptoms 4 months postoperatively. x - rays revealed a large anteroinferior acromial osteophyte that had not been present preoperatively and was deemed to represent a malunited osteo - periosteal sleeve from the mackenzie approach. |
hemophilia is a coagulation disorder characterized by acute hemorrhages into the musculoskeletal system leading eventually to arthropathy and disability. deficiency of factor viii (hemophilia a) accounts for 85% of cases and 15% are due to factor ix deficiency (hemophilia b). acute bleeding increases the pressure in the synovial cavity and bone marrow which leads to severe pain and possible osteonecrosis or a pseudotumoral mass. intra - articular bleeding produces a direct chemical effect on synovium, cartilage, and bone. recurrent hyperemia of the joint in the growing child causes juxta articular osteoporosis and overgrowth of the epiphysis. patients with severe hemophilia may be at risk for developing reduced bone density in childhood and adolescence for a number of reasons as arthropathy and joint deformities result in prolonged immobilization and reduced physical activity which predisposes them for osteoporosis. this can lead to increasing tendency of bone fragility and fractures in patients after trivial trauma. osteoporosis is a multifactorial disease with particular considerations to calcium, magnesium (mg), and other trace elements as copper (cu) and zinc (zn), as they are essential in bone metabolism as cofactors for specific enzymes for optimal bone matrix development and bone density sustenance. therefore, we aimed to assess the presence and severity of osteoporosis in patients with hemophilic arthropathy by determining dual energy x - ray absorptiometry (dexa) and to correlate these findings with the extent of joint disease and serum levels of trace minerals as magnesium, copper, and zinc. their age ranged from 7 to 40 years compared to 20 controls matched for age and sex. those patients and controls were presenting to the internal medicine and physical medicine, rheumatology, and rehabilitation outpatient clinics of ain shams university hospitals. the diagnosis of hemophilia was made clinically and useful to classify them according to measured factor viii activity in the plasma as severe 5 units / dl, according to arnold and hilgartner. we excluded patients with cigarette smoking or alcohol abuse, patients with history of any chronic medical illness producing osteopenia / osteoporosis, thyroid or parathyroid disorders, history of chronic renal, hepatic, or gastrointestinal disease including parasitic infestations, patients with prolonged intake of steroids, antiepileptic medication, iron for anemia, ca or vit d supplementation, or any drug affecting bone metabolism. joint evaluation : lower limb joints (ankles and knees) were assessed by using the clinical evaluation score of the world federation of hemophilia (wfh) which includes seven criteria and a total possible score of 12. normal joints were scored as 0, the highest possible score for knees and ankles was 48. the sum of scores for both knees and ankles was used in the analysis. juvenile arthritis functional assessment report (gafar) : it is a single dimension scale developed by howe. based on 23 items used as a disability score to evaluate the functional status and daily living activities for each patient. the response to each activity was scored between 0 and 2 as follows : 0 = indicating that the activity could be done alone without any difficulty during the previous week ; 1 = indicating that it could be done some of time and 2 = indicating that it was almost never done alone. the score based on the 23 items was calculated as the sum of all items, assuming a range between 0 and 46 with lower score indicating better function. radiological evaluation : the x - ray were classified according to pettersson. for standardized examination. the total score for both knee joints was 26.bone densitometry : in all subjects, bone mineral density (bmd) was measured at the femoral neck and lumbar spine (l1l4) in the anterior and posterior projection using dexa. results were recorded for each patient as z score (difference in sd from the mean of a healthy age- and gender - matched sample). the roentgenographic examination included an anteroposterior projection and lateral views of both knee joints. the x - ray were classified according to pettersson. for standardized examination. bone densitometry : in all subjects, bone mineral density (bmd) was measured at the femoral neck and lumbar spine (l1l4) in the anterior and posterior projection using dexa. results were recorded for each patient as z score (difference in sd from the mean of a healthy age- and gender - matched sample). laboratory investigations : hemoglobin for anemia.complete liver and kidney function tests.serological screening for hbs ag and hcv.serum calcium (ca), phosphorus, and alkaline phosphatase to rule out osteomalacia and other metabolic bone disorders.stool analysis to rule out any parasitic infestations.serum magnesium level was assayed using advia 1650 (payer, siemens healthcare diagnostics) using modified xylidyl blue reaction, described by mann and yoe. normal range for serum is 1.92.5 mg / dl.determination of copper and zinc in serum was done by using the flawless atomic absorption spectrometry (perkin - elmer corp., norwalk, conn. serum was diluted 1 : 1 with deionized water for copper and 1 : 4 with 1% glycerol for zinc determination [13, 14 ]. results were expressed in g / ml ; normal levels for copper and zinc ranged from 0.71.0 g / ml and 0.81.2 g / ml, respectively. serum calcium (ca), phosphorus, and alkaline phosphatase to rule out osteomalacia and other metabolic bone disorders. serum magnesium level was assayed using advia 1650 (payer, siemens healthcare diagnostics) using modified xylidyl blue reaction, described by mann and yoe. determination of copper and zinc in serum was done by using the flawless atomic absorption spectrometry (perkin - elmer corp., serum was diluted 1 : 1 with deionized water for copper and 1 : 4 with 1% glycerol for zinc determination [13, 14 ]. results were expressed in g / ml ; normal levels for copper and zinc ranged from 0.71.0 g / ml and 0.81.2 g / ml, respectively. data were expressed as mean sd for quantitative measures and both number and percentage for categorical data. comparison between two independent groups of numerical parametric data was done using student 's t - test. comparison between two independent groups of nonparametric data was done using wilcoxon rank sum test. ranked spearman correlation test was done to study the possible association between each two variables among each group for nonparametric data. this study included 20 patients with hemophilic arthropathy and twenty healthy male subjects served as the control group. the patients ' age ranged from 7 to 40 years, with a mean of 21.7 11.2 years. the disease duration ranged from 2 to 33 years with mean of 14.7 10.5. the annual number of bleedings during the last 5 years ranged from 2 to 6 attacks per year with a mean of 4.45 1.43. as regards therapy, we use cryoprecipitate and/or purified plasma derived factor eight, around 5005000 units / kg / year according to requirements. the plasma factor level (viii) ranged from 210 units / dl, with a mean of 5.05 3.34 (table 1). according to the factor level, 8 patients (40%) diagnosed as mild grade and 12 patients (60%) diagnosed as moderate grade of hemophilia. the number of clinically affected joints ranged from 14 (both knees and ankles). the total joint score (tjs) ranged from 421 with mean of 13.5 6.2. functional assessment score (jafar) ranged from 09 with mean of 2.8 3.4. the total x - ray score ranged from 619 with a mean of 12.5 4.8 (table 1). total joint score correlated positively with total x - ray score (r = 0.58, p = 0.006). in addition, both tjs and total x - ray score correlated positively with disease duration (r = 0.46, p = 0.03 and r = 0.63, p = 0.002, resp.), and correlated negatively with the factor level of hemophilia (r = 0.64, p = 0.002 and r = 0.49, p = 0.02, resp.). functional assessment score correlated positively with tjs and total x - ray score (r = 0.81, p = 0.001 and r = 0.55, p = 0.01, resp.) and correlated negatively with factor level (r = 48, p = 0.03). presence of osteoporosis assessed by dexa (table 2) revealed highly significantly lower z scores of lumbar spine and neck of femur in hemophilic arthropathy patients versus controls (p 0.05). z score of neck of femur correlated negatively with total joint score (r = 0.547, p = 0.01), functional assessment score (r = 0.553, p = 0.01), and total x - ray score (r = 0.484, p = 0.03).while, there was no significant correlation between z score of lumbar spine and the clinical or radiological scores (p > 0.05). in hemophilic arthropathy patients, a highly significant decrease was found in serum levels of mg, cu, and zn compared to controls (p 0.05) (table 3). serum levels of zn correlated negatively with tjs, functional assessment score, and total x - ray score (r = 0.51, p = 0.01, r = 0.66, p = 0.001 and r = 0.94, p = 0.001, resp.). serum levels of cu and zn correlated positively with z score of neck of femur (r = 0.61, p = 0.004 and r = 0.83, p = 0.001, resp.), there was no significant correlation between serum levels of either calcium or magnesium and the severity of osteoporosis as measured by z score (p > 0.05). it has been suggested that the hemophiliacs may show a markedly lower bone mineral density (bmd) than the average population due to arthropathy that may induce lack of mobility as well as a pathological liver metabolism in those patients coinfected with a chronic hepatitis. the possible role of functional interaction between hematopoietic and bone tissues in the development of age related osteoporosis is discussed by gurevitch and slavin. blood loss creating developmental pressure on hematopoietic system enhances production of hematopoietic growth factors and subsequently intensifies proliferation of hematopoietic progenitor cells, increasing the number of osteoclasts which intensifies resorption of bone tissue. in addition, blood loss leads to the production of activity stimulating bone formation factors, extensive proliferation of osteogenic progenitor cells resulting in increased numbers of osteoblasts followed by new bone formation, and at same time increased production and maturation of osteoclasts which again enter the cycle of bone resorption together with exhaustion of osteogenic cell population for gradual development of osteoporosis. trace minerals may be important in maintaining bone quality through their role as metalloenzymes in the synthesis of collagen and other proteins that form the structure of bone. zinc inhibits the differentiation of osteoclasts and promotes osteoblast activity affecting the formation of hard tissues. it could also increase bone growth factors and bone matrix protein, which are involved in the stimulation of bone formation and proliferation of osteoblastic cells. it is shown to be mitogenic for osteoblasts, and its depletion causes cellular growth inhibition in vitro. copper (cu) is a cofactor for lysyl oxidase which is required in cross - linking of collagen and elastin. cu deficiency causes inhibition of bone growth and osteoporosis as observed in menkin 's disease, an inherent inability to absorb cu. the present study aimed to find out the presence of osteoporosis in patients with hemophilic arthropathy and its correlation with clinical disease severity and serum levels of trace minerals as zn, cu and mg. in the present study, we used 3 scoring systems ; clinical, functional and radiological scoring system to assess the extent of joint damage and related disability in 20 hemophilic patients. we scored only the lower limb major joints (knees and ankles), because these are the joints frequently affected by hemophilia, and damage of these joints will have a major impact on the patient 's daily activities involving weight bearing. this was in agreement with, gurcay. who studied thirty one young patients with hemophilia aged between 318 years using the 3 scoring systems. they found that the most commonly affected joints in hemophilia are the hinge joints : knee, elbow, and ankles. especially, the knees are most commonly affected in early childhood because of their weight bearing function. 60% of our patients diagnosed as moderate grade of hemophilia, our results showed that the total joint score correlated positively with total x - ray score. in addition, both total joint and total x - ray scores correlated positively with disease duration and correlated negatively with serum factor level of hemophilia. this was in accordance with the study done by gurcay. who diagnosed 21 from 31 patients (67.7%) as moderate grade and found that the clinical score correlated significantly with the radiological score. their results were supported by those of pettersson. who concluded that pettersson 's score correlated very well with the clinical profile of patients with hemophilic arthropathy. this may be primarily based on the duration of the disease and increased recurrent hemarthrotic attacks due to insufficient factor treatment. in the current study, there was positive correlation between the jafar disability score, total joint score, and total x - ray score.. reported a similar observation, as their analysis showed significant correlation between the disability and the radiological scores. also, other study found strong association between the disability score, radiological and clinical findings. the most difficult activities reported by our patients were walking 50 feet without help, standing up on tiptoes, and picking up something from the floor from a standing position. this was in accordance with gurcay. who concluded that these activities were limited mainly due to involvement of the joints of lower extremities which may easily result in functional disability. in our study, presence of osteoporosis assessed by dexa revealed highly significant lower z scores of lumbar spine and neck of femur in hemophilic arthropathy patients versus controls. this was in accordance with the study done by wallny. who found a relationship between hemophilia and osteoporosis and increased severity of hemophilia studied 19 patients with severe hemophilia a and showed significantly lower bmd values in comparison with controls. in addition, z score of neck of femur correlated negatively with total joint, functional assessment, and total x - ray scores. this was in accordance with barnes. who found a statistically significant association between areal bmd z scores and objective lower limb joint evaluation results. also, other studies [5, 15 ] found that patients with severe hemophilia are at risk of developing reduced bmd. lastly, khawaji. stated that with increasing severity and number of affected joints, bmd significantly decreases. there was no significant correlation between z score of lumbar spine and either clinical or radiological scores. this was in accordance with nair. as the bmd of femoral neck showed over all better correlation with the examined variables than the bmd of the lumbar spine. similar observation was seen between our study and the previous studies done that no significant difference was found in z scores of lumbar spine and neck of femur between patients with or without hepatitis c virus. in our study, we found a highly significant decrease in serum levels of mg, cu, and zn among hemophilic patients compared to controls, while there was no statistically significant difference as regards serum ca levels. moreover, serum levels of cu and zn correlated positively with z score of neck of femur and serum levels of zn correlated negatively with total joint, functional assessment, and total x - ray scores. on the other hand, there was no significant correlation between serum levels of either ca or mg and severity of osteoporosis as measured by z score. also, mir. found a significantly lower serum zn concentration in men, with hip osteoporosis and concluded that zinc had a positive association with bmd in men and its deficiency was more common in osteoporotic individuals. in addition, mutlu. found that serum mg and zinc were significantly lower in osteopenic women than in normal women. but they also concluded that trace element supplementation especially with magnesium, zinc, and perhaps copper, may have beneficial effects on bone density. on the other hand, gur. stated that mg, cu, and zn levels in serum of patients with postmenopausal osteoporosis were lower than those in controls demonstrating that deficiency of these trace minerals plays a major role in the development of osteoporosis when serum ca and phosphorus were normal. also, odabasi. reported significant differences between osteoporotic patients and controls as regards mg concentration. in conclusion, in hemophilic arthropathy patients, osteoporosis represents a frequent concomitant observation which may complicate the future treatment of these patients. trace minerals like mg, cu, and zn are essential in bone metabolism as cofactors for specific enzymes that are essential for organic bone matrix synthesis. screening of young hemophiliacs for reduced bone density is recommended with measuring the levels of mg, cu, and zn for better assessment and management of the disease. | objective. to find out the presence of osteoporosis in hemophilic arthropathy patients and its correlation with clinical severity and serum levels of magnesium, copper, and zinc. methods. joint score, functional assessment score, bone densitometry, and serum magnesium, copper and zinc were done in twenty male hemophilic arthropathy patients and twenty controls. results. there was highly significant lower z scores of lumbar spine and neck of femur in patients versus controls (p < 0.011). z score of neck of femur correlated negatively with total joint score (p = 0.013) and functional assessment score (p = 0.011). serum levels of copper and zinc correlated positively with z score of neck of femur (p = 0.004, p = 0.001, resp.). conclusion. osteoporosis represents a frequent concomitant observation in hemophiliacs. screening of young hemophiliacs for osteoporosis is recommended with measuring serum levels of magnesium, copper, and zinc for better management of the disease. |
irritable bowel syndrome (ibs) is a heterogeneous, idiopathic, gastrointestinal syndrome characterized by abdominal pain or discomfort, diarrhea, and/or constipation. a questionnaire survey conducted in students from a chinese university found that only 5.7% of respondents could be diagnosed with ibs, but epidemiological surveys in india, iran, japan, and other asian countries suggest that ibs is more prevalent in elderly people than in young adults [24 ]. questionnaires and related testing of elderly people in hong kong diagnosed 13.1% with ibs, and epidemiological investigation in the songjiang district of shanghai found a prevalence of 9.4% in adults aged 6079 years old and 10.2% in those over 80 years of age. although the etiology of ibs is currently unknown, several hypotheses have been proposed for its pathogenesis. the development of ibs may involve visceral abnormalities, genetic susceptibility, a neuro - immunological disorder, and/or psychological factors. ibs shows a familial tendency, and the mutations involved in genetic susceptibility to ibs have recently received particular attention. however, identifying genetic markers of ibs is complicated by the multi - factorial pathophysiology of this syndrome. it is hypothesized that exposure to certain environmental factors, such as infection or severe psychological stress, may induce ibs in genetically susceptible individuals. polymorphisms in the 5-hydroxytryptamine (5-ht) transporter or receptors, adrenergic system, and mitochondrial genome have been highlighted as potentially contributing to abnormalities in ibs patients, including enhanced perception of gut stimuli, altered gastrointestinal motor function, and psychiatric symptoms. several neurotransmitter receptors involved in the regulation of gastrointestinal motility, such as the 5-ht receptor, adrenergic receptor, and cannabinoid receptor, are g protein - coupled receptors. one g protein subunit, g protein beta polypeptide 3 (gn3), is a component of several g protein complexes, and thus polymorphisms in the gn3 gene impact signal transduction from several receptors. a common single nucleotide polymorphism substitution of c with t at position 825 within exon 10 of the gn3 gene (rs5443) results in a truncated gn3 splice variant with enhanced g protein activation. this polymorphism has been reported to be overrepresented in patients diagnosed with some forms of ibs [1217 ]. sympathetic dysfunction of the adrenergic nerves has also been associated with the pathogenesis of some cases of ibs [1820 ]. dysfunction of catechol - o - methyltransferase (comt), an enzyme capable of degrading catecholamines such as dopamine, norepinephrine, and epinephrine, appears to play a role in the pathogenesis of some cases of ibs [1820 ]. a common single nucleotide polymorphism substitution at position 1947 in exon 4 of the comt gene results in the substitution of valine 158 with methionine in the comt protein (a / a genotype), which reduces the activity of comt by about 4-fold. however, comt with val 158 (g / g genotype) appears to have higher activity. recent studies have demonstrated that comt gene polymorphisms are correlated to chronic pain and depression. although ibs patients often have abdominal pain accompanied by anxiety or depression, few studies have investigated the relationship between comt gene polymorphisms and ibs. preliminary investigations have found that individuals with 2 copies of the gene producing comt with valine at position 158 are over - represented in groups diagnosed with ibs. we sought to determine whether these single nucleotide polymorphisms in gn3 and comt were associated with ibs in elderly chinese patients. due to the likely multi - factorial nature of ibs, we sub - divided patients diagnosed with ibs according to their symptoms and determined the frequency of these snps in these groups and an age - matched control group unaffected by gastrointestinal disease. between march and december 2012, 66 patients over 60 years old were enrolled at huashan hospital (shanghai, china). all patients were diagnosed with ibs according to the rome iii diagnostic criteria and were further subdivided into 3 groups based on symptom presentation : 1) ibs with constipation (ibs - c), 2) ibs with diarrhea (ibs - d), and 3) ibs with both symptoms (ibs - m). patients with liver, gallbladder or pancreatic disease, diabetes, hyperthyroidism, cancer, or any other disease were excluded by examination of medical records, physical examination, ultrasound, and blood biochemical analysis. patients with history of abdominal surgery or adverse drug reactions, including constipation and diarrhea, were also excluded from this study. we enrolled 115 age - matched subjects without ibs as a control group during the same period at routine annual physical examination. subjects in the control group defecated once or twice a day, or once every 2 days, producing yellow, soft, solid stools, and did not experience hard bowel movements, abdominal pain, or discomfort. all participants provided written informed consent, and the study was approved by the ethics committee of huashan hospital, fudan university [2012(229) ]. a professionally trained physician evaluated all participants with the geriatric depression scale (gds), a set of 30 polar questions used to quantify participant emotional state during the preceding week. for each question, a positive response is assigned 1 point, and a negative response is assigned no points. genomic dna was extracted using qiaamp mini - spin columns (qiagen, usa) according to the manufacturer s instructions and separated by agarose gel electrophoresis for snp determination. the snp sequences of gnb c825 t (rs 5443) and comt val158met (rs 4680) were based on the gene bank databases. primers and probes were designed using abi primer express software v2.0 and synthesized by invitrogen, as detailed in table 1. the pcr reaction was carried out in a steponeplus real - time pcr system (applied biosystems, foster city, ca, usa) and subjected to 95c for 2 min, then 40 cycles of 95c for 10 s, followed by 53c for 45 s. fluorescence was measured by the real - time pcr system and data was converted by sds 2.0 software. the chi - square test and hardy - weinberg equilibrium theory were used to compare the genotype and allele frequency between the ibs and control groups, and between ibs subgroups. chi - square test and fisher s exact test were used to assess the relationship between clinical factors and genotype. unconditional logistic regression analysis and crossover analysis statistical analysis was performed with spss 13.0 statistical software and p - values 0.05). gn3 genotyping determined that the c allele frequency was 85.6% in the ibs group and 89.1% in the control group, and the t allele frequency was 14.4% in the ibs group and 10.9% in the control group (table 3). the cc genotype of gn3 was found in 75.8% of those enrolled in the ibs group (50 instances) in comparison to 80% of the control group (92 instances). the ct genotype was present in 19.7% (13 instances) of the ibs group and 18.3% (21 instances) of the control group, and the tt genotype was present in 4.5% (3 instances) of the ibs group and 1.7% (2 instances) of the control group (table 4). there was no statistically significant difference between allele frequency (p=0.323) or the genotype distribution (p=0.534) between the ibs and control groups. comt genotyping determined that the g allele frequency was 87.1% in the ibs group and 93.5% in the control group, and the a allele frequency was 12.9% in the ibs group and 6.5% in the control group (table 5). the gg genotype of comt was found in 86.4% of those enrolled in the ibs group (57 instances) in comparison to 87.8% of the control group (101 instances). the ga genotype was present in 1.5% (1 instance) of the ibs group and 11.3% (13 instances) of the control group, and the aa genotype was present in 12.1% (8 instances) of the ibs group and 0.9% (1 instance) of the control group (table 6). the a allele was found significantly more frequently in the ibs group than in the control group (p=0.040), and the genotype distribution also differed significantly between the ibs group and the control group (p 0.05, table 11). interaction analysis indicated no interaction between the gn3 gene and the comt gene in ibs patients (p>0.05, table 12). participant demographics are shown in table 2. the genomic distribution of the gn3 c825 t and comt val158met polymorphisms in the control group did not significantly deviate from the hardy - weinberg equilibrium (p > 0.05). gn3 genotyping determined that the c allele frequency was 85.6% in the ibs group and 89.1% in the control group, and the t allele frequency was 14.4% in the ibs group and 10.9% in the control group (table 3). the cc genotype of gn3 was found in 75.8% of those enrolled in the ibs group (50 instances) in comparison to 80% of the control group (92 instances). the ct genotype was present in 19.7% (13 instances) of the ibs group and 18.3% (21 instances) of the control group, and the tt genotype was present in 4.5% (3 instances) of the ibs group and 1.7% (2 instances) of the control group (table 4). there was no statistically significant difference between allele frequency (p=0.323) or the genotype distribution (p=0.534) between the ibs and control groups. comt genotyping determined that the g allele frequency was 87.1% in the ibs group and 93.5% in the control group, and the a allele frequency was 12.9% in the ibs group and 6.5% in the control group (table 5). the gg genotype of comt was found in 86.4% of those enrolled in the ibs group (57 instances) in comparison to 87.8% of the control group (101 instances). the ga genotype was present in 1.5% (1 instance) of the ibs group and 11.3% (13 instances) of the control group, and the aa genotype was present in 12.1% (8 instances) of the ibs group and 0.9% (1 instance) of the control group (table 6). the a allele was found significantly more frequently in the ibs group than in the control group (p=0.040), and the genotype distribution also differed significantly between the ibs group and the control group (p 0.05, table 11). interaction analysis indicated no interaction between the gn3 gene and the comt gene in ibs patients (p>0.05, table 12). in this study we found that comt, encoding an enzyme with reduced activity, was significantly associated with ibs in elderly chinese patients, and was particularly significantly associated with ibs accompanied by diarrhea and ibs that had persisted for longer than 5 years. we also found that ibs patients were significantly less likely to possess heterozygous comt genes than the control patients. the frequency of comt 158met in the ibs group was significantly higher than that in the control group, suggesting that the predisposes elder people to ibs, and the val allele protects against ibs. our results are consistent with previous reports that found that sympathetic dysfunction of the adrenergic nerves is associated with the pathogenesis of some cases of ibs [1820 ], but inconsistent with another study that found the comt 158val allele to be significantly associated with pathogenesis of ibs and increased frequency of defecation. we found no significant difference in the percentage of patients with a gds score above 10 in the ibs group and the control group, no significant difference in the genotype distribution or allele frequency of gn3-c825 t between the ibs and the control group, and no interaction between the gn3 gene and the comt gene in ibs patients. no difference in the genotype distribution and allele frequency of gn3-c825 t was detected when ibs patients were further subdivided according to symptom presentation and persistence of symptoms. our findings concerning gn3-c825 t further support the results of case - control studies conducted in the united states in 2007 and in korea in 2012. however, several additional studies found different results in other populations. a 2011 study in a greek population found the gn3 - 825 t allele encoding a g protein that mediates enhances activation to be closely associated with the occurrence of ibs. a 2001 study conducted in a caucasian population found that gn3 - 825c was associated with increased defecation frequency, and korean studies in 2010 and 2012 found gn3 - 825 t to be associated with ibs with constipation in adults and children, and gn3 - 825c to be associated with ibs with diarrhea in children. we did not detect a statistically significant association of particular gn3 or comt genotypes with ibs with constipation, but our analysis may have been limited by sample size because we enrolled only a small number of patients (7) who experienced ibs with constipation. the inconsistent results of these studies may be partially explained to be relatively small sample sizes. for example, both kim and saito subdivided small populations (60 and 50 ibs patients, respectively) into cc, ct, and tt genotype subgroups, resulting in a smaller size in each group. the studies that found association between gn3-c825 t and ibs were conducted in ethnic koreans and greeks. fang. performed a correlation study between the gn3c825 t polymorphism and functional dyspepsia (fd) in a han chinese population, and found that this polymorphism is not a risk factor for the onset of fd. currently, both fd and ibs are thought to share a similar pathogenesis, and both are categorized as functional gastrointestinal diseases (fgid). therefore, one can speculate that the c825 t polymorphism of gn3 may not be a risk factor for the pathogenesis of ibs in the chinese population. we found that the comt val158met single nucleotide polymorphism was associated with susceptibility to ibs, and the was over represented in patients diagnosed with ibs with diarrhea or ibs that had persistsed for longer than 5 years. however, no evidence for a correlation between gn3 c825 t single nucleotide polymorphisms and the pathogenesis of ibs was found among this population of elderly ibs patients. to further validate genetic markers of ibs, larger cohorts will need to be enrolled, as identifying genetic markers of ibs is complicated by the multi - factorial pathophysiology of this heterogeneous syndrome. | backgroundseveral polymorphisms have been reported to be associated with irritable bowel syndrome (ibs), including c825 t, the single nucleotide polymorphism (snp), responsible for a truncated g protein 3 subunit (gn3), and the vall158met substitution in catechol - o - methyltransferase (comt). we investigated the association between these mutations and the prevalence of ibs in 66 elderly chinese patients.material/methodssixty-six patients (over age 60 years) were diagnosed with ibs according to the rome iii criteria, and divided into 3 groups based on symptom presentation. the groups consisted of 7 patients with constipation, 46 patients with diarrhea, and 13 patients with both or neither symptoms. we enrolled 115 age - matched individuals without ibs as the control group. all patients were evaluated by using the geriatric depression scale, disease progression was recorded, and gn3 and comt were genotyped by pcr.resultsthere was no significant difference in gn3 c825 t genotype distribution and allele frequency between the 2 groups. in contrast, compared with control subjects, comt 158met was significantly more prevalent in the ibs group (p=0.040) and significantly more prevalent in patients with diarrhea (p=0.029). 158met was also more prevalent in those patients who had experienced symptoms for over 5 years (p=0.022).conclusionsin elderly chinese patients, the 158met snp in comt is associated with ibs pathogenesis, but the gn3-c825 t snp is not associated with ibs pathogenesis. |
progression of chronic obstructive pulmonary disease (copd) assessed by decline in forced expiratory volume in one second (fev1) is closely related to active tobacco smoking. it has been shown that smoking cessation decelerates the fev1 decline in patients with mild - to - moderate copd.1,2 it is also widely assumed that acute exacerbations contribute to disease progression.3 exacerbations increase respiratory symptoms, impair exercise capacity, and cause a deterioration in quality of life.4,5 however, most studies evaluating the functional and clinical changes associated with an acute exacerbation and its recovery have included current smokers, making it difficult to separate the effects of exacerbations from those of smoking.36 studies in ex - smokers are scarce and have yielded conflicting results.79 analysis of the lung health study data by kanner indicate that acute exacerbations evaluated by self - reported episodes of lower respiratory infections resulting in physician visits during the previous year in ex - smoking patients do not contribute to fev1 decline, whereas in a follow - up of 102 patients for 3 years, makris reported that acute exacerbations produced a decline in fev1 in ex - smokers, albeit of a lesser magnitude that in active smokers. on the other hand, there is no information about the effect of acute exacerbations on patient - centered outcomes in ex - smoking copd patients. the aim of the present study was to evaluate if frequent acute exacerbations in ex - smoking copd patients are associated with disease progression by assessing functional and clinical indices over 2 years of follow - up. a cohort of 105 consecutive ex - smoking patients with a long history of copd according to global initiative for chronic obstructive lung disease (gold) criteria10 was enrolled in a 2-year follow - up study, based on scheduled visits every 6 months. recruitment criteria included : age older than 40 years ; smoking history greater than ten packs / year ; cessation of smoking for at least 6 months before recruitment, confirmed by urine cotinine levels ; absence of an acute exacerbation in the previous month at least ; absence of any physical condition precluding the ability to perform a 6-minute walking test (6mwt) ; or a short life expectancy. patients with asthma, bronchiectasis, sequelae of tuberculosis, or known malignancy were excluded. the institutional review board at our institution approved the study, and written informed consent was obtained from all patients. for diagnosis of acute exacerbation, the following definition was applied : a sustained worsening of the patient s condition from the stable state and beyond normal day - to - day variation that is acute in onset and necessitates a change in regular medication.11 a sustained worsening of the patient s condition from the stable state and beyond normal day - to - day variation that is acute in onset and necessitates a change in regular medication.11 patients were instructed to contact one of the investigators if they had an acute increase in symptoms (dyspnea, cough, sputum, and/or purulent sputum) for 2 consecutive days with or without symptoms of upper respiratory tract infection or fever and to attend our clinic to confirm the presence of an acute exacerbation.12 severity of exacerbation was graded according to the health care utilization classification. thus, exacerbation was considered : mild if it required increases in regular inhaled medication ; moderate if courses of antibiotics and/or systemic corticosteroids were needed ; and severe if the patient required hospital admission.13 exacerbations were treated at our facility or at other health institutions. those exacerbations treated at other institutions were registered during the scheduled visits, and their severity was categorized according to the treatment received using the above classification. evaluations were performed at recruitment and at every scheduled visit while patients were in a stable condition. at each visit, assessments included anthropometry, dyspnea, health status, peripheral capillary oxygen saturation (spo2), lung function, 6mwt, and comorbidities.14 magnitude of dyspnea was assessed using the modified medical research council scale (mmrc).15 lung function included spirometric testing and inspiratory capacity before and after 400 g of albuterol following international guidelines,16 and was standardized as percentages of predicted values by using prediction equations.17,18 measurements were carried out by the same laboratory personnel and with the same equipment over the 2 years of follow - up. health status was assessed with two questionnaires, ie, the spanish version of the st george s respiratory questionnaire (sgrq)19 and the spanish version of the chronic respiratory disease questionnaire (crq).20 the 6mwt was measured according to current guidelines21 and its values were expressed as a predicted percentage using reference values from troosters.22 the bode (body mass index, airflow obstruction, dyspnea, exercise performance) index23 was calculated according to celli. we considered a drug as being the main therapy if the patients reported its use for at least more than 50% of the follow - up period. disease progression was assessed by lung function decline and progressive impairment of symptoms, functional capacity (6mwd), health status, and bode index. the results are expressed as the mean one standard deviation or median and interquartile range in tables and text, and as the mean one standard error in figures. the kolmogorov - smirnov and shapiro - wilk tests were used for checking distribution normality. baseline variables were compared by the student s t - test for independent samples, the mann whitney u - test, or chi - square test according to the type of variable and its distribution. for analyses, patients were grouped into two categories according to the annual rate of total number of exacerbations experienced. since the median exacerbation frequency for the whole group was two per year, those patients experiencing more than the median annual exacerbation rate (two or more acute exacerbations per year) were termed frequent exacerbators whereas those with less than the median were considered infrequent exacerbators. differences in changes across time in functional, clinical, and health status indices between infrequent exacerbators and frequent exacerbators were assessed by linear mixed effects models using hierarchical linear modeling (hlm). using hlm, we examined patterns of change for all measurements (ie, fixed effects) and if there were variations among individuals in these patterns of change (ie, random effects) based on the frequency of exacerbations (ie, covariate). baseline values for all measurements were used as intercept, allowing us to examine change from these while controlling for the initial values. for these analyses, we used data for all subjects who had at least two measurements. although linear and quadratic changes were examined (based on number of measures over time), the simplest pattern of change was selected, ie, the most parsimonious model, for further analyses. all analyses were conducted with the hlm version 6.08 statistical package (scientific software international, inc. a cohort of 105 consecutive ex - smoking patients with a long history of copd according to global initiative for chronic obstructive lung disease (gold) criteria10 was enrolled in a 2-year follow - up study, based on scheduled visits every 6 months. recruitment criteria included : age older than 40 years ; smoking history greater than ten packs / year ; cessation of smoking for at least 6 months before recruitment, confirmed by urine cotinine levels ; absence of an acute exacerbation in the previous month at least ; absence of any physical condition precluding the ability to perform a 6-minute walking test (6mwt) ; or a short life expectancy. patients with asthma, bronchiectasis, sequelae of tuberculosis, or known malignancy were excluded. the institutional review board at our institution approved the study, and written informed consent was obtained from all patients. for diagnosis of acute exacerbation, the following definition was applied : a sustained worsening of the patient s condition from the stable state and beyond normal day - to - day variation that is acute in onset and necessitates a change in regular medication.11 a sustained worsening of the patient s condition from the stable state and beyond normal day - to - day variation that is acute in onset and necessitates a change in regular medication.11 patients were instructed to contact one of the investigators if they had an acute increase in symptoms (dyspnea, cough, sputum, and/or purulent sputum) for 2 consecutive days with or without symptoms of upper respiratory tract infection or fever and to attend our clinic to confirm the presence of an acute exacerbation.12 severity of exacerbation was graded according to the health care utilization classification. thus, exacerbation was considered : mild if it required increases in regular inhaled medication ; moderate if courses of antibiotics and/or systemic corticosteroids were needed ; and severe if the patient required hospital admission.13 exacerbations were treated at our facility or at other health institutions. those exacerbations treated at other institutions were registered during the scheduled visits, and their severity was categorized according to the treatment received using the above classification. evaluations were performed at recruitment and at every scheduled visit while patients were in a stable condition. at each visit, assessments included anthropometry, dyspnea, health status, peripheral capillary oxygen saturation (spo2), lung function, 6mwt, and comorbidities.14 magnitude of dyspnea was assessed using the modified medical research council scale (mmrc).15 lung function included spirometric testing and inspiratory capacity before and after 400 g of albuterol following international guidelines,16 and was standardized as percentages of predicted values by using prediction equations.17,18 measurements were carried out by the same laboratory personnel and with the same equipment over the 2 years of follow - up. health status was assessed with two questionnaires, ie, the spanish version of the st george s respiratory questionnaire (sgrq)19 and the spanish version of the chronic respiratory disease questionnaire (crq).20 the 6mwt was measured according to current guidelines21 and its values were expressed as a predicted percentage using reference values from troosters.22 the bode (body mass index, airflow obstruction, dyspnea, exercise performance) index23 was calculated according to celli. we considered a drug as being the main therapy if the patients reported its use for at least more than 50% of the follow - up period. disease progression was assessed by lung function decline and progressive impairment of symptoms, functional capacity (6mwd), health status, and bode index. the results are expressed as the mean one standard deviation or median and interquartile range in tables and text, and as the mean one standard error in figures. the kolmogorov - smirnov and shapiro - wilk tests were used for checking distribution normality. baseline variables were compared by the student s t - test for independent samples, the mann whitney u - test, or chi - square test according to the type of variable and its distribution. for analyses, patients were grouped into two categories according to the annual rate of total number of exacerbations experienced. since the median exacerbation frequency for the whole group was two per year, those patients experiencing more than the median annual exacerbation rate (two or more acute exacerbations per year) were termed frequent exacerbators whereas those with less than the median were considered infrequent exacerbators. differences in changes across time in functional, clinical, and health status indices between infrequent exacerbators and frequent exacerbators were assessed by linear mixed effects models using hierarchical linear modeling (hlm). using hlm, we examined patterns of change for all measurements (ie, fixed effects) and if there were variations among individuals in these patterns of change (ie, random effects) based on the frequency of exacerbations (ie, covariate). baseline values for all measurements were used as intercept, allowing us to examine change from these while controlling for the initial values. for these analyses, we used data for all subjects who had at least two measurements. although linear and quadratic changes were examined (based on number of measures over time), the simplest pattern of change was selected, ie, the most parsimonious model, for further analyses. all analyses were conducted with the hlm version 6.08 statistical package (scientific software international, inc., lincolnwood, il, usa). two patients died and another three were lost to follow - up before the first scheduled visit and were consequently excluded from the analyses. during follow - up, seven patients died and four declined to continue in the protocol, mainly due to transport difficulties (figure 1). compared with patients who completed the study, these eleven patients were older, had more severe disease according to gold criteria and bode index, and nine reported frequent exacerbations during the year before recruitment. the baseline characteristics of the 100 patients included in the analyses are shown in table 1. during follow - up, two hundred and fifty - six patients (61%) were seen and treated at our institution, and the remaining 163 (39%) at other institutions. according to the classification employed, thirteen subjects had one hospitalization, one subject had two hospitalizations, and a third subject had three hospitalizations. the number of exacerbations per patient over the 2 years of follow - up was variable (figure 2). seventeen patients did not experience an exacerbation, whereas five experienced 14 or more exacerbations during the observation period. median exacerbation frequency was two and one per year in patients classified as frequent exacerbators and infrequent exacerbators, respectively (p<0.001). at baseline, patients with frequent exacerbations showed significant differences in the number of acute exacerbations during the previous year, fev1 % predicted, forced vital capacity (fvc) % predicted, inspiratory capacity % predicted, and spo2, as well as a greater impairment in mmrc, health status (sgrq and crq) and the bode index, as compared with infrequent exacerbators (see table 2). the slopes for all these variables, representing changes over the 2-year follow - up period, were not significantly different between the two groups (table 3). thus, fev1 decreased 5713 ml / year (1.7% predicted / year) and 8515.8 ml / year figure 3 shows lung function indices at baseline and at each scheduled visit in both groups of patients. over the study period, frequent exacerbators had consistently lower values for inspiratory capacity, fvc, fev1, and fev1/fvc than infrequent exacerbators, but changes across time did not differ between the two groups. similar behavior was observed for functional exercise capacity (6mwt) and health - related quality of life (figure 4). patients with frequent exacerbations were treated more frequently with inhaled corticosteroids, whereas short - acting bronchodilators were used as the main therapy more often by infrequent exacerbators (table 4). two hundred and fifty - six patients (61%) were seen and treated at our institution, and the remaining 163 (39%) at other institutions. according to the classification employed, thirteen subjects had one hospitalization, one subject had two hospitalizations, and a third subject had three hospitalizations. the number of exacerbations per patient over the 2 years of follow - up was variable (figure 2). seventeen patients did not experience an exacerbation, whereas five experienced 14 or more exacerbations during the observation period. median exacerbation frequency was two and one per year in patients classified as frequent exacerbators and infrequent exacerbators, respectively (p<0.001). at baseline, patients with frequent exacerbations showed significant differences in the number of acute exacerbations during the previous year, fev1 % predicted, forced vital capacity (fvc) % predicted, inspiratory capacity % predicted, and spo2, as well as a greater impairment in mmrc, health status (sgrq and crq) and the bode index, as compared with infrequent exacerbators (see table 2). the slopes for all these variables, representing changes over the 2-year follow - up period, were not significantly different between the two groups (table 3). thus, fev1 decreased 5713 ml / year (1.7% predicted / year) and 8515.8 ml / year figure 3 shows lung function indices at baseline and at each scheduled visit in both groups of patients. over the study period, frequent exacerbators had consistently lower values for inspiratory capacity, fvc, fev1, and fev1/fvc than infrequent exacerbators, but changes across time did not differ between the two groups. similar behavior was observed for functional exercise capacity (6mwt) and health - related quality of life (figure 4). patients with frequent exacerbations were treated more frequently with inhaled corticosteroids, whereas short - acting bronchodilators were used as the main therapy more often by infrequent exacerbators (table 4). the main findings of this prospective observational study in patients with copd who had quit smoking and were followed up for 2 years are : frequent moderate exacerbations did not accelerate the decline in fev1 nor worsen other functional indices as compared with infrequent exacerbations, and there were no significant differences in patient - centered outcomes over time between the two groups. our results also confirm that exacerbations are more frequent in patients with more severe disease and that previous history of exacerbations is a predictor of frequent exacerbations.24 to our knowledge, this is the first study performed solely in ex - smoking copd patients that included several clinical indices in addition to lung function to evaluate the effect of exacerbations on disease progression. our findings are consistent with results reported in other studies, where no significant differences were found in the fev1 decline between frequent and infrequent exacerbators.4,7,9,25,26 some of these studies included current smokers.4,25,26 two studies have previously assessed the effects of exacerbations on fev1 decline in ex - smokers, with conflicting results.7,9 kanner performed a secondary analysis of the lung health study and found that, in intermittent and continuous smokers, frequent lower respiratory infections were associated with a greater 5-year averaged annual rate of fev1 loss (52 ml / year and 69 ml / year, respectively) than in sustained quitters (12 ml / year). makris evaluated the effect of acute exacerbations in current and ex - smokers during a 3-year prospective study including 58 ex - smokers and 44 current smokers. our results are in agreement with those of the ex - smoker group reported by kanner but differed from those reported by makris. this may be explained by differences between the present study and the study by makris with regard to the number of ex - smoking patients (100 versus 58, respectively) and length of follow - up (2 versus 3 years, respectively), but mainly in the annual rate of hospitalizations (0.10 versus 0.35 per year, respectively), indicating more severe exacerbations in their study. the role of exacerbation frequency in fev1 decline is largely based on findings of studies that have included active smokers.36 even so, differences in fev1 decline between frequent exacerbators and infrequent exacerbators have been small : 8 ml / year was reported by donaldson in a group of 32 patients followed for 4 years and, according to makris frequent exacerbations added 1.4% predicted / year to fev1 decline. in the present study, which included only ex - smoking patients, the decline in fev1, although not significant, was smaller in frequent exacerbators (54.7 ml / year) than in infrequent exacerbators (85.4 ml / year). this unexpected nonsignificant faster decline in infrequent exacerbators could be explained by their higher baseline fev1 value, in agreement with previous data from casanova.27 it is also consistent with the findings from the analysis of data obtained from the placebo arms of several recent clinical series by tantucci and modena,28 which showed that the loss of lung function, assessed as expiratory airflow reduction, seems more accelerated and therefore more relevant in the initial phases of copd. other possible explanations for the lack of a more rapid fev1 decline in frequent exacerbators are frequent treatment with systemic corticosteroids due to acute exacerbations as well as more regular use of inhaled corticosteroids than in infrequent exacerbators. moreover, the effects of acute exacerbations on fev1 decline are difficult to assess due to the heterogeneity of the patient behavior over time. several recent studies, independent of the frequency of exacerbations, show a heterogeneous fev1 decline in copd.2931 according to these studies, fev1 may remain stable in some patients, increase in others, or experience a slow or accelerated decline. accelerated fev1 decline was associated with a high baseline fev1, low body mass index, active smoking, bronchodilator reversibility, and greater magnitude of emphysema. our results confirm the heterogeneous fev1 decline reported by these authors, independently of the frequency of exacerbations.2931 in addition to the lack of effect of acute exacerbations on fev1 decline, the present results also demonstrate no effects on other physiological or clinical outcomes in ex - smoking copd patients. to our knowledge, the effects of exacerbations on patient - centered outcomes in ex - smoking copd patients have not been previously assessed. the few data available derive from the study by cote in a cohort of 205 patients (95% men), including active smokers, who were followed for 2 years after their first acute exacerbation. although no significant changes in fev1 decline were observed, they found a significant deterioration of mmrc, 6mwt, and bode index in their frequent exacerbators. in addition to differences in cohort characteristics, our results differ from theirs in the percentage of patients who required hospital admissions. fifty of their patients with frequent acute exacerbations were hospitalized, of whom 39% required one hospitalization, 35% required two hospitalizations, and 26% required three or more hospitalizations. in our cohort, 13 patients required hospitalization and only four of them required a second hospitalization. as it is known that severe acute exacerbations contribute to deterioration and mortality in copd patients,32 the greater number of patients and hospital admissions reported by cote may explain the clinical deterioration of their patients. the most advanced therapy (long - acting muscarinic antagonist + long - acting beta-2 agonists + inhaled corticosteroids), mostly received by the frequent exacerbators given their greater copd severity, could partly explain the lack of significant differences in the measured indices. our study has some limitations : the sample size was relatively small ; most exacerbations were moderate and treated in an outpatient setting ; severe exacerbations, which are known to contribute to mortality and hospitalization, were infrequent ; no mild acute exacerbations, ie, those needing only an increase in bronchodilator therapy, were registered, probably because they resolved without medical assistance ; and follow - up was relatively short. however, we believe that these limitations do not affect our main findings, indicating no effect of moderate acute exacerbations on fev1 decline or clinical progression of copd in ex - smokers. in summary, our findings suggest that acute moderate exacerbations in ex - smoking patients do not contribute to copd progression assessed by fev1 decline and by the bode index during a follow - up of 2 years. they also confirm that changes in fev1 over time are heterogeneous independent of exacerbation frequency. although patients with frequent exacerbations had greater baseline impairment of functional and clinical indices than those with infrequent acute exacerbations, their parameters behaved similarly over time. replication of these findings may help us to better understand how the effects of frequency of exacerbations among ex - smoking copd patients impacts their disease behavior. | backgroundin addition to smoking, acute exacerbations are considered to be a contributing factor to progression of chronic obstructive pulmonary disease (copd). however, these findings come from studies including active smokers, while results in ex - smokers are scarce and contradictory. the purpose of this study was to evaluate if frequent acute moderate exacerbations are associated with an accelerated decline in forced expiratory volume in one second (fev1) and impairment of functional and clinical outcomes in ex - smoking copd patients.methodsa cohort of 100 ex - smoking patients recruited for a 2-year follow - up study was evaluated at inclusion and at 6-monthly scheduled visits while in a stable condition. evaluation included anthropometry, spirometry, inspiratory capacity, peripheral capillary oxygen saturation, severity of dyspnea, a 6-minute walking test, bode (body mass index, airflow obstruction, dyspnea, exercise performance) index, and quality of life (st george s respiratory questionnaire and chronic respiratory disease questionnaire). severity of exacerbation was graded as moderate or severe according to health care utilization. patients were classified as infrequent exacerbators if they had no or one acute exacerbation / year and frequent exacerbators if they had two or more acute exacerbations / year. random effects modeling, within hierarchical linear modeling, was used for analysis.resultsduring follow - up, 419 (96% moderate) acute exacerbations were registered. at baseline, frequent exacerbators had more severe disease than infrequent exacerbators according to their fev1 and bode index, and also showed greater impairment in inspiratory capacity, forced vital capacity, peripheral capillary oxygen saturation, 6-minute walking test, and quality of life. however, no significant difference in fev1 decline over time was found between the two groups (54.713 ml / year versus 85.415.9 ml / year in frequent exacerbators and infrequent exacerbators, respectively). this was also the case for all other measurements.conclusionour results suggest that frequent moderate exacerbations do not contribute to accelerated clinical and functional decline in copd patients who are ex - smokers. |
their general metabolic pathway in mammals involves cytochrome p-450 (cyp) enzyme - mediated oxidation to form hydroxylated metabolites and epoxides and further conjugation with glucuronide or glutathione to create corresponding conjugates, leading further to methylsulfonyl metabolites catalyzed by glutathione s - transferase (gst) (1). pcbs with vicinal hydrogens at the 3 and 4 positions of the aromatic ring as well as 2,5- or 2,3,6-substituted chlorines are most likely to be metabolized to methyl sulfones (2,3). it is important to study the fate of individual pcb congeners because the properties, toxicities, and behavior of pcbs vary widely in the environment. the coplanar 3,3,4,4-tetrachlorobiphenyl (cb77, iupac number) is a dioxin - like congener, with a relatively high toxicity compared to most tetrachlorobiphenyls. the main monohydroxy metabolites of cb77 in mammals includes 4-hydroxy-3,3,4,5-tetrachlorobiphenyl (4oh - cb79), 5-hydroxy-3,3,4,4- tetrachlorobiphenyl (5oh - cb77), a trace of 6-hydroxy-3,3,4,4-tetrachlorobiphenyl (6oh - cb77), and 2-hydroxy-3,3,4,4-tetrachlorobiphenyl (2oh - cb77). the methylsulfonyl metabolites of cb77 were documented in rats, mice, and fish, although cb77 is not one of the preferred structures for methyl sulfone metabolism (58). according to the green liver model, metabolism in plants is similar to the detoxification mechanism in animal livers because of similar enzyme systems (9). to date, metabolism of pcbs in plants is generally reported in in vitro studies using cell or tissue cultures, which have the advantages of rapidly growing biomass and axenic conditions where microorganisms play no role in plant metabolism (1012). different plant species, including paul s scarlet rose, tobacco, solanum spp., horseradish, and alfalfa have been screened for their ability to biotransform pcbs using cell cultures. mono- and dihydroxy - chlorobiphenyls were identified as the metabolites of mono- and dichlorobiphenyls (13). other research showed only monohydroxy - pcbs as the metabolites of di-, tri-, tetra-, and penta - chlorobiphenyls (14). metabolism depends on the plant species and specific pcb congeners to which the plant tissue cultures are exposed. in general, lower chlorinated congeners are metabolized more rapidly than those with higher chlorine substitutions. however the position of chlorine atoms and molecular structures are also important factors affecting pcb metabolism (10). in the study of wilken., only three in 12 cell cultures, tomato (lycopersicum esculentum), lettuce (lactuca sativa), and paul s scarlet rose (rosa spp.), were able to hydroxylate cb77, and the main identified metabolites were 2-oh - cb77, 6-oh - cb77, and 5-oh - cb77 (15). more recently, methoxy- and hydroxy - methoxy - pcbs were reported as the metabolites of tobacco cells biodegrading dichlorobiphenyls (16). despite the advantage of cell and tissue cultures over whole plants as a fast screening tool, the extrapolation of metabolism from in vitro to in vivo is tenuous. to better understand the biodegradation of pcbs in ubiquitous plants in nature, we find in vivo studies are necessary. in our previous studies (17), it was found that whole hybrid poplars take up and bind cb77 strongly in root tissues, and metabolism was suggested but not confirmed. to study the biotransformation of cb77 in whole plants and the applicability of phytoremediation for such pcb congeners, we hydroponically exposed intact plants to cb77, and the presence of metabolites in hydroponic solution and plant tissues were tracked through time. to our knowledge, this is the first in vivo study on hydroxylation of pcb within intact plants. the standards of cb77, hydroxy metabolites (4-oh - cb79, 5-oh - cb77, 6-oh - cb77), and the derivatization reagent (diazomethane) were all synthesized by the national institute of environmental health sciences (niehs) superfund basic research program center at the university of iowa. cb77 was synthesized from 3,4-dichlorobenzene boronic acid and 3,4-dichlorobromobenzene using the suzuki coupling reaction (18). the 4-oh - cb79 metabolite was synthesized using the suzuki coupling of 3,4-dichlorobenzene boronic acid and 2,6-dichloro-4-bromoanisole, followed by demethylation of the methoxy pcb with boron tribromide (19). the 5-oh - cb77 and 6-oh - cb77 metabolites were prepared using the cadogan coupling of 3,4-dichloroaniline with 2,3-dichloroanisole and subsequent demethylation with boron tribromide (20). all four compounds were > 99% pure according to gas chromatographic analysis (based on relative peak area). a solution of diazomethane in diethylether was synthesized from n - methyl - n - nitroso - p - toluenesulfonamide (diazald) using an aldrich mini diazald apparatus (milwaukee, wi) following manufacturer instructions. the synthesization and use of diazomethane must be completed in a fume hood because of high toxicity. stock solutions of cb77 and hydroxy - pcbs at 1 mg ml as compounds were prepared in acetone. working solutions were prepared by gradual dilution of the stock solution. because of no commercial methylsulfonyl - cb77 standards available, 3-methylsulfonyl-2,2,5,5-tetrachlorobiphenyl (3-meso2-cb52) and 4-methylsulfonyl-2,2,5,5-tetrachlorobiphenyl (4-meso2-cb52) with the same amounts of chlorine substitutions as cb77 were purchased from accustandard as references. internal standard 2,2,3,4,4,5,6,6-octachlorobiphenyl (cb204, iupac number) was purchased from cambridge isotope laboratories, inc. all standards and solutions were stored hermetically in amber glass vials at room temperature in the dark. silica gel (70230 mesh, fisher scientific, inc.) two kinds of cleanup silica gel (concentrated and 90% sulfuric acid silica gel) were prepared by blending 100 g of activated silica gel with 50 g of concentrated h2so4 and 50 g of 90% (w / w) h2so4, respectively. acetone (hplc grade), methyl tert - butyl ether (mtbe) (hplc grade), and hexane (pesticide grade) were purchased from fisher scientific. all other chemicals and reagents used in this experiment were of analytical reagent grade or higher purity. cuttings from male clones of the adult imperial carolina hybrid poplar tree (populus deltoides nigra, dn34) and switchgrass (panicum vigratum, alamo) were used in this study. each cutting was fit snugly with a predrilled screw cap and predrilled ptfe - faced septum. then, the cuttings were grown hydroponically in half strength hoagland nutrient solution (21). after 25 days, healthy whole poplar plants were selected to carry out the experiment. seeds of switchgrass were germinated on a bed of perlite with 1/10 strength hoagland solution. the grasses were transferred into half strength hoagland solution (without perlite) when they grew 3 cm high. after enough biomass developed, bunches of grass (five to six individual grasses in a bunch) were grown in 500 ml glass jars for 1 week and then used for exposure. the exposure reactors were 500 ml glass screw top conical flasks for poplars, 500 ml glass screw top jars for switchgrass, which have a sampling port with a ptfe - faced silicon septum and predrilled screw cap. hoagland nutrient solution was made by using autoclaved deionized water saturated with oxygen by bubbling with compressed air flowing through a sterilized 0.20 m filter to prevent microbial contamination. then, each autoclaved reactor was filled with 400 g of nutrient solution and a suitable amount of cb77. each reactor was planted with 56 switchgrass plants or a single 8 in. individual poplar plant. three of nine poplar plants with shoots cutoff acted as the excised poplar controls, three of them were air - dried to act as dead poplar controls, and the remaining three were autoclaved before exposure to create autoclaved poplar controls. an untreated whole poplar control and an untreated switchgrass control, without cb77 in the hydroponic solutions, were used to detect any pcb contamination emanating from laboratory air during the experiment. all reactors were wrapped with aluminum foil, which supported roots in the dark environment and eliminated photolysis of cb77. the temperature during plant growth was maintained at 23 1 c, and the photoperiod was 16 h a day under fluorescent lighting with a light intensity between 120 and 180 mol m s. transpiration of whole plants was determined daily by weighing the reactors. oxygen - saturated, autoclaved deionized water was injected into the reactors to replace transpiration losses. roots and hydroponic solutions of whole plants and controls were sampled and analyzed after 1 and 5 day exposures. the extraction, separation, and cleanup procedures for pcbs, hydroxy - pcbs, and methylsulfonyl - pcbs were modified from the method for plasma (22). in brief, the hydroponic solutions were amended with 10 drops of 37% hcl and 5 ml of 2-propanol, and then extracted with hexane / mtbe (1:1, v / v) for 30 min twice. roots samples were mixed with 2 ml of 37% hcl and 5 ml of 2-propanol, homogenized, and extracted with 3 ml of hexane / mtbe (1:1, v / v) per gram of sample under vigorous shaking overnight. the hexane phase was partitioned with 500 l of naoh solution (0.5 m in 50% ethanol) and transferred to a clean vial after phase separation. the combined hexane extract contained pcbs, methylsulfonyl - pcbs, and any other neutral metabolites (neutral fraction), while hydroxy - pcbs were left in alkaline solution. the alkaline solution was acidified with 125 l hcl (2 m) and extracted with 1 ml of hx / mtbe (9:1, v / v) twice. the extract was amended with a few drops of methanol and 0.5 ml of diazomethane to derivatize the oh - pcb to meo - pcb. the derivatization reaction was performed in a refridgerator at 48 c for 3 h. excess diazomethane was evaporated under a gentle nitrogen stream. then, the derivatization products, methyoxy pcb, were cleaned with acid silica gel column a (1 g of concentrated sulfuric acid silica gel with 0.1 g activated silica gel at the bottom) and eluted by 10 ml of dichloromethane. the neutral fraction was partitioned with 0.5 ml of anhydrous dimethyl sulphoxide (dmso) for 10 min. the dmso phase containing methylsulfonyl - pcbs was then mixed with 1 ml of water and extracted with 3 ml of hexane. the pcb fraction was cleaned by silica gel column a with 10 ml of dichloromethane for elution. the methylsulfonyl - pcbs were cleaned by silica gel column b (0.5 g anhydrous na2so4 on top, 0.5 g of 90% sulfuric acid silica gel in the middle, and 0.1 g of activated silica gel at the bottom) and eluted by 15 ml of dichloromethane. eluates were evaporated to dryness and dissolved in hexane for gas chromatograph (gc) analysis. qualitative and quantitative analysis of hydroxylated and methylsulfonyl metabolites was performed on an agilent 6890 gas chromatograph equipped with split detector system, gc / ms / ecd. analytes were separated in a hp-5 fused silica capillary column (5% phenyl methyl siloxane, 60.0 m 250 m 0.25 m), and then split to a mass spectrometry and electron capture detector. the gc was set as follows : injection port, 270 c with splitless mode ; high purity helium carrier gas at a constant flow rate of 1.9 ml min ; ecd, 360 c ; 95% argon and 5% methane makeup gas flow at 30 ml min ; electron impact (ei) ionization ms source, 250 c, scan from m / z 50 to 500. the oven program started at 75 c and was held for 3 min, then was increased at 5 c min to 150 c and held for 1 min, and then heated at 2 c min to 300 c and held for 5 min. identification and quantification of daughter pcbs derived from dechlorination were performed by an agilent 6890n gas chromatograph coupled to a waters micromass quattro micro gc mass spectrometer (gc / ms / ms) (milford, ma) operating under electron impact (ei) positive mode at 70 ev and multiple reaction monitoring (mrm), and the trap current was 200 a. the gc was equipped with an agilent 7683 series autosampler (23). analytes were separated in a supelco spb - octyl capillary column (30 m 250 m 0.25 m) with helium at a constant flow rate of 0.8 ml min. the oven temperature was programmed from 75 c and held for 5 min, heated to 150 at 15 c min and held for 1 min, and then heated to 280 at 2.5 c min and held for 3 min. the mass recoveries and reproducibility of cb77 and 6-oh - cb77 were investigated by measuring the blank aqueous concentrations and root samples spiked with standards. results in table 1 show that the analytical method worked well without the occurrence of chemical or biological transformations during sample analysis. identification of the hydroxy metabolites of cb77 in plant roots and nutrient solutions was based on a comparison of the retention times and mass spectra of derivatives of analytes (in samples) to those of the standards. the derivatives of hydroxy - cb77, 6-meo - cb77, were coeluted with 4-meo - cb79, but they were separated from 5-meo - cb77 on the hp-5 capillary column. according to their mass spectra, they all have the molecular ion [m ] at m / z 322 but have different fragmentation patterns due to the different positions of methyoxy substitutions. the 6-oh - cb77 metabolite was identified in roots and nutrient solutions of exposed whole poplar plants and excised poplar controls, but it was not found in autoclaved poplar controls, dead poplar controls, or untreated whole poplar controls (table 2). the derivative of 6-oh - cb77, 6-meo - cb77 (figure 1), has a visible molecular ion and typical fragmentation clusters surrounding ions at m / z 267 [m - ch3-cl ] and 207 [m - coch3 - 2cl ] in the mass spectrum, and the ions around 207 possess the highest abundance. there were no hydroxy - pcb metabolites detected in whole switchgrass, indicating that switchgrass was not able to hydroxylate cb77 during the experimental period. all metabolites were identified and quantitatively analyzed by gc / ms / ecd and gc / ms / ms.. coeluted pcb congeners that were not able to be separated by the used gc column. chromatogram of 6-meo - cb77 after derivitization from 6-oh - cb77 in a root sample, detected by the split detector system (gc / ms / ecd). compound corresponds to the peaks highlighted in panel a and b. the autoclaved poplar controls were dead plants, minimizing the action of microbes and eliminating the role of plant enzymes. in the unautoclaved the metabolite 6-oh - cb77 was not detected in either control, but it was detectable in the live poplars, suggesting that hydroxylation in poplars was caused by the plant itself, rather than by microorganisms in the root zone. excised poplar controls had no significant evapotranspiration, but the plant tissue was viable during the exposure period. the 6-oh - cb77 metabolite was detected in the excised controls, and the concentration was similar to that in exposed whole poplars, indicating that transpiration was not required for metabolic transformation of cb77 in plant tissues. hydroxylation occurred very rapidly in poplars with 6-oh - cb77 detected in roots and solutions after 1 day exposure. the concentrations of 6-oh - cb77 increased slightly over time but without a significant difference between 1 and 5 day exposures, indicating that further transformation in poplars is possible if the exposure period was lengthened. there was no significant difference between the concentrations of hydroxy metabolites under different dosages, although the lower dosage of cb77 resulted in slightly higher concentrations of metabolites. other researchers reported that cb77 at a low dose was metabolized more rapidly than that at high dose in scup (7). the mass and concentrations of hydroxylated metabolites in roots were far higher than those in nutrient solution. it is concluded that small amounts of 6-oh - cb77 exuded from roots into the rhizosphere compartment during the exposure period. pcbs can be biotransformed to a multitude of sulfur - containing pcb metabolites, including thiol, methylthio-, and methylsulfonyl - pcbs under the function of cyp and gst enzymes in animals via forming glutathione conjugates of the pcbs (1,2426). most studies have focused on methylsulfonyl - pcbs (2527), which have been found in various tissues of mammals and fish (28). methylsulfonyl - cb77 and two possible sulfur - containing intermediates (figure 2) are of concern in this study. because the methylsulfonyl metabolites of cb77 are not commercially available, two standards of methylsulfonyl - cb52 with the same degree of chlorine substitutions as cb77 were selected as references. similar mass spectra were detected in which typical molecular ion [m ] at m / z 370 and fragmentation clusters surrounding at m / z 307 [m - ch3-ccl ], 279 [m - cso2ch3 ], and 184 [m - so2ch3 - 3cl ] were displayed. taking account of the possibility of various mass spectrum patterns of methylsulfonyl - cb77, molecular ions were powerful fragments to identify meso2-cb77. however, no similar mass spectra were found in the exposed poplar and switchgrass samples. there were also no molecular ions of cb77-thiol and methylthio - cb77 in any mass spectra of plant samples. it appears that neither poplar trees nor switchgrass are able to metabolize cb77 to methyl sulfones in 5 days. according to the literature (2,3,29), the structure of cb77, without not only vicinal 3,4 hydrogen substitutions but also 2,5-dichloro- or 2,3,6 trichloro - phenyl rings, makes it difficult to biotransform to methyl sulfones. various microbial dechlorination products of pcbs have been observed in anaerobic sediments (10). magee. found that the crude extract of nitrate reductase from leaves of the plant, medicago sativa, was capable of dechlorination of cb153 by observing the decrease of cb153 after its incubation with the plant crude extract (30). anaerobic microbial dechlorination was avoided in the experiments of this study by autoclaving the reactors and solution, saturating the hydroponic solution with oxygen at the beginning of the exposure and replacing the transpiration loss with sterilized and oxygen saturated water. thus, the hydroponic solution was aerobic during the short period of exposure (1 and 5 days). a large proportion of the exposed whole plants were observed to contain significant concentrations of the metabolites of 2,3,5,6-cb (cb73), 2,2,5,5-cb (cb52), 3,3,4-cb (cb35), 2,3,3-cb/2,4,4-cb (cb20/28), 4,4-cb (cb15), and 4-cb (cb3) (cb20 and 28 are coeluted congeners on the supelco spb - octyl capillary column. both of them have the possibility of occurrence.). for various controls, cb3 and 35 were the congeners detected most frequently. the concentrations of cb35 in autoclaved poplar control roots (with minimized roles of microbes and without the role of poplars) were far lower than that in dead poplar roots (with the presence of microbes but without viable poplar tissue) indicating that the symbiotic association of microbes in the root zone of the plant aid in the formation of cb35. the concentrations of cb35 in dead poplar roots were similar to that in excised poplar roots and exposed poplar roots (all with functioning root - associated microbes and poplar roots) at a lower dosage for 5 days, indicating that viable poplar tissue is not necessary for the formation of cb35. however, by comparison of the exposed whole poplars to the excised, dead, and autoclaved poplar controls, the formation of cb3, 15, 20/28, 52, and 73 were due to the role of the viable poplar (plant) tissues on dechlorination of the parent compound cb77. according to the daughter pcbs detected in the reactors, the dechlorination of cb77 progressed from the tetra - chlorine (3,3,44-cb) congener to the di-(4,4-cb) congener to the mono - chlorine (4-cb) congener. in addition, tetra - chlorine congeners, cb73, cb52, and trichlorine congeners cb20/cb28 were detected by gc / ms / ms, which were presumed to be the products of chlorine rearrangement of cb77 during metabolism by plants. the chlorine shift was suggested by our experiment in which a relatively high concentration of cb73 was detected after a whole poplar plant was exposed to cb52 for 5 days. the rearrangement of chlorines on the biphenyl rings was clearly observed in this study, although the mechanism is not understood. the concentrations of the dechlorination products and chlorine - shift metabolites in switchgrass roots were lower than those in exposed poplar roots. this suggests that the enzymatic dechlorination and rearrangement capability of poplar is better than that of switchgrass. generally, meta- and para - chlorines in pcb congeners are more susceptible to dechlorination (31). the 2,3,4,3,4-cb congener would biodegrade following the sequence as 2,3,4,3,4-cb, 2,4,3,4-cb, 2,4,3-cb, 2,3-cb, and 2-cb (32,33). intermediates following dechlorination of cb77 by a palladium / iron nanoparticles system were determined to be (a) from 3,3,4,4-cb (cb77) to 3,3,4-cb (cb35), 3,3-cb (cb11), 3-cb (cb2), and biphenyl ; (b) from 3,3,4,4-cb to 3,4,4-cb (cb37), 4,4-cb (cb15), 3,4-cb (cb12), 3,4-cb (cb13), 4-cb (cb3), and biphenyl (34). although the metabolism of pollutants in vivo is very complex and requires a wide range of enzymatic capability, a proposed mechanistic scheme (figure 3) is shown for how poplars and associated microbes might interact to affect the observed transformation products from this study. all of the compounds shown in figure 3 were confirmed by gc / ms / ms and gc / ms / ecd. the rearrangement of chlorines during metabolism of cb77 in poplar was suggested by the metabolites detected, cb52 and cb73 (figure 3, compounds in the dotted square), during our experiments. the para / meta and meta / ortho chlorine shifts are hypothesized in the transformation from cb77 to cb52, from cb77 to cb73, from cb52 to cb73, and from cb35 to cb20. the meta / ortho shift was reported in the formation of pcdfs from pcb pyrolysis (35,36). however, these chlorine rearrangements have not been previously reported in microbial, mammal, or plant degradation studies of pcbs. metabolites in the dotted square were also positively confirmed by gc / ms / ms in the poplar roots. our experiment showed that relatively high concentrations of cb15 and cb3 were detected after a whole poplar plant was exposed to cb28 for 5 days. taking the daughter polychlorinated biphenyls in the reactors into account, we established the stepwise dechlorination pathway from cb28 to cb15 to cb3. however, the key intermediate, cb37, which should have occurred in the dechlorination from cb77 to cb15, was not detected. possible hypotheses for the observed lack of cb37 are the following : (i) cb37 disappeared very quickly due to further transformation, including the formation of cb15 by dechlorination. (ii) two meta chlorines on coplanar cb77 are of the same bond length, bond energy, and charge, which makes these two chlorines depart easily and form cb15 directly. (iii) cb77 was metabolized to cb28 directly under an enzymatic mechanism by poplar, which might involve dechlorination and rearrangement in one step. there are two reported hydroxylation pathways for introducing hydroxyl substitution to the aromatic ring of pcbs (1). for example, cb77 could be metabolized to a 5,6-epoxide, which would form both 5-oh - cb77 and 6-oh - cb77. the second way is via an enzymatic hydroxyl substitution to a carbon on the aromatic ring directly to form the corresponding hydroxylated metabolite. these two hydroxylation pathways could be involved in poplars, but only the 6-oh - cb77 was detected in this paper. | polychlorinated biphenyls (pcbs) are widely distributed persistent organic pollutants. in vitro research has shown that plant cell cultures might transform lower chlorinated congeners to hydroxylated pcbs, but there are few studies on in vivo metabolism of pcbs by intact whole plants. in this research, poplar plants (populus deltoides nigra, dn34) and switchgrass (panicum vigratum, alamo) were hydroponically exposed to 3,3,4,4-tetrachlorobiphenyl (cb77). metabolism in plants occurred rapidly, and metabolites were detected after only a 24 h exposure. rearrangement of chlorine atoms and dechlorination of cb77 by plants was unexpectedly observed. in addition, poplars were able to hydroxylate cb77 and the metabolite 6-hydroxy-3,3,4,4-tetrachlorobiphenyl (6-oh - cb77) was identified and quantified. hybrid poplar was able to hydroxylate cb77, but switchgrass was not, suggesting that enzymatic transformations are plant specific. sulfur - containing metabolites (from the action of sulfotransferases) were investigated in this study, but they were not detected in either poplar or switchgrass. |
the most frequently used neck disability questionnaires are the neck pain and disability scale (npad) and neck disability index (ndi), which are validated in several languages [4, 16, 21, 22, 38 ]. to evaluate the effect of treatment programs for neck disorders it is necessary that questionnaires are responsive, i.e., have the ability to detect clinical important changes over time. there is a need to define minimum changes in scores on questionnaires that are relevant from patients-, clinicians- or socioeconomic perspectives. to determine relevant change two concepts of interpretability are described [1, 3, 8, 10, 34 ]. in a distribution - based approach the statistical characteristics of the sample are used to express the observed change in a standardized metric [8, 10, 34 ]. the most commonly used measure is the minimal detectable change (mdc) [3, 57, 9, 10, 16, 22, 29, 32, 36, 38, 40 ]. the mdc assesses the minimal magnitude of change required to be confident that the observed change reflects real change and not measurement error [1, 8, 10, 30, 34 ]. a major limitation of distribution - based approaches is that they are statistical measures which by themselves do not provide a good indication of the clinical relevance of the observed change [8, 10, 34 ]. the anchor - based approach assesses which change on a questionnaire corresponds with an important change defined on an external criterion or anchor [for example global perceived effect (gpe) ] [8, 10, 17, 34 ]. the most common method in this approach is the calculation of the minimal important change (mic) determined by the receiver operator characteristic (roc) curve cut - off point [68, 10, 23, 29, 33, 34, 40 ]. a major limitation of the anchor - based approach is the absence of a gold standard for the external criterion. a further limitation is that it does not take measurement precision into account and therefore does not necessarily imply statistical significance [8, 10, 34 ]. hence, studies which apply both approaches are relevant for clinicians and researchers [8, 10, 34 ]. moreover, there is a need of studies that assess relevant changes and compare the responsiveness of neck disability questionnaires applied at the same time to the same sample of patients using the same methods to investigate which questionnaire is most appropriate. there are no studies assessing the mdc and the mic as concepts of interpretability of relevant change for both npad and ndi. the aim of this study was to investigate relevant change on the npad and ndi and to investigate which questionnaire is most responsive in a single sample of patients with non - specific chronic neck pain (cnp) in an outpatient tertiary rehabilitation setting. patients with cnp were recruited from referrals from general practitioners or medical specialists for diagnostic procedures as well as advices and rehabilitation treatment in a tertiary university center for rehabilitation in the netherlands. to be admitted for a multidisciplinary pain rehabilitation, patients had to agree with the time - contingent approach to restore activities and to facilitate return to work. inclusion criteria for this study were non - specific cnp (> 3 months duration), admitted for outpatient rehabilitation, age between 18 and 65 years, and sufficient knowledge of the dutch language to complete questionnaires. neck pain was labeled as non - specific or mechanical when the neck pain was produced or aggravated by neck movements or sustained neck postures and no specific underlying pathology could be established [2, 13 ]. exclusion criteria were status post neck surgery, co - morbidity severely diminishing physical or mental capacity, pregnancy, addiction to drugs, and extensive psychological or behavioral problems. specific neck pain and exclusion criteria were assessed based on clinical examination with help of red flags and orange flags and based on the information of the referrals [25, 26, 31 ]. prior to the first visit (t0) a questionnaire to assess patient and clinical characteristics was filled out. during t0 a review of the medical history and a physical examination was performed. a second visit (t1) was scheduled, prior to the start of the multidisciplinary rehabilitation program. during t1 the patients filled out the npad and ndi. after completion of the program varying from 3 to 5 months (t2), patients filled out the npad, ndi, and the gpe. data were gathered as part of care as usual between november 2006 and october 2010. each item has a vas of 100 mm with numeric anchors at 0, 1, 2, 3, 4, and 5 (each 20 mm apart). item scores range from 0 (no pain or activity limitation) to 5 (as much pain as possible or maximal limitation). higher scores indicate greater disability. the npad has shown to be a reliable and valid measure of disability in different languages [4, 16, 18, 19, 21, 22, 38 ]. each item has six different assertions expressing progressive levels of pain or limitation in activities. item scores range from 0 (no pain or limitation) to 5 (as much pain as possible or maximal limitation). the ndi has shown to be a reliable and valid measure of disability in different languages [6, 7, 1822, 29, 36, 38, 40 ]. for the gpe patients were asked to rate their overall perception of change since beginning treatment ranging from 3 (completely recovered) to zero (no change) to 3 (worse than ever). the reliability of the gpe was moderate to good in patients with neck pain and chronic arthritis [14, 24 ] and the validity was fair to moderate in patients with neck pain [6, 7, 24, 29, 40 ]. patients were considered improved when they scored completely recovered (3) or much recovered (2) and stable when they scored slightly recovered (1) no change (0) or slightly worsened (1). baseline (t0 and t1) variables were compared between these groups using t tests for independent samples and chi - square tests for categorical data. mdc was calculated as 1.96 2 standard error of measurement (sem). the sem was calculated in stable patients as sd (1 r) where r is the test retest reliability coefficient expressed in icc value and sd is the standard deviation of the baseline scores [30, 34 ]. roc curves were constructed to determine mic for npad and ndi [11, 30 ]. the roc cut - off point was calculated by identifying the point on the roc curve nearest to the upper left - hand corner, which is considered to be the best cut - off for which the sum of the percentages of false positives and false negatives classifications ([1 sensitivity ] + [1 specificity ]) is smallest. responsiveness was assessed by examining areas under roc - curve (auc) and correlations between change scores of npad and ndi, and gpe. auc was obtained to describe the ability of the npad and ndi to distinguish improved patients from stable patients. auc of 0.50 indicates the questionnaire has no diagnostic accuracy beyond chance, whereas a value of 1.00 would indicate perfect accuracy. auc of at least 0.70 was considered adequate. a visual method called anchor - based mic distribution method was used to integrate anchor - based and distribution - based approaches. for the improved and stable group the distribution of the change scores on the npad and ndi were depicted in a graph [11, 12 ]. all statistical analyses were performed with spss software, version 18.0. the critical value for significance was p 3 months duration), admitted for outpatient rehabilitation, age between 18 and 65 years, and sufficient knowledge of the dutch language to complete questionnaires. neck pain was labeled as non - specific or mechanical when the neck pain was produced or aggravated by neck movements or sustained neck postures and no specific underlying pathology could be established [2, 13 ]. exclusion criteria were status post neck surgery, co - morbidity severely diminishing physical or mental capacity, pregnancy, addiction to drugs, and extensive psychological or behavioral problems. specific neck pain and exclusion criteria were assessed based on clinical examination with help of red flags and orange flags and based on the information of the referrals [25, 26, 31 ]. prior to the first visit (t0) a questionnaire to assess patient and clinical characteristics was filled out. during t0 a review of the medical history and a physical examination was performed. a second visit (t1) was scheduled, prior to the start of the multidisciplinary rehabilitation program. during t1 the patients filled out the npad and ndi. after completion of the program varying from 3 to 5 months (t2), patients filled out the npad, ndi, and the gpe. data were gathered as part of care as usual between november 2006 and october 2010. each item has a vas of 100 mm with numeric anchors at 0, 1, 2, 3, 4, and 5 (each 20 mm apart). item scores range from 0 (no pain or activity limitation) to 5 (as much pain as possible or maximal limitation). higher scores indicate greater disability. the npad has shown to be a reliable and valid measure of disability in different languages [4, 16, 18, 19, 21, 22, 38 ]. each item has six different assertions expressing progressive levels of pain or limitation in activities. item scores range from 0 (no pain or limitation) to 5 (as much pain as possible or maximal limitation). higher scores indicate greater disability. the ndi has shown to be a reliable and valid measure of disability in different languages [6, 7, 1822, 29, 36, 38, 40 ]. for the gpe patients were asked to rate their overall perception of change since beginning treatment ranging from 3 (completely recovered) to zero (no change) to 3 (worse than ever). the reliability of the gpe was moderate to good in patients with neck pain and chronic arthritis [14, 24 ] and the validity was fair to moderate in patients with neck pain [6, 7, 24, 29, 40 ]. patients were considered improved when they scored completely recovered (3) or much recovered (2) and stable when they scored slightly recovered (1) no change (0) or slightly worsened (1). baseline (t0 and t1) variables were compared between these groups using t tests for independent samples and chi - square tests for categorical data. mdc was calculated as 1.96 2 standard error of measurement (sem). the sem was calculated in stable patients as sd (1 r) where r is the test retest reliability coefficient expressed in icc value and sd is the standard deviation of the baseline scores [30, 34 ]. roc curves were constructed to determine mic for npad and ndi [11, 30 ]. the roc cut - off point was calculated by identifying the point on the roc curve nearest to the upper left - hand corner, which is considered to be the best cut - off for which the sum of the percentages of false positives and false negatives classifications ([1 sensitivity ] + [1 specificity ]) is smallest. responsiveness was assessed by examining areas under roc - curve (auc) and correlations between change scores of npad and ndi, and gpe. auc was obtained to describe the ability of the npad and ndi to distinguish improved patients from stable patients. auc of 0.50 indicates the questionnaire has no diagnostic accuracy beyond chance, whereas a value of 1.00 would indicate perfect accuracy. auc of at least 0.70 was considered adequate. a visual method called anchor - based mic distribution method was used to integrate anchor - based and distribution - based approaches. for the improved and stable group the distribution of the change scores on the npad and ndi were depicted in a graph [11, 12 ]. all statistical analyses were performed with spss software, version 18.0. the critical value for significance was p < 0.05. during the recruitment period 391 patients with cnp were referred to the center for rehabilitation. a total of 129 patients, of which 4 were with status post neck surgery, were admitted for multidisciplinary outpatient rehabilitation. a total of 125 patients fulfilled inclusion criteria for this study. during the waiting period 14 patients decided not to start with the rehabilitation program because of practical reasons unrelated to the study. after the start of the rehabilitation program 35 patients decided not to continue because of lack of further interest or practical reasons. the clinical characteristics of these patients and of the 35 dropouts are presented in table 1. after the rehabilitation program 6 patients were completely recovered as assessed with gpe, 39 much recovered, 17 slightly recovered, 10 no change, 3 slightly worsened, 0 much worsened and 1 worse than ever. in total 45 (60 %) patients were labeled as improved and 30 (40 %) patients as stable. baseline differences between improved and stable patients were non - significant, as were baseline differences between improved and stable patients on the one hand and dropouts on the other hand. more male patients dropped out than female patients.table 1baseline characteristics of improved and stable patients, and dropoutsimproved patients (n = 45)stable patients (n = 30)dropouts (n = 35)age (years)37.7 (12.3)39.5 (12.0)39.2 (10.1)duration of chronic pain (months)18.5 (9.358.5)24.0 (9.069.0)18.0 (7.548.0)sick leave in the past year (weeks)18.5 (19.4)16.1 (17.6)17.5 (20.6)ndi (050)21 (5.5)21 (8.1)23 (8.6)npad (0100)50 (12.3)53 (16.5)56 (20.0)vaspain (0100)52 (20.1)52 (18.6)54 (24.8)female (%) 677749pain radiating to (%) shoulder(s)828386 upper arm(s)514054 forearm(s)332046 hand / fingers271740 between shoulder blades475350pins and needles below elbow (%) 363338concomitant complaints (%) headache846371 dizziness362735 concentration problems291712 nausea111312 fatigue695362 low back pain333149self reported cause of neck pain (%) motor vehicle accident564037 other trauma91317 spontaneously / unknown779 stress476 work related91014 other162317previous treatment for neck pain (%) 919394education low700 intermediate697982 high242118work status (self employed / employee) (%) 4/7810/8017/60involved in litigation (%) 402729values are means (sd) unless otherwise indicatednpad neck pain and disability scale, ndi neck disability index, vas visual analog scalemedian and interquartile range for duration of pain (months) baseline characteristics of improved and stable patients, and dropouts values are means (sd) unless otherwise indicated npad neck pain and disability scale, ndi neck disability index, vas visual analog scale median and interquartile range for duration of pain (months) the results for npad and ndi at baseline and follow - up and the change scores in the stable and improved groups are shown in table 2. the icc values calculated for stable patients were 0.52 (95 % ci 0.330.67) for npad and 0.86 (95 % ci 0.790.91) for ndi. the sem values of the stable patients were 11.4 for npad and 3.0 for ndi. these values resulted in mdc values of 31.7 points for npad and 8.4 for ndi.table 2baseline, follow - up and mean change scores of npad and ndi for the total, improved, and stable group of patientsbaseline mean (sd)follow - up mean (sd)change mean (sd)95 % cip valuenpad total (n = 76)51 (14.0)36 (19.0)15 (17.4)11.119.2<0.001 improved (n = 45)50 (12.3)29 (17.3)21 (16.1)15.725.7<0.001 stable (n = 30)53 (16.5)46 (16.9)7 (16.2)0.313.10.04ndi total (n = 76)21 (6.6)15 (7.2)6 (5.9)4.27.0<0.001 improved (n = 45)21 (5.5)13 (6.2)8 (6.3)5.79.6<0.001 stable (n = 30)21 (8.1)18 (7.7)3 (4.2)1.24.40.001npad neck pain and disability scale, ndi neck disability indexone patient scored worse than ever and was not included in the improved or stable group, but was included in the total group baseline, follow - up and mean change scores of npad and ndi for the total, improved, and stable group of patients npad neck pain and disability scale, ndi neck disability index one patient scored worse than ever and was not included in the improved or stable group, but was included in the total group roc curves for npad and ndi are presented in fig. the roc cut - off mic was for npad 11.5 points (sensitivity 0.74 ; specificity 0.70) and for ndi 3.5 points (sensitivity 0.74 ; specificity 0.66). changes should exceed these values of mdc and mic cut - offs (31.7 and 11.5 for npad and 8.4 and 3.5 for ndi) to be interpreted as relevant. the auc for npad was 0.75 (95 % ci 0.620.87) and for ndi 0.75 (95 % ci 0.640.87). the correlation between change scores of npad and ndi, and gpe were, respectively, 0.48 (95 % ci 0.290.64) and 0.49 (95 % ci 0.300.64).fig. 1receiver operator characteristic (roc) curves of npad and ndi change scores receiver operator characteristic (roc) curves of npad and ndi change scores the anchor - based mic distribution graphs for npad and ndi are presented in fig. 2a and b. these figures illustrate the effect of using mic cut - off for change scores in the distribution of true and false positives and negatives. when the change score equals mic, 26 % of the anchor - based improved patients have a lower change score. they are considered false negatives because the sensitivity of npad and ndi = 0.74. when the change score equals mic, 30 % (npad) and 34 % (ndi) of the anchor - based stable patients have higher change scores. they are considered false positives because specificity of npad = 0.70 and of ndi = 0.66.fig. 2distribution of npad - change scores in anchor - based improved and stable patients with indication of mic at 11.5. at this point sensitivity = 0.74 and specificity = 0.70 (a). distribution of ndi - change scores in anchor - based improved and stable patients with indication of mic at 3.5. at this point sensitivity the gray parts of the improved and stable patients represent the true positives (dark gray) and the true negatives (light gray), respectively. mdc is indicated at 31.7 for npad and at 8.4 for ndi distribution of npad - change scores in anchor - based improved and stable patients with indication of mic at 11.5. at this point sensitivity = 0.74 and specificity = 0.70 (a). distribution of ndi - change scores in anchor - based improved and stable patients with indication of mic at 3.5. at this point sensitivity the gray parts of the improved and stable patients represent the true positives (dark gray) and the true negatives (light gray), respectively. this study demonstrated that relevant change on both npad and ndi assessed with mdc or mic resulted in different cut - offs with different amounts of certainty that the patient is improved. furthermore, it demonstrated that the responsiveness of npad and ndi was similar when using the aucs and the correlations between change scores and the gpe. there is no consensus regarding the number of sems required to express statistically clinically relevant change : 1 sem, 1.65 sem or 1.9 sem. we used the 1.96 sem to correspond with 95 % ci. in the present study mdc for npad and ndi was 31.7 and 8.4, respectively. in a previous npad study [mean baseline score 39.8 (sd 23.3) ] the icc was 0.97, the sem 3.8 scale points, and follow up 12 weeks ; therefore, the mdc of 10.5 was low compared with the present study. the iccs of 0.52 for npad and 0.86 for ndi in the present study measured on stable patients was compared with the iccs of 0.76 for npad and 0.84 for ndi in a previous study in the same setting with a retest interval of 18 days. larger instability of the npad may be explained by differences in operationalizations of neck disability between items of the npad and the ndi [35, 37 ]. post hoc analysis showed that the amount of variation of the npad could be attributed to significant differences in seven individual items (2, 6, 812) of the questionnaire. with an icc of 0.76 for npad previous ndi studies report for mdc ranges between 1.7 and 13.4 [57, 29, 33, 36, 39, 40 ]. apart from different patient populations, the observed differences are most likely the result of different formula for the mdc calculation (1.96 or 1.65 2 sem) and large ranges in sem (0.604.4) in these ndi studies [57, 29, 33, 36, 39, 40 ]. mic is defined as the smallest change that is important to patients [8, 10, 17, 33, 34 ]. how to classify the smallest important change and patients as improved or stable with gpe scale levels, is an arbitrary decision [57, 10, 29, 36, 3840 ]. in most studies using gpe as external standard, a 15-point scale was used with 3 (moderately better) as cut - off to distinguish improved from stable patients [6, 7, 29, 39, 40 ]. we classified patients as improved when their score completely recovered or much recovered to reflect important improvement similar to other studies [11, 29 ]. mic for ndi has been reported to range from 3.5 to 9.5 [57, 29, 33, 39, 40 ]. differences between these studies and the present study could be the result of several factors, such as different external criteria (prognostic estimate of change, health transition item of sf-36 and gpe by patient [6, 7, 29 ] or by patient and therapist), the number of scale levels of the external criteria, the combination of scale levels to form the improved and stable group, characteristics of population (such as age, nature and acuity of neck condition, patient setting, baseline scores), treatment, and period of follow up [57, 29, 39, 40 ]. the auc was used to determine the probability that the improved patient can be correctly distinguished from the stable patient. in this study npad and ndi both have an auc of 0.75 which is a satisfactory result and in line with results found by other studies (range 0.570.90) [6, 7, 22, 29, 32, 33, 39, 40 ]. the auc of 0.90 for the ndi was reported in a study using a prognostic estimate of change as external criterion made by clinicians at patient s initial visit. in one study responsiveness of npad and ndi clinicians should be aware of the fact that choosing either the mdc or the mic cut - off gives different values and amounts of certainty on whether the observed change is relevant. smaller values for the mic were observed in almost all neck pain studies including the present study [57, 29, 39, 40 ]. using the anchor - based mic the proportion of false positives and false negatives, the probability of false positives is reduced and the probability of false negatives is increased. applying the more conservative mdc, the certainty that the change score is relevant and larger than the measurement error, is high. the amount of certainty needed may depend on the consequences in patient care and could be a case by case decision. for example for risk, full neck surgery or an expensive time - consuming multidisciplinary rehabilitation the more conservative mdc cut - off could be used, while in primary care setting the more liberal mic cut - off could be used. on the other hand, socio - economic factors such as chance of returning to work as result of a therapy may be also of importance as external criterion for relevant change. in the present study, the visual anchor - based mic distribution method was used whereby the distribution of the change scores on the npad and ndi was depicted in curves. the narrower the curves and the smaller the overlap of the curves, the smaller the chance of misclassification. both aspects of the curves largely depended on the correlation between change scores of npad and ndi and gpe as anchor. in the present study these correlations were similar to those of most other npad (range 0.420.59) [16, 22, 38 ] and ndi (range 0.190.58) studies [6, 7, 22, 32, 3840 ]. the gpe as external criterion to operationalize relevant change has been criticized because it consists of only one question and patient s ability to recall their previous health status is questionable [15, 27 ]. any anchor - based approach is as good as the used external criterion and the methodology to define relevant change. the present study is conducted in a university setting and is therefore representative of patients with cnp in a tertiary referral center. percentage of females was similar to that of other responsiveness studies [47, 16, 21, 22, 29, 32, 33, 36, 38, 40 ]. mean age in our study was lower (38.5 years) compared with other responsiveness studies [47, 16, 21, 22, 29, 32, 33, 36, 38, 40 ]. a potential limitation of this study is that the sample consisted largely of patients with moderate neck pain and disability. although this may be expected in this tertiary rehabilitation setting generalizability beyond this setting can not be assumed. the dropouts did not introduce bias because this study was aimed to measure the questionnaires and not the effect of the rehabilitation program. the strength of this study is that relevant changes were assessed with mdc and mic on the npad and ndi and that a head - to - head comparison of the responsiveness of npad and ndi was performed. further study of mdc, mic, and responsiveness of npad and ndi is necessary to assess the measurement properties in other patient groups and also in comparison with other external criteria for relevant change. relevant change of both npad and ndi assessed with mdc and mic resulted in different cut - offs with different amounts of certainty that the patient is improved. this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. | purposeto investigate relevant change on the neck pain and disability scale (npad) and neck disability index (ndi) and which questionnaire is the most responsive in patients with non - specific chronic neck pain (cnp).methodsseventy - six patients with non - specific cnp in an outpatient tertiary rehabilitation setting were dichotomized into improved and stable based on global perceived effect (gpe) scores. to investigate relevant change minimal detectable change (mdc) and minimal important change (mic) with the receiver operator characteristic (roc) cut - off point were assessed. comparison of responsiveness was performed using areas under the roc curve (auc) and correlations between change scores of npad and ndi, and gpe.resultsmdc and mic on npad (scale 0100) were 31.7 and 11.5 points, respectively. mdc and mic on ndi (scale 050) were 8.4 and 3.5 points, respectively. changes should exceed this mdc or mic cut - off to be interpreted as relevant. auc was 0.75 for both npad and ndi. correlations between change scores of npad and ndi, and gpe were, respectively, 0.48 (95 % ci 0.290.64) and 0.49 (95 % ci 0.300.64).conclusionsrelevant change on both npad and ndi assessed with mdc and mic resulted in different cut - offs and consequently with different amounts of certainty that the patient is improved. responsiveness of npad and ndi was similar. |
peritonitis is the most troublesome complication in patients on continuous ambulatory peritoneal dialysis (capd). gram - positive organisms, especially staphylococcus aureus and staphylococcus epidermidis, are the most frequent causative pathogens the prevalence of vancomycin resistant enterococci (vre) in hospitalized patients is reported to be 0.4 - 14%. there are few agents with in vitro activity against pathogens that have developed resistance to vancomycin. newer drugs such as quinupristin, dalfopristin and linezolid show in vitro and in vivo activity against enterococci including vre. we report (i) a case of peritonitis, in a capd patient, caused by two different species of enterococci with a different antimicrobial susceptibility pattern, especially for vancomycin and (ii) the effectiveness of linezolid for the treatment of vancomycin resistant enterococcal peritonitis. a 63-year - old male was hospitalized in bp koirala institute of health sciences, dharan, nepal with the complaint of bilateral swelling of lower limbs for 7 days, clouding of capd fluid for 5 days and fever for 2 days. the patient, having history of stage 2 hypertension for 20 years and type 2 diabetes mellitus with nephropathy (with end stage renal disease) for 15 years, was on capd. in addition, he also had history of one episode of peritonitis and recurrent urinary tract infections in the past. gram staining of the capd fluid showed plenty of polymorphs with gram - positive cocci (gpc) in pairs and short chains. the organism in culture was presumptively identified as enterococcus sp ecies based on the colony morphology, absence of hemolysis, gram reaction, catalase negativity and bile esculin positivity. the isolate was further characterized with a set of biochemical tests using standard microbiological methods and identified as enterococcus faecalis. on performing antimicrobial susceptibility by kirby - bauer disc diffusion method in compliance with clinical and laboratory standards institute guidelines, the isolate exhibited sensitivity to ampicillin, azithromycin, linezolid, ofloxacin and vancomycin while it remained resistant to amikacin and ceftriaxone. the minimum inhibitory concentration (mic), performed by agar dilution method, of the isolate for vancomycin was 0.5 g / ml. the mic value confirmed its sensitivity to vancomycin. the patient was administered intraperitoneal vancomycin and was discharged after 5 days of admission. after a week of discharge, the patient was readmitted with no resolution of symptoms. gpc with few pus cells were observed in direct microscopy and isolate obtained this time was identified as enterococcus faecium. the isolate remained resistant to amikacin, ampicillin, azithromycin, ceftriaxone, ofloxacin and vancomycin while it was sensitive to linezolid.. following catheter removal, patient 's condition improved with the intravenous administration of linezolid and he was discharged from the hospital. first, in the previous two situations where the cultures were performed, two different species of enterococci were recovered, one species at a time, from the same patient. e. faecalis, recovered initially, was vancomycin sensitive while e. faecium, isolated in the second time, was resistant to vancomycin. second, the e. faecium isolate was not only resistant to vancomycin but also to other important group of antibiotics. the appearance of two different species of enterococci at two different times may be explained by a fact that the patient must already have harbored two different species of enterococci but only e. faecalis having the predominant role in infection for the first time. after administration of the drug, since e. faecalis was sensitive to vancomycin, e. faecium being resistant to vancomycin predominantly replaced e. faecalis under selective pressure and was recovered in the culture. vancomycin - resistant strains of enterococci are generally resistant not only to vancomycin but also to other antibiotics and can cause severe infections in compromised hosts. the e. faecium isolate in the above case was not only resistant to vancomycin, but also to amikacin, ampicillin, azithromycin, ceftriaxone and ofloxacin. the widespread and often inappropriate use of broad spectrum antibiotics in the hospital is recognized as an important contributing factor to the spread of resistance. the patient had frequent exposure to antibiotics such as aminoglycoside, cephalosporin and glycopeptide in the past. moreover, it has been documented that enterococci have shown to produce biofilm in the peritoneal catheters. dissemination of these strains from catheter into the peritoneum may induce recurrent peritonitis in pd patients and are refractory to routine antibiotic treatments. as observed in in vitro sensitivity of e. faecium isolate to linezolid, patient responded well with its intravenous administration. this fact highlights that newer drugs such as linezolid remain a good option for treatment of vancomycin resistant enterococcal peritonitis. the aforementioned case report emphasizes the importance of accurate identification or speciation of enterococci as the antimicrobial susceptibility pattern may differ among its various species. newer drugs such as linezolid remain a good treatment option for vancomycin resistant enterococcal peritonitis. however, prudent use of antimicrobials and strict adherence to the infection control practices are crucial not only in the effective management of enterococcal infection but also for the prevention of development and spread of resistance. | peritonitis in a continuous ambulatory peritoneal dialysis patient by two different species of enterococci is a rare condition. we report a case of peritonitis from which vancomycin sensitive enterococcus faecalis and vancomycin resistant enterococcusc faecium were isolated. it also emphasizes the effectiveness of linezolid for the treatment of vancomycin resistant enterococcal infection. |
obesity results from the accumulation of excess adipose tissue ; however, it is not a single disorder but a heterogeneous group of conditions with multiple causes. major causes of the increasing prevalence of obesity include behavioral and environmental factors, such as excessive consumption of energy - dense foods and a sedentary lifestyle. still, it is now recognized that a series of underexplored physiological and environmental predispositions underlies the traditional risk factors for obesity and its associated metabolic disorders. in this regards, gut microbiota has recently been proposed as an environmental factor responsible for the weight gain and the altered energy metabolism that accompanies the obese state. gut microbiota affects host metabolism by increased energy extraction, immune system modulation, and altered lipid metabolism. furthermore, both the physical presence of bacteria and the metabolites from bacteria are responsible for these effects. an evaluation of the evidence indicating that the gut microbiota plays a role in energy balance and obesity - associated diseases such as diabetes and cardiovascular diseases (cvds), as well as factors that mediate these hard endpoints, is presented in this paper. microbiota is a collection of microorganisms including bacteria, archaea, viruses, and some unicellular eukaryotes., it has been estimated that 10 microorganisms reside in various parts of the body such as the surface of skin and in the gastrointestinal, genitourinary, and respiratory tracts. the gastrointestinal tract, which has the largest number of microorganisms in humans, is comprised of specialized compartments such as the mouth, esophagus, stomach, small intestine, large intestine (colon), rectum, and anus. each of these compartments has unique physiological functions and anatomical structures. as a result, the chemical environment and habitable microorganisms differ tremendously in each compartment (figure 1). in the colon, up to 10 microorganisms this is by far the highest density found in humans [24 ], and the vast majority of microorganisms belong to the phyla of firmicutes, bacteroidetes, actinobacteria, and proteobacteria, with relatively lower numbers belonging to fusobacteria, verrucomicrobia, and tm7 [48 ]. fungi and archaea may also be resident [6, 9, 10 ], but comprise less than 1% of the total inhabitants. altogether, a human gastrointestinal microbiota comprises more than 10,000 different phylotype s, most of which have not been characterized by a culturing technique or even sampled to date [8, 11 ]. however, this notion has been challenged by a recent finding by goodman. who reported that 99% of the bacteria characterized in the phylum, class, and order level could also be found in the biomass from an anaerobic culture of original fecal samples. several studies reported by members of the gordon 's lab showed that the gut microbiota differs at the phylum level depending on weight status [7, 16, 17 ]. in agreement with results from animal studies, it appears that human obesity is associated with a low abundance of intestinal bacteroidetes and high abundance of firmicutes. duncan. showed that no difference was found in the proportions of bacteroidetes and firmicutes in the feces of lean and obese subjects. in another study, overweight and obese subjects had a ratio of bacteroidetes to firmicutes in favor of bacteroidetes. recently, jumpertz. applied the state - of - the - art pyrosequencing technique to examine the bacterial 16s rrna genes and reported that no phylum level difference was found in between the obese and lean fecal microbiota. therefore, the phylum level difference of the gut microbiota between obese and lean individuals might not be universally true. compositional changes of the human gut microbiota in response to weight change have been examined by many groups. monitored the fecal gut microbiota in 12 obese subjects participating in a weight - loss program by consuming restricted diets for a year. following weight loss, the proportion of bacteroidetes increased while the number of firmicutes reciprocally decreased. fecal microbiota compositions of overweight and obese adolescents also were determined in the evasyon study group. after 10 weeks of energy restriction and exercise, participants who lost more than 4 kg body weight showed a significant increase in the population of bacteroides fragilis group, as determined by a quantitative pcr technique. another evasyon cohort study also reported that an increase in bacteroides / prevotella group positively correlated with the amount of weight loss. to date, the most effective treatment for morbid obesity is gastric bypass surgery. the consequences of gastric bypass surgery in the composition of the fecal microbiota have been studied. zhang. found that obesity was associated with an increase of family prevotellaceae prior to the surgery, and following surgery prevotellaceae was reduced to the level of lean individuals whereas other bacteria such as family enterobacteriaceae and genus akkermansia were enhanced. in another clinical study, the number of fecal e. coli species was increased at 3 and 6 months after having gastric bypass surgery. the impact of gastric bypass surgery on the gut microbiota profile in animals was an increase in the proportion of enterobacter hormaechei by 200- and 42.8-fold at 2 and 8 weeks after the surgery, respectively. since both e. coli species and e. hormaechei belong to the family enterobacteriaceae, a proportional increase of enterobacteriaceae could be a common outcome of gastric bypass surgery. however, it is not known whether changes in enterobacteriaceae populations are linked to the rapid weight loss and improvement of insulin sensitivity resulting from gastric bypass surgery. obesity caused by a leptin mutation in mice (ob / ob) is associated with altered gut microbiota profiles [16, 17 ]. analyzed more than 5000 bacterial 16s rrna gene sequences from the cecal content of ob / ob mice, their lean ob/+ and + /+ siblings, and their ob/+ mothers. homozygous ob / ob mutation coincided with 50% fewer bacteroidetes and a proportional increase in firmicutes in the gut. the gut microbiota profile of mice with a leptin receptor mutation (db / db) has recently been published. in accordance to ob / ob mice, db / db mice cecal microbiota was characterized with higher abundance of phylum firmicutes and lower abundance of phylum bacteroidetes compared to the lean mice. furthermore, certain genera such as odoribacter, prevotella, and rikenella only present in the ceca of db / db mice, whereas enterorhabdus presents in the ceca of lean mice. however, it is possible that the alterations in the bacterial composition in ob / ob and db / db mice are secondary to hyperphagia. a careful pair - feeding experiment would be needed to show if the amount of food intake contributes to the signature of gut microbiota associated with genetic obesity in mice. while the host genotype has been proven to affect microbiota, the effect of diet, specifically dietary fat, also plays an important role in determining bacterial composition. a high - fat - fed animal displays a significant shift in both bacterial and metagenomic profiles as compared to an animal on a normal, chow diet. western diet - associated cecal microbiota is characterized with a reduction in the relative abundance of bacteroidetes and an increase in the relative abundance of firmicutes. in particular mollicutes, a class of firmicutes, has been found to be significantly more prevalent in conv mice fed a western - type diet. an increase of genes involved in the import and processing of sugars in the gut metagenome was also found in western diet - fed mice. examination of the role of the western diet - associated cecal microbiota in facilitating weight gain has revealed that ex - gf mice receiving the western diet - associated gut microbiota gained significantly more body fat than the mice receiving the chow diet - associated gut microbiota. murphy. reported that c57bl/6j mice responded to hf feeding with a progressive increase in abundance of firmicutes over time. ravussin. also reported that in weight - matched animals, high - fat feeding was associated with an increase in the family of lachnospiraceae and genera bacteroides and mucispirillum whereas low fat feeding was associated with an increase in genus allobaculum. the literature evidence further suggests that quality of the diet instead of the weight of animals is stronger modulator to the composition of the gut microbiota. it was found that diet explained 57% of the bacterial variation in the gut while genetic background only accounted for 12% of the variation in animals, which suggests the primacy of diet in determining the composition of the gut microbiota. hildebrandt. reported a similar finding in that mice deficient in relm gene had moderate impacts on the gut microbiota profile whereas high - fat feeding caused greater changes in the composition of the gut microbiota. metagenomic analysis of normal weight (relm deficient) and obese mice (wildtype) fed a high - fat diet found that the diet, rather than weight or genetic status, correlated with an increase in nutrient - transport genes and a decrease in amino acid and carbohydrate metabolism genes. these data support the influence of the diet composition in the diversity and profiles of the gut microbiota in mice ; however, the mechanism by which high - fat diets change the microbiota 's functionality requires further study. within the context of obesity, it appears that genetics may determine initial gut composition, but dietary fat is a potent modulator. the interaction between the gut environment and diet (modulated by bacterial composition) may explain why genetically identical mice respond differently to a high - fat diet some are prone and some resistant to weight gain. sprague - dawley male rats prone to weight gain exhibited ileal inflammation, decreased intestinal alkaline phosphatase activity (enzyme which detoxifies the bacterial component known to cause inflammation, lipopolysaccharide, or lps), and increased innate immune system activation in the luminal wall as compared to the obesity - resistant rats. both obesity - prone and obesity - resistant rats had an overall decrease in bacteria on the high - fat diet ; however, enterobacteriales increased in the obesity - prone rats on a high - fat diet. in a study of genetically identical male rats, infusing a low level of lps for 4 weeks caused the same amount of weight gain as a high - fat diet. rats that had a knockout of an immunoprotein (cd14), which is necessary to cause an inflammatory reaction to lps, were immune to weight gain. together the data show that rats naturally prone to weight gain on a high - fat diet have intestinal inflammation, inflammation alone can cause weight gain in normal rats, and the absence of inflammation protects rats against weight gain from a high - fat diet. therefore, it is hypothesized that the inflammatory mileu is integral in the development of obesity. a series of experiments comparing the germ - free (gf) to conventional (cv) mice concluded that the development of diet - induced obesity requires the colonization of complex gut microbiota [3436 ]. microbiota transplantation experiments showed that the accumulation of body fat depends on the type of the gut microbiota, which supports the role of the gut microbiota in the development of obesity [17, 27 ]. mechanistically, the acceleration of fat mass gain in conventionalized mice can be partly explained by the suppression of hepatic de novo lipogenesis and by the inhibition of triglyceride storage in the white adipose tissue. the latter effect is thought to be caused by an excessive production of fiaf (fasting - induced adipocyte factor or angptl4) in the intestine of the gf mice. fiaf inhibits lipoprotein lipase (lpl) thereby blocking the disassociation of fatty acids from triglycerides for uptake into tissues and upregulating fatty acid oxidation and uncoupling proteins, potentially reducing the amount of fat storage in gf mice. the importance of fiaf as a mediator by which the gut microbiota regulates body weight was demonstrated in gf fiaf - knockout mice. unlike the gf wild - type animals, gf mice lacking fiaf responded to a high - fat diet with excessive weight gains. although the function of fiaf in blocking lpl activity is clear, the extent to which fiaf from the intestine as compared to adipose tissue is causing this effect has not been elucidated. initial studies characterizing fiaf showed that it is located primarily in white and brown adipose tissue, as well as in the liver during fasting, and has a very low expression in small intestine. moreover, fleissner. found an increase in intestinal fiaf mrna in gf mice but no increase in secreted plasma protein levels compared to conv mice. in a global fiaf - knockout model, all fiaf including that in the adipose tissue and in the intestine is eliminated, which diminishes the ability to determine the tissue specific contribution of fiaf to the phenotype therefore, available evidence does not indicate that intestine - derived fiaf has a significant impact on regulating the triglyceride storage in the adipose tissue, and perhaps the lack of weight gain in gf mice is due to other mechanisms. determining the physiological contribution of intestinal fiaf as well as its regulation by specific bacterial populations and metabolites warrants further investigation. another mechanism by which the gut microbiota affects body weight is by increasing energy harvesting from dietary fibers. the intestinal microbiota breaks down indigestible polysaccharides (i.e., fiber) to short - chain fatty acids (scfas) providing 80 to 200 kcal per day or about 410% of daily energy intake in normal adults. metagenomic analysis of ob / ob cecal microbiota revealed that ob / ob - specific gut microbiota was enriched with bacterial genes capable of utilizing and fermenting dietary fibers. the greater cecal scfa concentrations of acetate and butyrate and lower fecal energy contents of ob / ob than in lean animals suggest additional absorption of scfa was absorbed by the intestine of the ob / ob mice. murphy. independently evaluated whether energy harvesting is different between ob / ob and lean mice and found a similar result to that obtained by turnbaugh. with 7-week - old mice but not at 15-weeks - old animals as older mice showed a similar amount of cecal scfas and fecal energy. the concept of changing energy harvesting by the gut microbiota has also been tested in humans. reported that the amount of stool energy in proportion of ingested calories was positively correlated with the abundance of phylum bacteroidetes and negatively correlated with the abundance of phylum firmicutes in the feces. an estimate of 150 kcal difference can be achieved with a change of 20% relative abundance of firmicutes and corresponding decrease of bacteroidetes in the stool of lean individuals. thus, excessive calories taken in the form of scfas from microbiota metabolism of fiber may be a contributing factor in the obese state. food intake and energy expenditure are two key factors that determine energy balance in humans and animals. in humans, the role of gut microbiota in food intake has not been tested, and in animals the results are inconclusive. as nicely summarized by wostmann, earlier work from the 1960s and 70s showed that food intake is lower in gf than in conv rats. bckhed. showed that gf c57bl/6j mice consumed more chow than conv mice ; however, no relationship was found between chow intake and the presence or absence of the gut microbiota in c3h mice (and personal communication with professor michael blaut). on a western diet, reported food intake in gf and conv mice was similar, regardless of their genetic background ([34, 40 ] and personal communication with professor michael blaut). on a semisynthetic high - fat diet, we observed that gf c57bl/6j mice consumed a less amount of food than conv mice in a 10-week feeding experiment, but the same relationship was not observed in c3h mice. inconsistent findings of food intake in gf mice might be due to the species of animals (rat versus mouse), strain of mouse (c57bl6/j versus c3h), the quality of the diet (chow, western, or high - fat diet), and the sample size in different studies. similar to food intake, the role of the gut microbiota on energy expenditure in humans is not known, and only limited information is available from animal models. early work showed that basal metabolic rate, cardiac output, and body temperature of gf rats were lower than those of their conv counterparts, indicating that microbiota may affect energy expenditure in animals. recent observations also indicated that the oxygen consumption was 25 to 40% less in gf than in conv c57bl/6j mice. the comparison of energy expenditure in different gnotobiotic mice colonized with different gut microbiota would be needed to demonstrate the role of gut microbiota in energy expenditure. few studies have reported the relationship between the gut microbiota composition and disease states, such as diabetes and cardiovascular diseases (cvds), in humans. larsen. showed that 16s rdna sequences representing the class of bacteroidetes were slightly higher in the diabetic subjects than in nondiabetic subjects, although the difference was not statistically significant. in this study a lower proportion of class clostridia and higher proportion of class betaproteobacteria were associated with diabetes. another study compared the fecal microbiota profile of three groups of subjects (lean control, obese diabetic, and obese nondiabetic) with a quantitative pcr technique and found diabetes was associated with a reduction of faecalibacterium prausnitzii species. a case - control study with 16 type 2 diabetics and 12 healthy controls found decreased b. vulgatus and bifidobacterium in the diabetic group. the interaction between the oral and gut microbiota and cvd, in particular atherosclerosis, has recently been discovered. interestingly, high abundance of certain bacteria was found in the atherosclerotic plaques and in the mouth microbiota, but no relationship was found between the plaque microbiota and the gut microbiota of affected patients. the role of the gut microbiota in type 1 and 2 diabetes has been researched in mouse models. wen. showed that the development of type 1 diabetes in myd88-deficient nod mice, a model of type 1 diabetes, was dependent on the presence or absence of the gut microbiota. indeed, nearly all gf myd88-deficient nod mice developed diabetes, whereas the ex - gf mice with the same genetic background colonized with a consortium of 6 bacteria strains had much reduced incidence. obesity and chronic low - grade inflammation are common aspects of both type 2 diabetes and cvd. we and others postulated that gut microbiota could contribute to the onset of insulin resistance. in one study, a 2-week treatment with broad range antibiotics, norfloxacin and ampicillin, significantly reduced the number of the cecal microbiota in ob / ob mice, and the treated ob / ob mice exhibited marked reductions in fasting blood glucose levels and glucose intolerance. increase of liver glycogen and decrease of liver triglyceride stores were accompanied with improved glycemic control. this study showed that metabolic health was improved in ob / ob mice with reduced gut bacteria. to further examine if glucose tolerance of mice is affected by the presence or absence of the gut microbiota, we fed both gf and conv c57bl/6j mice with a high - fat diet, and our results showed that gf mice did not develop diet - induced obesity and glucose intolerance. since all results based on comparisons between the gf and conv mice can not be extrapolated to represent normal physiology, evidence from microbiota transplantation studies will be needed to conclude if the gut microbiota composition predisposes the host to diet - induced glucose intolerance. changes in the number of bacteria, proportion of certain phylotypes, and bacterial activities of the microbiome are sensed by the host. the main pathways by which the host and bacteria interact are when bacteria or the bacterial metabolites enter the host 's circulation (figure 2). there are multiple systems in our body to sense environmental cues, but the aforementioned bacterial components and metabolites have been implicated to the gut microbiota sensing with evidence related to the development of metabolic diseases. our innate immune system is capable of sensing various type s of bacterial components via pattern recognition receptors (prrs). in general, there are two type s of prrs, toll - like receptors (tlrs) and nod - like receptors (nlrs). tlrs are highly conserved transmembrane receptors, and each tlr recognizes specific ligands and is capable of activating inflammatory responses. in humans, some tlrs (tlr1, 2, 4, 5, 6, and 10) are expressed in the cell surface, but other tlrs (3, 7, 8 and 9) locate in the membrane of endolysosomal compartments. twenty - two nlrs have been identified in humans, but only two of them (nod1 and nod2) are well characterized. in contrast to tlrs that are associated with membranes, nod1 and nod2 are located in cytoplasm. prrs recognize the structures of bacteria (e.g., lps, lipoproteins, and peptidoglycan) in order to signal the immune system of a pathogen. prrs not only engage in pathogen sensing, but are also implicated in the development of metabolic diseases such as insulin resistance and cardiovascular diseases. therefore prrs are perfect candidates for sensing the changes of the gut microbiota and more importantly mediating the subsequent inflammatory and metabolic responses. although the cause of metabolic inflammation is unclear, there is evidence to suggest that lps originating from gram - negative bacteria in the gut induces low - grade inflammation and insulin resistance. the presence of lps is constantly monitored by the host via the tlr4, which also recognizes compounds of nonmicrobial origin such as saturated fatty acids. evidence has suggested that metabolic endotoxemia (lps, 50100 pg / ml) can cause chronic low - grade inflammation and mild disturbances in energy metabolism implicated in the development of cvd and type 2 diabetes [55, 56 ]. a series of animal experiments has helped elucidate the relationship between gut microbiota, lps, diet, and insulin sensitivity. first, when lps was chronically infused to mice, it resulted in mild obesity and hepatic insulin resistance. mice deficient in tlr4 were protected from high - fat - diet - induced obesity and insulin resistance [5759 ]. second, the authors showed increased endotoxemia in mice consuming a high - fat diet [33, 60 ]. finally, it was found that ob / ob mice or high - fat - diet - fed c57bl/6j mice treated with ampicillin and neomycin had altered gut microbiota composition and reduced endotoxemia with improved glucose tolerance. furthermore, a reduction of lps by prebiotic (oligo - fructosaccharide) treatment significantly enhanced the whole body glucose tolerance and inflammatory markers in the liver and adipose tissue of mice fed a high - fat diet [62, 63 ]. together, these data suggest that lps enters circulation more readily while being on a high - fat diet, increased plasma lps has a detrimental effect on glucose metabolism, and altering the microbiota can alleviate endotoxemia and insulin resistance. therefore, available evidence suggests that lps plays a critical role in the development of obesity - and obesity associated insulin resistance and low - grade inflammation. human studies have provided support for this hypothesis as well. in a cross - sectional study of 50 human subjects, fasting lps levels in type 2 diabetics were significantly higher than the age, bmi, and sex - matched nondiabetic controls. furthermore, treatment of a subgroup of newly diagnosed type 2 diabetics with insulin - sensitizing drugs for 10 weeks decreased both endotoxin and insulin levels, such that the greater the change in endotoxin, the greater the change in insulin sensitivity. risk of incident diabetes was significantly associated with higher endotoxin (lps) activity, such that those in the highest quartile of lps activity had a 52% increased risk of having diabetes as compared to the lowest quartile. elevated lps levels in type 1 diabetic and kidney vascular disease (igagn) patients were associated with higher serum triglycerides, earlier onset of diabetes, increased diastolic blood pressure, and elevated marker of inflammation, monocyte chemoattractant protein-1. in sum, endotoxemia is a key player in the pathogenesis of diabetes and microbes may have a central role. metabolic endotoxemia has been linked to the development of cvd as well. in a 5-year epidemiological study of 516 middle - aged men and women, those with plasma lps levels over 50 pg / ml had a threefold increase in risk of developing atherosclerosis while the subpopulation of smokers or ex - smokers with the same level of lps had a 13-fold increase. in another study, endotoxin and tnf were elevated systemically in those with acute heart failure as compared to stable heart failure or normal controls. nevertheless, intervention studies which lower lps plasma levels and cause a subsequent decrease in cvd risk have not been conducted to our knowledge, and such result would clarify the importance of lps in the etiology of cvd. the transport of lps into blood can be through a number of pathways (figure 3). compromised intestinal tight junctions or a leaky gut enhances the possibility of bacteria translocation and uptake of the bacterial products. the bacterial products induce an inflammatory response, which is central to the pathophysiology of cvd and diabetes and provides a link between diseases of the gut and the vasculature. indeed, individuals with inflammatory bowel disease were at a higher risk of developing coronary artery disease despite having lower rates of traditional risk factors (dyslipidemia, hypertension, obesity, diabetes) than their age - matched controls in a longitudinal cohort study. in a study mentioned previously, obesity - prone rats were found to have increased gut permeability and higher plasma lps as compared to obesity - resistant mice. to further explore the mechanism by which leaky gut is related to the microbiome and diet, cani. found that male c57bl6/j mice fed a high - fat diet had increased intestinal permeability and decreased expression of genes encoding for tight junction proteins, but administration of antibiotics with the high - fat diet effectively blunted these negative effects [33, 61 ]. in follow - up experiments, it was confirmed that the obese mouse controls had the highest levels of intestinal permeability ; prebiotics were found to improve markers of intestinal permeability via glucagon - like peptide-2-mediated and cannabinoid - receptor-1-receptor-(cb1-) mediated pathways [70, 71 ]. another pathway by which lps may enter the blood stream is its integration into chylomicrons ; however, the mechanism has not been thoroughly elucidated. it is hypothesized that a certain amount of lps is present within the enterocyte, attached to or within chylomicrons. when chylomicron formation occurs during fat absorption, lps is then transported into the lymph circulation with chylomicrons. it has been shown in humans that increased chylomicron formation due to a high - fat meal causes greater lps transport postprandially as compared to a low - fat meal or no meal [60, 74 ]. levels of scd14 and il-6, both markers of acute inflammation, were elevated after a mixed meal containing lipid in association with higher lps levels. in both animal and cellular models, chylomicron formation stimulated by a high - fat challenge significantly enhanced the transport of lps from the intestinal lumen or enterocyte to circulation [73, 75 ]. this high - fat challenge did not affect the gut tight junctions in mice, and chemically inhibiting chylomicron formation in the presence of a high - fat stimulus effectively blocked lps translocation. together these data suggest that there is transcellular and chylomicron - dependent transport of lps, which is induced by high - fat intake. while evidence for a role of lps in chronic disease is intriguing, issues related to lps measurement and lps contamination make the study of this mechanism difficult ; therefore, developing a fully quantitative lps assay with a greater dynamic range should be considered for future study. nevertheless, the role of the gut microbiota in pathogenesis of diabetes and cvd should not be overlooked and understanding the contribution of inflammation from gut microbiota may provide insight on new therapies to decrease disease risk. tlr5 is highly expressed in epithelial cells of the intestinal mucosa and is involved in mediating immune response through recognition of bacterial flagellin. discovered that tlr5-knockout (t5ko) mice and wild - type littermates harboured different profiles of the gut microbiota. the changes of t5ko gut microbiota were mainly at the species level, which is in contrast with the ob / ob mouse model of obesity, where the alternation of the microbiota was characterized by a phylum - level shift. in t5ko mice, altered gut microbiota profile was associated with metabolic syndrome characterized with increased visceral fat deposition, dyslipidemia, hypertension, and insulin resistance. the cause of metabolic syndrome by the t5ko gut microbiota was demonstrated with a transplantation study, and the results show that gf wild - type mice receiving the t5ko gut microbiota developed metabolic syndrome. similarly, tlr2 recognizes a wide array of molecules including structural lipids, lipoproteins, and lipopeptides found on the surface of bacteria. ligand - induced dimerisation of tlr2 with either tlr1 or tlr6 triggers a cascade of kinase activations and eventually activation of nfb. several groups have shown that lacking tlr2 prevents mice from developing to high - fat - diet - induced obesity, hepatic steatosis, and insulin resistance [81, 82 ]. the liver, instead of skeletal muscle and white adipose tissue, is the major tissue with increased insulin sensitivity in tlr2-deficient mice. however, disrupting tlr2 in the skeletal muscle and white adipose tissue with tlr2 antisense oligonucleotides also improved the whole - body insulin sensitivity of mice. together, data indicate several tlrs not only can sense the bacterial structures but also can mediate insulin resistance once activated. nucleotide oligomerization domain (nod) 1 and 2 are intracellular sensors of bacterial peptidoglycan (pgn). nod 1 preferentially responds to pgn fragment, containing meso - diaminopimelic acid (meso - dap) [84, 85 ], which was found widely in gram - negative but also in some gram - positive bacteria, whereas nod2 recognizes monosaccharide with a dipeptide stem such as muramyl dipeptide (mdp), which was found in both gram - positive and gram - negative bacteria [86, 87 ]. the role of nods in controlling immune responses to bacterial ligand is reviewed by clarke and weisser, and these responses include the activation of innate defense, mediating antimicrobial peptide production, influencing phagocytosis of neutrophils and macrophages. in addition to their role in regulating innate and adapted immunity, activation of nods has been implicated in causing insulin resistance. in isolated human preadipocytes, a treatment with nod1 ligand dap led to an activation of nf - kb and increase of il-6 secretion. in 3t3-l1 adipocytes, dap induces insulin resistance with a decrease in insulin - stimulated akt phosphorylation (ser 473 and thr 308) and a reduced phosphorylated irs-1 (tyr 632). impaired insulin signalling by dap or a synthetic nod1 ligand fk565 translated to a significant reduction of insulin stimulated glucose uptake in 3t3-l1 adipocytes [89, 90 ]. in vivo, the fk565 treatment resulted in whole - body insulin resistance with decreased glucose infusion rate, reduced glucose disposal rate, and diminished suppression of hepatic glucose production by insulin during a hyperinsulinemic euglycemic clamp. the fk565-induced insulin resistance was absent in mice deficient in functional nod1, which suggests the functional role of nod1 activation in causing whole - body insulin resistance. similar to nod1, activation of nod2 also has been shown to induce insulin resistance. tamrakar. reported that addition of nod2 ligand mdp dose dependently suppressed basal and insulin - stimulated 2-dg glucose uptake in the cultured l6-glut4myc myotubes. these data suggest that bacterial pgn can cause insulin resistance in tissues but each insulin - sensitive tissue responds to a different bacterial pgn. the liver and adipose tissue primarily react to nod1 ligand whereas skeletal muscle preferentially reacts against nod2 ligand. together, available data suggest that bacterial pgn fragments can cause insulin resistance and prrs such as nod1 and nod2 are responsible for triggering a cascade of events leading to inflammatory responses and insulin resistance. bacterial metabolites encompass a group of molecules found in circulation that are a product of bacterial metabolism. the following bacterial metabolites : scfa, tmao, and hippurate, have been shown to affect diabetes or cvd risk. gut microbes ferment indigestible material in the colon to produce scfas, which is affected by the composition of the gut microbiota. applied hnmr spectroscopic analysis and examined the metabonomic profiles of feces from mice colonized with different gut microbiota. mice colonized with a human baby microbiota with a supplement of l. paracasei differed from conventional and conventionalized mice with less fecal butyrate and propionate and more succinate. however, near identical metabonomic pattern was observed between the conventional and conventionalized mice. in another study, conventional rats were treated with penicillin and streptomycin, and the amounts of fecal acetate, n - butyrate, and propionate were greatly reduced by the treatment. these results clearly demonstrated the changes of fecal scfa concentrations as a result of gut microbiota modulation. turnbaugh. have looked at the role gut microbiota profile plays in the production of scfa at two levels : the capacity to produce scfa and the type of scfa produced. by looking at metagenomic differences between lean and obese mice, a higher concentration of butyrate and acetate in the caeca of obese mice although the increase in scfa production capacity may be directly linked to the difference in microbiota profile between lean and obese conv mice, other confounding factors such as calorie and fiber intake associated with the hyperphagic phenotype of ob / ob mice could be driving the upward changes in scfa production and should be considered in future studies. as mentioned earlier, gut - derived scfas supply energy to the host. in addition, scfas are suggested to play a role in the regulation of blood lipids and therefore may have an effect on cvd risk. the three primary scfas produced by microbiota fermentation are acetate, propionate, and butyrate with the former two being absorbed into portal circulation and the latter used as an energy source for colonocytes. acetate (or acetyl - coa) is a substrate for cholesterol synthesis and is hypothesized to increase in plasma cholesterol, whereas propionate may decrease cholesterol by inhibiting acetyl - coa synthetase activity, the enzyme that converts acetate to acetyl - coa. therefore, propionate is thought to inhibit the cholesterol - raising effect of acetate ; however, the hypothesis is controversial. human studies have provided evidence to support the acetate : propionate hypothesis. in a cross - sectional study of normolipidemic men and women, the acetate : propionate ratio was positively associated with total and ldl cholesterol in men, but not women, even after adjusting for age and bmi. lactulose, a synthetic, nonabsorbable sugar, which is metabolized by microbiota to produce high levels of acetate, included in the diet of 6 healthy volunteers for 2 weeks, resulted in a significant increase in total and ldl cholesterol and apolipoprotein b with a trend towards increased serum acetate as compared to the control diet. furthermore, in a human comparative study using rectal infusions of acetate, propionate, or both, acetate increased serum cholesterol whereas propionate did not affect serum cholesterol, and the combination of the two did not cause an increase in serum cholesterol. it should be noted that acetate is always produced to a greater extent than propionate and butyrate, and in vivo levels of propionate in the portal vein are quite low ; therefore, the optimal acetate : propionate ratio needed to lower serum cholesterol and how that translates to fiber intake in humans require further study. animal and in vitro studies have provided supporting evidence concerning acetate and propionate 's role in cholesterol and fatty acid synthesis. the role of propionate in lipid metabolism and overall health is well reviewed by hosseini.. however, it is well known that there are many differences between the lipid metabolism of rodents and humans ; therefore, more research must be done in models with similar lipid metabolism, such as hamsters. in addition to their other physiological roles, scfas are signalling molecules which may help explain some mechanisms by which gut microbiota affects obesity and chronic diseases. recently it has been discovered that scfas act as a ligand for g - protein - coupled receptors gpr41 (ffa3) and gpr43 (ffa2) [98100 ]. ffa2 exhibits binding potency to scfas in the order of carbon chain length (c2 = c3 > c4 > c5 = c1), whereas ffa3 prefers to bind scfas with longer chain length (c3 = c4 = c5 > c2 = c1) [98100 ]. ffa2 mrna can be detected in various tissues, and the highest expression is found in immune cells such as neutrophils, monocytes, peripheral blood mononuclear cells, b cells, and polymorphonuclear cells [98100 ]. expression of ffa2 mrna was also detected in the skeletal muscle, adipose tissue, distal ileum, and colon. a high level of ffa3 expression can be found in the adipose tissue, pancreas, spleen, lymph nodes, bone marrow, blood mononuclear cells, and 3t3-l1 and 3t3-f442 preadipocytes [98, 99 ]. infusion of acetate reduced circulating free fatty acids in c57bl/6j and ob / ob mice, but the antilipolytic effect was abolished in ffa2-knockout mice, suggesting that ffa2 is partially responsible for the results of acetate infusion. therefore, activation of ffa2 by short - chain fatty acids might be one of regulatory mechanisms controlling the basal lipolysis and circulating fatty acid concentrations. leptin is an adipokine produced by the adipose tissue, and one of its functions is to negatively control the food intake. recently, xiong. demonstrated that scfas such as propionate and butyrate increased the expression of the leptin gene. conversely, knockdown of ffa3 by sirna almost completely inhibited the ability of propionate to induce leptin gene expression. ffa2 is expressed in peptide - yy-(pyy-) containing enteroendocrine cells, presumably l cells. since l cells are also responsible for the production and secretion of glucagon - like peptide (glp-1), it is plausible that scfas may affect insulin secretion via multiple pathways and mechanisms. in support of this hypothesis, mice deficient in ffa2 showed reduced insulin secretion in an oral glucose tolerance test (ogtt), which further suggests the involvement of ffa2 in glucose - stimulated insulin secretion. phosphatidylcholine is a phospholipid integral to cell membranes and present in higher - fat foods. gut microbiota releases choline from dietary phosphatidylcholine where it is then metabolized to trimethylamine (tma). tma is transported to the liver via the portal vein where it is oxidized by flavin monooxygenase-3 to trimethylamine - n - oxide (tmao). wang. showed that increasing levels of plasma tmao, choline, and betaine had dose - dependent relationships with the presence of cvd in a cohort of 1,876 men and women, after controlling for established risk factors and medication use. the same group further demonstrated that atherosclerosis - prone mice fed either high - choline or tmao diets had higher tmao plasma levels and enhanced total aortic root atherosclerotic plaque area without any differences in plasma lipids or glucose. while the microbiota is necessary for methylamine production (gf mice do not excrete tma), dietary interventions can modulate the metabolic outcomes. conv mice fed high - fat diets had higher conversion of choline to methylamines as compared to those on low - fat diets. elevated plasma tmao also is a signature biomarker of a high - meat diet as compared to low - meat and vegetarian diets in humans. tmao not only is a new biomarker for developing cvd but also a novel biomarker for food choice behavior. low - molecular - weight aromatic compounds and polyphenols from the diet are metabolized by intestinal bacteria, resulting in a production of benzoic acid. then, in the liver mitochondria, benzoic acid is conjugated with glycine to form hippurate, which subsequently is excreted into urine. therefore interestingly, urinary hippurate, acetate, and propionate were found lower in obese and insulin resistant zucker (fa / fa) rats than in wild - type (-/-) or heterozygous mutation (fa/-) rats. in humans, the amount of urinary hippurate discriminates the morbidly obese and insulin - resistant patients from age - matched control subjects. in particular, obese urinary metabolic profile was characterized with a lower level of hippurate than that of lean controls. recently, hippurate has been suggested as a biomarker discriminating people with elevated blood pressure from normotensive subjects. in the intermap study, which collected 24-hour urine samples from 4630 participants in china, japan, uk, and usa, together, data suggest that metabolites of the gut microbiota origin hold a great promise as a diagnostic marker for people at the risk of obesity or cardiovascular diseases. however, since the intakes of dietary polyphenols found in fruits and vegetables can potentially affect the amount of urinary hippurate, both dietary habits and gut microbial metabolic activities should be considered when examining the association between urinary hippurate and metabolic diseases. obesity and its associated diseases such as type 2 diabetes and cvd are increasing at an alarming rate. during the past years, the discovery of the roles of the gut microbiota in energy homeostasis raised the question of whether commensal intestinal bacteria are friends or foes in maintaining a healthy weight. as summarized in this paper, published results so far do not offer a clear answer to this question. factors specific to individuals, such as dynamic changes of microbiota and behavioral and genetic predispositions, likely work in a concert to determine the response of an individual to increased adipose stores. thus, gut metagenome should be considered as a risk factor joining the classic factors such as host genetics and environmental facts for the development of metabolic diseases (figure 4). low - grade inflammation, increased oxidative stress, dyslipidemia, hypertension, and insulin resistance have all been linked with obesity. thus far, many questions regarding the microbiota and obesity have been researched, such as do microbiota in the gut influence body weight, how do different gut microbiota profiles affect mucosal immune sensing, how is the production of bacterial metabolites regulated in the gut, and what are the impacts that bacterial metabolites have on the human body ? however, most studies emphasize on the description of the gut microbiota in different populations and associations between bacterial phylotype s and certain metabolic outcomes. therefore, it is paramount that the causality of metabolic diseases by disturbed gut microbiota be clearly demonstrated. high - priority questions that remain to be addressed are whether the gut microbiota should be considered as a pharmaceutical or nutritional treatment target for diabetes and cvd and what would be the ideal gut microbiota profile to prevent or to delay the onset of metabolic diseases. for future study in this field, it is reasonable to rely on a classical approach to identify pathogens in the gut that could help explain metabolic diseases. however, taking a more global approach and regarding the gut microbiota as an ecosystem would be more appropriate. specifically, research addressing the functionality rather than composition of the gut microbiota should be encouraged. numerous human metagenome projects, such as metahit (eu and china), microbes (inra, france), meta - gut (china), the canadian microbiome initiative (canada), and the nih human gut metagenome initiative (usa), are currently on - going. metaproteomics of human gut microbiota has been published, and nontargeted metabonomic profiling has recently been used to study the interactions between the gut microbiota and its host [92, 110 ]. omics platforms are undoubtedly powerful tools to study the role of the gut microbiota in health and diseases. hopefully, in the near future it will be possible to consolidate data from multiple omic platforms and identify whether certain profiles of gut microbiota or particular microbiota functionalities are a friend or foe of metabolic health. this would allow the design of proper diagnostic tools and therapeutic strategies to treat the consequences caused by dysbiosis between the gut microbiota and its host. in our opinion, gut microbiota may play an intriguing role in the development of obesity and obesity - related diseases, and, although microbiota seems strongly associated with obesity, a clear causal relationship remains to be established. | the gut microbiota refers to the trillions of microorganisms residing in the intestine and is integral in multiple physiological processes of the host. recent research has shown that gut bacteria play a role in metabolic disorders such as obesity, diabetes, and cardiovascular diseases. the mechanisms by which the gut microbiota affects metabolic diseases are by two major routes : (1) the innate immune response to the structural components of bacteria (e.g., lipopolysaccharide) resulting in inflammation and (2) bacterial metabolites of dietary compounds (e.g., scfa from fiber), which have biological activities that regulate host functions. gut microbiota has evolved with humans as a mutualistic partner, but dysbiosis in a form of altered gut metagenome and collected microbial activities, in combination with classic genetic and environmental factors, may promote the development of metabolic disorders. this paper reviews the available literature about the gut microbiota and aforementioned metabolic disorders and reveals the gaps in knowledge for future study. |
curing type acetoxycolor tranlucentdensity approximately 1.04 g / ccskin formation time approximately 5 min at 30ccuring time approximately 24 h. for 2 mm thickness at 30cmovement accommodation approximately 0.61 n / mmelasticity approximately 260% curing type acetoxy density approximately 1.04 g / cc skin formation time approximately 5 min at 30c curing time approximately 24 h. for 2 mm thickness at 30c movement accommodation approximately 15% tensile strength step 1 : ear impression is made using irreversible hydrocolloid alginate and custom made wax rim. wet gauze pieces are layered on top of alginate for the interlocking of the plaster to be poured [figure 1 ]. making of ear impression step 2 : undercut areas in the impression are identified and blocked out with wax. block out impression partly with wax and cast poured step 3 : working model is retrieved from the impression carefully and base trimmed [figure 3 ]. working model with dental stone prepared step 4 : custom tray using soft tray sheets prepared. a complete ear cast is made by making alginate impression for a person related to the patient [figure 4 ]. custom tray using soft tray sheets prepared for complete ear cast step 5 : skin tone of the patient is matched to the oil colors. colors mixed with silicone step 6 : silicone material is dispensed to the under cuts of the custom tray and filled completely [figure 6 ]. material dispensed with syringe into soft sheet custom tray step 7 : separating medium is applied on working cast and under cuts are filled with silicone material. it is pressed against the soft sheet custom tray carrying the silicone material [figure 7 ]. silicone filled tray pressed against working cast step 8 : prosthesis is tried on the model and trimmed before insertion on patient [figure 8 ]. over a period of time various materials such as wood, leather, polyurethane and polyvinyl chloride have been used to produce esthetic properties to prosthesis, but silicone has proved to be the material of choice because of its lifelike effects and flexibility. methyl methacrylate resin is been used as a maxillo - facial material, but its use is limited due to its rigidity. the flexibility and staining of silicone this industrial silicone has the property of elasticity and has the potential to incorporate stains and colors to mimic natural skin. the advantage of this industrial silicone material is the low cost factor, soft nature, flexibility, easy to fabricate and having good esthetic property and it will be an ideal material for the post - graduate student to train himself and acquire skills during his pre - clinical training program, which includes maxilla facial prosthodontics. since the cost is not prohibitive and as this material is quite similar to the current silicones, it can be recommended. | this article describes an industrial elastic silicone as a material for the laboratory fabrication of ear prosthesis. it has been tested for toxicity in lab animals by the sgs india pvt. ltd and approved as a material to pass the parameter of abnormal toxicity. this material therefore can be safely recommended for laboratory exercise to fabricate facial prosthesis. the high cost of the maxillo facial silicone materials prohibits their use for facial prosthesis in pre - clinical training of post - graduate students in maxillofacial prosthodontics. for this reason, pre - clinical laboratory exercise in facial prosthesis is inadequate. a few institutions use polymethyl methacrylate resins which are rigid and do not have elastic characteristics of silicone, which is used for facial defects. this cost - effective industrial silicone material which mimics the elastic and color characteristics of the conventional silicones can be recommended for preclinical exercises. |
during 20082013, a total of 68 satellite or global positioning system radio collars were deployed on african buffalo captured in southern gonarezhou np ; in northern kruger np, south of the limpopo river ; and in zimbabwe, north of limpopo river (on sengwe communal land). of the 68 buffalo, 47 were adult females, selected because their behavior is representative of core herd movements. two adult males were also equipped with global positioning system devices because males are believed to move between herds (7) ; however, these devices failed after a few weeks because the collars fell off. nineteen subadult female buffalo 2.54.5 years of age (age determined by teeth eruption) were also selected because individuals from this group are believed to disperse from their native herds (8 ; r. bengis, pers. blood samples were taken and stored appropriately for disease screening, and individually numbered ear - tags were applied. during the study, extraordinarily long - distance movements for 3 subadult females were plotted by satellite telemetry readings (figure 1). in january 2014, a 2.5-year - old female buffalo collared in south africa walked a maximum direct distance of 95 km. in 6 days, she crossed into zimbabwe, then into mozambique, and into zimbabwe again to enter gonarezhou np, with localizations within the home range of the buffalo herd in which btb was first diagnosed in a female buffalo in 2008 (6). this subadult buffalo later left the park and visited a commercial farm area before reentering gonarezhou np. in february 2014, another 4-year - old female buffalo walked a direct distance of 64 km in 8 days. finally, in march 2013, a 4.5-year - old female captured in july 2011 was sighted in a location deep into communal land at a distance of 96 km from her capture site. in contrast to these young female buffalo, no adult females collared in this study moved such long distances outside their home range during 20082014. the long - distance travel of these 3 subadult females occurred over a few days during the rainy season (figure 2) and included movements outside the gltfca boundary. cumulative home - range area of 15 buffalo collared with global positioning system devices in kruger national park, south africa, in november 2013. only buffalo with collars that functioned for an entire year are displayed. data for 2 subadult female buffalo (paths displayed in figure 1) are shown (id65 and id68) separately from data for the 13 other buffalo (subadult and adult females). our findings strengthen the hypothesis that btb was spread from kruger np to gonarezhou np through buffalo - to - buffalo transmission by subadult females that dispersed from their native herds. buffalo populations in kruger and gonarezhou nps are connected through long - distance movements of individuals, specifically prebreeding heifers. although this movement is important for buffalo conservation in tfcas, it could also facilitate the spread of animal diseases, including zoonoses, across borders. in 2010 and 2011, btb, rift valley fever, and brucellosis were detected in kruger np buffaloes, although a previous study failed to detect brucellosis in the gonarezhou np population (9) (table). buffalo id65, which was initially captured in kruger np, was seen among a breeding herd in gonarezhou np, indicating the possibility of direct, buffalo - to - buffalo transmission of btb by dispersing infected individuals, without the need for bridge hosts (e.g., other wild or domestic ungulate species) (10). individuals and herds belong to the same population (see figure 1, adult female home range). calf, 4.5 y. laboratory tests used : for bovine tuberculosis, gamma - interferon test and isolation ; for brucellosis, rose bengal and complement fixation tests ; for rift valley fever, indirect elisa test. published results (9) for the same diseases tested in the gonarezhou national park buffalo population in zimbabwe are shown in parentheses. difference between kruger and gonarezhou population results for brucellosis was significant by fisher exact test for equality of proportion for small samples (p<0.02). additional ecological information on buffalo dispersion is needed : frequency of dispersion events ; size, age, and sex composition of the dispersing groups ; and information about whether dispersed individuals later return to their home ranges. subadult females appear to be particularly prone to dispersing behavior, unlike adult females, and we speculate that they may do so in small groups of individuals that are approximately the same age (8). no record exists of subadult female buffalo mixing with male bachelor groups, which are also known to connect to adjacent herds (7).. one possible explanation may be an out - breeding mechanism (11) that occurs before the start of reproduction ; subadult females may leave their native herd to begin their reproduction in a distant herd to minimize in - breeding. furthermore, abundant resources (i.e., water and grazing areas) available during the rainy season maximize the probability of success of such behavior. we found that subadult females were infected with btb, brucellosis, and rift valley fever (table), diseases with different mechanisms of transmission. age and social position in the herd may influence individuals rate of exposure to pathogenic infections and consequently may affect the dynamics of infection within and between herds. our results indicate that subadult female buffalo could play a role in the spread of diseases among distant populations, across protected areas and international borders, and during the rainy season. this seasonal pattern contrasts with the timing of most wildlife and livestock contact between adult females, which has been observed to occur predominantly during the dry season in the study area (4). buffalo have been observed far outside the boundaries of protected areas, even outside the gltfca, in communal land where livestock farming is the main livelihood ; these observations considerably widen the wildlife livestock interface area where disease spread can occur (12). livestock interfaces can encompass large areas, rather than being a fence or strip of land at the edge of protected areas. these data should assist in refining disease modeling by showing the importance of temporal and spatial considerations and by redefining variables (e.g., age and sex) involved in risk for pathogen spillover or emergence (i.e., identifying super - spreaders) (13). our results suggest that the spillover of btb and other zoonoses at the wildlife livestock human interface constitutes a risk to animal and human health in the gltfca (9,14). the health issue in tfcas can not be overlooked and must be part of any management decision. combining ecological and epidemiologic knowledge is necessary to understand disease dynamics in these complex agro - ecosystems. | we report on the long - distance movements of subadult female buffalo within a transfrontier conservation area in africa. our observations confirm that bovine tuberculosis and other diseases can spread between buffalo populations across national parks, community land, and countries, thus posing a risk to animal and human health in surrounding wildlife areas. |
while nigeria has only two percent of the global population, it contributes 10% to the global maternal mortality burden. the average national maternal mortality rate is 545 per 100,000 while in the north eastern zone, it is 1,549 per 100,000. annually, an estimated 52,900 nigeria women die (about 1,000 per week) while pregnant, during childbirth or within 42 days of giving birth out of a global 529,000.1 this is the equivalent of an aeroplane, full of precious nigerian mothers crashing with no survivor, every day, for an entire year!. antenatal care (anc) reduces the incidence of maternal mortality. most maternal deaths occur in poor countries and most of these can be attributed to low level of supply and utilisation of skilled maternal health services.23 skilled attendants at delivery care has been considered as the single most effective means for reducing maternal mortality and morbidity in low and middle income countries.4 however, studies have suggested that the cost of health services is a major determinant of demand for healthcare, particularly for maternity healthcare.5 estimates of out - of - pocket costs for maternity care shows that they constitute a significant percentage of household income.6 the cost of obtaining skilled obstetric care at a health facility is prohibitively high for many poor households and constitutes a major barrier to increase utilization and access to safe maternal care.78 it is, therefore, not surprising that in rural communities, majority of births take place at home. for example, in rural bangladesh, 85% of births take place at home.9 home delivery is preferred as it is associated with low cost.10 almost four times as many women in the rural areas had no anc compared to urban areas.11 the cost of anc and delivery services (obstetric care) could be catastrophic in some communities. for example, in a study in matlab, a rural community of bangladesh, the cost of a normal delivery alone was found to be equivalent to 18% of the annual income of the household heads.5 on a similar note, in south delhi, india, the average cost of a normal vaginal delivery in government health facility was $ 61.1, which constitutes 10% of the annual household income of the lowest income groups.12 on the contrary, in other communities, studies have shown that the cost of anc and delivery services were not catastrophic. for example, in rajasthan, india, the average rural household income was rs 23,527 while average amount spent on anc and delivery were rs 397 and rs 836, respectively. these costs constituted 1.7% and 3.6% of household annual income respectively.13 also in dhaka, a rural community of bangladesh, the mean cost of normal delivery was $ 31.9, which was 2.2% of the annual income of the poor households.14 health expenditure has been defined as catastrophic if it exceeds 10% of the annual household income remaining after subsistence needs have been met.1517 catastrophic health expenditure depletes household income and contributes to the vicious cycle of poverty and disease. it forces poor households to reduce other basic expenses such as food, shelter, or their children 's education.18 in other words, it forces household members to cut their consumption of other minimum needs, triggers productive asset sales or high levels of debt, and leads to impoverishment. worst still, it adversely affects the health seeking behaviour of poor households, forcing them to access less than the required amount of treatment or cheap, inappropriate treatment. in the case of delivery, it forces women to deliver at home instead of a health facility, thereby, missing the care of a professional birth attendant. in nigeria, like in most developing countries, there is regional differential in poverty and financial access to healthcare with the north east and north west being worst hit. this has made some governments in these regions to come up with a free maternal and child healthcare package to solve the problems of financial access to healthcare services. however, giwa local government area, where this study was conducted, is yet to introduce or benefit from such a package, probably due to financial constraints. it was in this regard that this study was carried out with the aim of estimating the total cost to the user of anc services and how this affects the use of the service in fatika village, a rural health facility in giwa local government area in kaduna, north -western nigeria. this was necessary since only few studies have investigated the total cost of anc services in rural health facilities of developing countries. a cross - sectional descriptive study conducted during community diagnosis posting of final year medical students of ahmadu bello university, zaria, from 28 february, 2011 to 25 march, 2011. the study was conducted in fatika community, a rural settlement in giwa local government area of kaduna state, north - western nigeria. it is 234 kilometers north of abuja and 162 kilometers south - west of kano. the population of the village was re - projected from 2,576 in 1996 to 3,586 in 2011 using kaduna state 's annual growth rate of 2.8 percent.19 the inhabitants are predominantly hausas and farming is the predominant occupation. a total of 135 women participated in the survey. only currently pregnant women and those who delivered recently (within 6 weeks postpartum) were included. all anc attendees received care at the village 's health facility, which is managed by the local government authority. respondents used their money to pay the official prices of various services at the facility. an ingredients approach, whereby the cost incurred by respondents for each of the inputs for anc, was used to estimate the total cost of anc. yakawada comprehensive health centre is the nearest referral centre to the village and is more than 30 kilometres away. none of the respondents received anc at yakawada comprehensive health centre probably because of the distance, economic implications and the distress of transporting a pregnant woman in labour there, coupled with the near absence of commercial vehicles. data was collected by trained final year medical students using an interviewer administered questionnaire in which respondents were asked about their socio - demographic profiles, monthly income and estimates of cost of anc services (drugs, investigations and delivery, transportation). the questionnaire was pretested on 42 pregnant women in danmahawayi village, a community with similar characteristics with the study area. ethical clearance for the study was obtained from ahmadu bello university teaching hospital 's ethical committee. appropriate entry permission to conduct the study was sought from giwa local government area, kaduna state and from fatika community leaders. after the data collection, all completed questionnaires were checked properly for any error and edited. the data obtained were cleaned and analyzed using statistical package for social sciences software (spss), version 19. a total of 135 women participated in the study, of whom 43 (39.4%) have delivered within past 6 weeks of study date. most of the respondents (62.3%) are in the 20 - 31 years age group. seventy one percent (71.9%) of the respondents have between 1 and 5 living children. most of the spouses of respondents (52.6%) earn less than n10, 000($65) per month and have only one wife (64%). a significant proportion of respondent 's spouses (36%) have more than one wife (polygamy). socio - demographic characteristics of respondents as shown in table 2, anc attendance was high among respondents (80.7). however, majority of the respondents (56%), who attended anc or are attending it, have no formal education. also, majority of the respondents (78%), who did not attend or do not attend anc, had no formal education. distribution of respondents by anc attendance and educational attainment table 3 shows the estimated amount of money spent on anc and delivery services by respondents. the average amount spent on booking and initial laboratory investigations were n77 (half a dollar) and n316 ($ 2), respectively. per anc visit, the average amount spent on drugs and transportation were n229 ($ 1.5) and n139 ($ 0.9), respectively. most of the respondents that have delivered (60.4%) spent between n1,000-n1,500 ($ 6.5-$9.6) for delivery. estimated cost of anc services and delivery in naira table 4 shows the estimated monthly income of spouses of respondents, who attended or are attending anc, with 38.5% earning less than n10,000 per month. only 12% earn more than n30,000 per month. estimated monthly income of husbands of anc attendees in naira the community is predominantly hausa and early marriage is a common practice among them.13 hence, a significant proportion of the respondents (20.7%) were aged between 14 - 19 years. in northern nigeria, female education is considered of secondary importance due to socio - cultural practices, religious beliefs and misconceptions. parents generally consider sending their daughters to school as a complete waste of time and resources, especially in rural communities where the latter are scarce. this resulted in poor girl - child enrolment into formal schools.2021 it, therefore, accounted for the low proportion (39.3%) of respondents with formal education. this finding is not surprising, since the educational level of a woman, her age, number of children, and other socio - demographic profiles such as her husband 's income and type of marriage have been shown to influence the use of antenatal and delivery services.11 majority of the spouse of the respondents (52.6%) earn less than n10,000 ($ 64.5) per month, which translates to less than $ 2 per day. their average estimated monthly income is n14, 000 ($ 90) which translates to n120, 000 ($ 774) per annum. all 15 women with secondary school education attended anc (100%), also the only two women with tertiary education both attended anc. however, only 83% and 74% of those with primary and those without formal education attended anc, respectively. the average number of living children was three and most of the respondents had between 1 and 5 children. this is in keeping with the finding that mothers with less than six children are likely to seek for professional anc than those with six or more children.11 there was a statistically significant association between level of formal education and anc attendance (x = 4.25, df = 1, p = 0.039) [table 2 ]. the possible explanation for this is that level of education improves health seeking behaviour.22 however, it is contrary to findings among chinese women where there was a negative relationship between education and seeking for treatment in a hospital.23 this difference could be due to the fact that the chinese women were not pregnant and so there was no perceived risk of danger. the average cost of a single anc visit (drugs and transportation only) from the study was n368 (us$2.4) while the average number of anc visits was four. since, focussed anc is not practiced in the health facility, four anc visits will cost n1, 472.00 (n368 4). on the other hand, the average cost of normal delivery was n1,500 (us$9.6). it, therefore, follows that the summation of average costs of anc (n1, 472.00), delivery (n1,500.00), and anc booking / laboratory investigations (n393) is n3, 365.00 ($ 22). this amount translates to 2% of the average annual income of household heads in the community. therefore, the cost of anc and delivery was not catastrophic. however, there was a statistically significant association between husband 's income and anc attendance (x = 2.451, df = 2, p = 0.048) [table 4 ]. this implies that anc attendance was directly related to the monthly income of household heads. in other words, thus, the cost of anc services is a possible barrier to anc attendance and a possible contributory factor to high prevalence of home deliveries in the community. this implies that the fees, though not catastrophic, still present a significant barrier to accessing anc and facility - based delivery services in the study area. this finding is contrary to that of a study in matlab, a rural community in bangladesh where the cost of a normal delivery alone was catastrophic, amounting to 18% of the annual income of the household heads.12 one limitation of the study is that it relied on recall of the clients with respect to estimations of costs incurred at point of service. another limitation was the small number (proportion) of women who delivered in the village 's health facility. cost of anc and delivery services were not catastrophic but were a barrier to accessing anc and facility - based delivery services in the study area. pro - poor policies and actions are needed to address this problem, as it will go a long way in reducing maternal mortality in this part of the country. | background : maternal mortality remains a leading cause of death among women of reproductive age. while nigeria has only two percent of the global population, it contributes 10% to the global maternal mortality burden. antenatal care (anc) reduces the incidence of maternal mortality. however, financial capability affects access to antenatal care. thus, the rural poor are at a higher risk of maternal mortality.materials and methods : a cross - sectional descriptive study involving 135 women (pregnant women and those who are 6 weeks postpartum). structured interviewer - administered questionnaires were used for data collection. data analysis was carried out using statistical package for social sciences software (version 17).results : the average amount spent on booking and initial laboratory investigations were n77 (half a dollar) and n316 ($ 2), respectively. per anc visit, average amount spent on drugs and transportation were n229 ($ 1.5) and n139 ($ 0.9) respectively. for delivery, the average amount spent was n1500 ($ 9.6). on an average, anc plus delivery cost about n3,365.00 ($ 22). there was a statistically significant association between husband 's income and anc attendance (x2 = 2.451, df = 2, p = 0.048).conclusion : cost of antenatal care and delivery services were not catastrophic but were a barrier to accessing antenatal care and facility - based delivery services in the study area. anc attendance was associated with the income of household heads. pro - poor policies and actions are needed to address this problem, as it will go a long way in reducing maternal mortality in this part of the country. |
early in life, parental interactions can serve as a buffer against the negative consequences of stress on physiology and learning (stanton and levine, 1990, kirschbaum., 2003, shionoya., 2007, taylor., 2008, however, if rearing conditions are suboptimal, parental stress or disruptions in the quality or reliability of care can be rapidly transmitted to the offspring and serve as a primary source of stress, driving changes in development and neurobehavioral outcomes (levine, 1967, rosenfeld., 1991,, 1993, liu., 1997, avishai - eliner., 2001, rice., 2008,, 2014, bath., 2016, heun - johnson and levitt, 2016). significant disruptions in the quality of early life care impact neural structure and functional plasticity of the brain (teicher., 2006, chen., 2008, chen., 2013) and have been identified as potential catalysts for negative health outcomes, including disturbance in cognitive development. for example, in humans, institutionalized rearing or abusive early environments have been associated with the development of significant impairments in general cognitive functioning with specific deficits identified in memory recall (bremner and narayan, 1998) and short term memory (bremner., 2000). these same experiences are associated with regional effects on brain development, with significant reductions in hippocampal volume and cortical thinning in frontal regions associated with memory function, attention, and spatial abilities (bremner., 1997). significant sex disparities have been identified for stress - associated pathology, with females being twice as likely as males to develop ptsd, depression, and anxiety disorders (weissman., 1996,, 1997a, breslau., 1997b, felitti., 1998, gater., 1998, burt and stein, 2002, kuehner, 2003, keita, 2007, breslau, 2009, de munck., 2009, hankin, 2009, olino. however, whether similar sex disparities exist for stress - associated cognitive disturbance have not been as well characterized. recent studies in animal models have provided mixed results, but have identified sex, developmental status, and timing of the stressor, as potential variables contributing to risk for cognitive outcomes. for example, some studies have reported significant impairment following various forms of els on spatial learning in female rats (marco., 2013 ; wang., 2016), while others found a male bias in impairment in both mice and rats (barha., 2007 ; mueller and bale, 2007 ; salomon., 2011 ; other studies from both mice and rats failed to identify sex differences in performance (benoit., 2015 ; nazeri., 2015), while further studies in rats found els to be associated with improved cognitive performance (barha., 2007 ; zuena., 2008 ; uysal., 2012 ; barbie - shoshani., 2016). thus, considerable confusion exists with regard to the effects of els on cognitive functioning, possibly due to the varied forms of stress, timing of stress implementation, and age at testing. it should be noted that the majority of studies focused on assessing outcomes at one or two time points in development, typically late adolescence or adulthood, or failed to take into account possible developmental changes in performance on cognitive measures. here, the effect of els, in the form of maternal bedding restriction from p4-p11 (rice., 2008, bath., 2016), was tested on the development of spatial learning in male and female mice across early development. to do this, els and control reared mice were tested on a novel object placement (nop) task, at postnatal days 21, 28, 38, 50, and/or 75. this allowed for assessment of time points that approximate childhood (p21), the pre - adolescent period (p28), the peri - adolescent period (p38), early adulthood (p50), and adulthood (p75) in mice. to minimize potential practice effects and diminish the contribution of any single litter on the overall results, a large number of litters were sampled (26), and no mouse was tested at more than 2 developmental time points. males showed early emergence and persistent impairments in performance on the nop task, while females showed an earlier but transient impairment in nop performance. the current data suggest that stress may have sex and developmental selective effects on the emergence of cognitive disturbance, and such factors may be critical in understanding the contribution of stress and sex to developmental pathology. breeding stock of male and female c57bl/6n mice were acquired from charles river labs and all mice used for the current studies were derived from litters that had been bred in house. animals were maintained under normal housing conditions on a 12h:12h light cycle with ad libitum access to food and water. all animal procedures were approved by the brown university institutional animal care and use committee and consistent with the national institutes of health guide for the care and use of laboratory animals. four days following the birth of a litter, the dam and pups in the fragmented maternal care condition were transferred from their standard home cage, to a home cage with a wire mesh floor and a 2 4 cm cotton nestlet as their only source of bedding (as described in bath., 2016). dam and litters remained in these modified housing conditions for seven days, and were then returned to standard housing, containing cob bedding and a 4 4 cm nestlet. litters were composed of both male and female pups, and litters ranged in size from 5 to 8 pups per litter. previous work in both mice and rats have shown that the bedding restriction manipulation leads to a fragmentation in maternal care and elevations in stress hormones in the dam immediately following the stressor (avishai - eliner., 2001, rice., 2008, bath., 2016, els housing leads to an increase in the number of departures by the dam from the nest, but no change in the duration or total time spent licking and grooming or arched back nursing (heun - johnson and levitt, 2016, molet., 2016). here, detailed assessment of maternal behavior was not carried out. instead, successful replication of core features of this paradigm were used to verify the efficacy of the manipulation, including diminished weight gain of pups, an effect that was observed in both male and female mice (fig. 1). at 20 days of age, prior to beginning the novel object placement task, all mice received an initial exposure to the open field testing apparatus (the same apparatus used for nop testing). open field testing occurred between the hours of 9 am and 12 pm, under approximately 1520lux of light and lasted for a total of 7 min. during this time, mice were video recorded and their activity was tracked with the aid of digital tracking software (noldus ethovision xt 8.5). to determine if els or sex significantly impacted locomotion within the testing apparatus, total distance traveled was quantified. percent time in the center of the open field was used to test if els altered anxiety - like behavior. one day prior to testing, mice were again habituated to the empty open field for 7 min to acclimate them to both handling as well as the testing environment. testing occurred between the hours of 9 am and 12 pm, under approximately 1520lux of light. testing consisted of an exploration phase (t1, exploration trial) and a recognition phase (t2, recognition trial). in the exploration trial, mice were placed in the open field with two identical objects (supplemental fig.. investigation of the objects was timed using automated tracking software (noldus ethovision xt 8.5), with investigation defined as the subject 's nose being directed at and within 1 inch of the object. after the t1 exploration phase iti duration was chosen based on prior work in rats, which found that 21 day old rats could complete the task with short < 1hr, but not long (24hr) intervals (jablonski., 2013, westbrook., 2014). during the iti, one object was moved to a new location and the arena and object were cleaned with ethanol and dried. the mouse was then returned to the arena for an additional 5-min trial and the time spent exploring the objects in the new (novel) and old (familiar) locations after the delay (t2, recognition trial) were assessed with the aid of automated tracking. all locations for the objects were counterbalanced among groups. because mice generally show a novelty preference, (e.g. more time spent exploring the object in the novel rather than familiar location) a bias toward investigation of the object in novel location was used to assess the subjects memory of objects in familiar relative to novel locations (aggleton., 1997, luine., 2003). for each developmental time point, the hippocampus (whole hippocampus from one hemisphere - side randomly selected) was collected from behaviorally nave animals from at least 2 different litters to eliminate the possibility of cohort effects on measures of gene expression (n = 5 animals per group / per age). brains were dissected on ice, hippocampus was isolated followed by homogenization in rnazol (molecular research center, cincinnati, oh) and stored at 80c until processing. first strand cdna synthesis was in accordance with new england biolabs mmulv protocols (neb, ipswitch, ma). predesigned and pre - validated taqman assays from applied biosystems (life technologies, norwalk, ct) for ki-67 (assay # mm01278616_m1) and dcx (assay # mm00438400_m1) were used and run in multiplex with housekeeping gene (18s cat # 4319413e). for each plate and assay, gene expression was calculated based upon a standard curve included on each plate. a cfx384 rt - qpcr system (biorad, hercules, ca) and associated software was used for all gene expression profiling. effects of sex, age, and treatment were calculated for gene expression, locomotor activity, object visits, and object exploration time during the t1 exploration and t2 recognition phase of the task using anova. for t2 recognition testing, a single sample t - test (tested against 50- defined as chance) was used to test for successful performance on the task. correlation analysis was used to test for relationships between t1 exploration (object visits and object investigation) and t2 recognition performance. for all tests alpha was set at p < 0.05. between p11 and p60, a significant effect of age was observed on weight (f(11,780) = 1719.732, p < 0.001 ; eta = 0.960), with all groups showing a significant increase in weight over development. a significant main effect of sex was also observed (f(1,780) = 73.120, p < 0.001 ; eta = 0.086), with females being smaller than males. in addition, a significant main effect of treatment was found for weight (f(1,780) = 350.234, p < 0.000 ; eta = 0.310 ; fig. 1a), with els animals weighing on average 17% less than control reared mice. the effect of els on weight gain was similar in both male and female mice, with no sex x age treatment interaction (f(11,780) = 0.467, p = 0.924 ; eta = 0.007). for corticosterone, basal serum levels significantly increased with age (f(1,50) = 71.797, p < 0.001 ; eta = 0.812), and a main effect of treatment was found (f(1,50) = 25.128, p < 0.001 ; eta = 0.334 ; fig. 1b) with els leading to a significant elevation in basal corticosterone levels in both male and female mice. in addition, there was a significant main effect of sex (sex : f(1,50) = 18.837, p < 0.001 ; eta = 0.274) with corticosterone levels in females being significantly higher than those observed in males for both control and els reared conditions (fig. 1b). follow - up analyses at each independent age show a significant effect of treatment that emerged at p16 (anova, f(1,12) = 14.780, p = 0.002 ; eta = 0.552), but did not reach significance at p12 (anova, f(1,14) = 2.515, p = 0.135 ; eta = 0.152). in previous work, els has been shown to impair early motor development (bath., 2016). differences in locomotor activity can influence performance on the nop task by altering the number of visits and amount of time investigating objects in the environment. to test for effects of sex (male and female) and treatment (control and els) on general locomotor activity prior to study enrollment, aged p20 mice were tested in the open field for 7 min. no main effect of treatment was found for total distance traveled (anova ; f(1,85) = 2.247, p = 0.138 ; eta = 0.026). there was also no effect of sex on distance traveled (anova ; f(1,85) = 0.000, p = 0.993 ; eta 0.000) with male and female control and els mice traveling similar overall distances (fig. 1c). to determine if els alters the expression of anxiety - like behavior at this young age, the percent time that mice spent in the center of the open field was assessed. there was no main effect of treatment (f(1,47) = 0.198, p = 0.658), sex (f(1,47) = 3.448, p = 0.070), or treatment sex interaction (f(1,47) = 0.032, p = 0.858) for this measure, indicating that els does not alter locomotor activity or anxiety - like behavior at this age (fig. prior work in mice has shown sex differences in nop performance when using a 24 h iti (frick and gresack, 2003). here, a 25-min iti was used and no difference was found between control reared male and female mice (f(1,110) = 1.065, p = 0.304, eta = 0.010), indicating that both male and female mice can perform successfully on this task. however, for mice reared under els conditions, a significant main effect of sex was found (f(1,127) = 6.581, p = 0.011, eta = 0.049) with males performing significantly worse than females. in follow - up analyses, the effect of treatment was assessed within sex, and showed that els led to impaired nop performance in male (f(1,135) = 4.663, p = 0.033 ; eta = 0.033), but not female mice (f(1,107) = 0.148, p = 0.701 ; eta = 0.001). to test for successful performance on the nop task at each age tested, a single sample t - test was used to test group performance against chance levels of investigation (50e.g. at p21, control reared male mice showed successful discrimination (t(10) = 2.827, p = 0.009). however, els male mice failed to differentiate between the object in the novel relative to familiar locations (t(16) = 0.224, p = 0.413), performing at levels indistinguishable from chance. at p28, performance of control reared male mice approached but did not reach significance (t(8) = 1.397, p = 0.100), while male mice reared under els conditions again failed to show successful discrimination (t(7) = 0.368, p = 0.362). at p38, control reared male mice again approached but did not reach significance with regard to successful performance (t(13) = 1.470, p = 0.083). however, at this same developmental time point els reared males failed to show significant discrimination (t(11) = 0.265, p = 0.398). at p50, control reared male mice were again successful at performing the task (t(13) = 2.085, p = 0.029), while els males again failed to show successful discrimination (t(23) = 0.827, p = 0.209). finally, at p75, control reared male mice showed successful performance (t(7) = 1.941, p = 0.047), while els male mice continued to fail to discriminate between the objects in the novel relative to familiar location (t(21) = 1.186, p = 0.125). for female mice, at p21, control reared female performance approached, but did not reach significance (t(11) = 1.509, p = 0.080). at this age, els female mice failed to differentiate between the object in the novel relative to familiar locations, performing at levels indistinguishable from chance (t(8) = 0.218, p = 0.417). at p28, both control reared females (t(12) = 2.848, p = 0.008) and els reared females (t(8) = 2.326, p = 0.024) successfully performed the task. at p38, control reared female mice could successfully discriminate between the novel and familiar object location (t(16) = 2.496, p = 0.012), however, els reared females failed to show significant discrimination at this time (t(7) = 0.706, p = 0.252). at p50, both control reared female mice (t(15) = 2.083, p = 0.028) as well as els female mice (t(15) = 1.923, p = 0.037) performed successfully on the task. finally, at p75, again, both control reared female mice (t(4) = 1.560, p = 0.097) as well as els female mice (t(11) = 3.482, p = 0.003) were successful in discriminating between the object in the novel relative to familiar location. a subset of mice (96) were tested at two separate developmental time points. comparing performance between first and second test, no effects of test repetition on performance were found (paired t - test- t(95) = 1.669, p = 0.098). mean performance on test 1 was 53.9 (sem = 2.1) while mean performance on test 2 was 58.2 (sem = 1.8). performance across age was also assessed in control and els reared animals to determine if age significantly contributed to changes in performance over development. no significant main effect of age was found for either control (f(4,110) = 0.438, p = 0.748, eta = 0.017) or els reared mice (f(4,127) = 1.345, p = 0.257, eta = 0.041), indicating no differences in performance across the ages tested. as mentioned above, deficits in performance can be due to failures in a number of different phases of this task beyond impaired memory or spatial abilities. in some instances, failures can emerge as a consequence of diminished initial investigation of the objects during habituation, a failure to approach the objects during the recognition phase, and/or increased or decreased locomotion which may impair overall investigation time and number or duration of object visits. data was collected on all of these metrics to test if any of these variables might explain impairments in performance observed in els male and female mice, as well as test for potential effects of age, sex, and treatment on these measures. group differences in total distance traveled during the t1 (exploration) as well as t2 (recognition) phases of the nop task were collected using automated tracking software (noldus ethovision), to investigate the effects of age, sex, and treatment on locomotor activity. during the exploration phase of the task, no effects of sex (anova ; f(1,162) = 0.280, p = 0.598 ; eta = 0.002) or of treatment (anova ; f(1,162) = 0.380, p = 0.539 ; eta = 0.002) were found for total distance traveled (fig. 3a and b). in addition, there was no sextreatment interaction (anova ; f(1,162) = 0.858, p = 0.356 ; eta = 0.005). however, a significant effect of age was found for distance traveled (anova ; f(4,162) = 6.873, p < 0.001 ; eta = 0.145 ; fig. 3a and b) with mice showing a general trend toward greater distance traveled with increasing age. however, there were no interactions between age and any other variables. during the t2 recognition phase of testing, no effect of sex (anova ; f(1,162) = 0.016, p = 0.901 ; eta = 0.001), treatment (anova ; f(1,162) = 1.813, p = 0.179 ; eta = 0.007), or sextreatment interaction (anova ; f(1,162) = 0.192, p = 0.661 ; eta = 0.001 ; fig. a significant effect of age was observed (anova ; f(4,162) = 8.124, p < 0.001 ; eta = 0.118 ; fig. 3c and d), with greater distance traveled in older mice, but no interaction between age and any other variable. els may impact the overall duration or frequency of object investigation during the exploration or recognition phase of testing, an indicator of object interest or willingness to explore objects. thus, total time spent engaged in object exploration for each phase of testing was assessed. for the exploration phase, similar overall levels of investigation spent investigating objects, no main effect of sex (anova ; f(1,162) = 0.577, p = 0.449 ; eta = 0.004), treatment (anova ; f(1,162) = 0.001, p = 0.978 ; eta = 0.001), or sex treatment interaction (anova ; f(1,162) = 0.003, p = 0.956 ; eta = 0.001 ; fig. however, a main effect of age was found (anova ; f(4,162) = 5.450, p < 0.001 ; eta = 0.119, fig. 4a and b), with a trend toward lower levels of investigation at later ages in development. near identical effects were observed for the number of object visits during the exploration phase of testing (data not shown). for the number of object visits, no main effect of sex (anova ; f(1,162) = 0.193, p = 0.661 ; eta = 0.001), treatment (anova ; f(1,162) = 1.003, p = 0.318 ; eta = 0.006), or sextreatment interaction (anova : f(1,162) = 0.035, p = 0.852 ; eta = 0.001) were found. for number of object visits during t1 exploration, a significant effect of age was found (anova ; f(4,162) = 2.969, p = 0.021 ; eta = 0.068) but no interaction between age and any other variables. for the t2 recognition phase of the task, a main effect of sex was found (anova ; f(1,242) = 6.714, p = 0.010 ; eta = 0.027) with males showing slightly higher overall levels of investigation, but no main effect of treatment (anova ; f(1,242) = 2.899, p = 0.090 ; eta = 0.012) as well as no treatmentsex interaction (anova ; f(1,242) = 1.238, p = 0.267 ; eta = 0.005 ; fig. 4c and d). for time spent investigating objects during the recognition phase of the task, there was no main effect of age (anova ; f(4,242) = 0.729, p = 0.573 ; eta = 0.012, fig. 4c and d), or interaction between age and any other variable. for the number of object visits during the recognition phase, a significant main effect of treatment was found (anova ; f(1,242) = 4.060, p = 0.045 ; eta = 0.016, data not shown), with els animals tending to visits objects more frequently. a marginal main effect of sex was also found for this measure (anova ; f(1,242) = 3.455, p = 0.064 ; eta = 0.014, data not shown), but no treatmentsex interaction (anova ; f(1,242) = 0.486, p = 0.486 ; eta = 0.002, data not shown). finally, there was no significant main effect of age on object visits (anova ; f(4,242) = 0.219, p = 0.928 ; eta = 0.004, data not shown) or interaction between age and any other variable. multiple methodologies exist for carrying out nop testing, with some groups suggesting that increasing object investigation time or equating object investigation time during the exploration phase can lead to enhanced performance during the recognition phase of the task. here, a fixed trial duration was used for both the t1 exploration and t2 recognition phases of testing. correlation analysis was used to investigate whether differences in object investigation time or number of visits to the objects during the t1 exploration phase significantly correlated with performance in the t2 recognition phase. interestingly, no correlation was found between object visits during the t1 exploration phase and performance during the t2 recognition phase (r(182) = 0.017, p = 0.822) and no correlation between object investigation time during the t1 exploration phase and performance during the t2 recognition phase (r(182) = 0.023, p = 0.760), indicating that increased investigation during the exploration phase did not lead to better performance in the recognition phase of the task. when correlations were carried out at each age (fig. 5a e), no significant relationships were found between time investigating the objects during the t1 phase and t2 performance at p21 (r(35) = 0.003, p = 0.750), p28 (r(33) = 0.092, p = 0.076), p38 (r(49) = 0.000, p = 0.953), p50 (r(36) = 0.029, p = 0.311), or p75 (r(19) = 0.022, p = 0.525) (see fig. previous work has suggested that els effects on hippocampus neurogenesis may contribute to sex differences in performance on the nop task (naninck., 2015). here, whole hippocampus was collected from one hemisphere of behavioral nave male and female control and els mice across development. using rt - qpcr, effects of development, sex, and treatment on markers of cell proliferation (ki-67) and cellular differentiation (doublecortin- dcx) were tested. for ki-67 levels in male mice, a significant main effect of age (f(7,65) = 16.247, p < 0.001 ; eta = 0.636), treatment (f(1,65) = 4.887, p = 0.031 ; eta = 0.070), and marginal treatment age interaction (f(7,65) = 1.870, p = 0.089 ; eta = 0.168) was found. specifically, a significant reduction in ki-67 levels across early development was observed, with reduced ki-67 expression in els hippocampus at p12 (p < 0.011) and p16 (p < 0.022). for dcx in male hippocampus, a significant effect of treatment (f(1,65) = 6.411, p = 0.014 ; eta = 0.090), age (f(7,65) = 54.231, p < 0.001 ; eta = 0.854), and a treatmentage interaction (f(7,65) = 2.313, p < 0.036 ; eta = 0.199) were observed, with decreasing expression over development and lower levels of expression in els mice at p8 (p = 0.070), p12 (p < 0.001), and p16 (p = 0.014). for female mice, specifically, for ki-67, a significant effect of age (f(7,64) = 75.712, p < 0.001 ; eta = 0.892), treatment (f(1,64) = 9.150, p = 0.004 ; eta = 0.125), and treatmentsex interaction (f(7,64) = 5.055, p < 0.001 ; eta = 0.356) were observed, with decreasing expression over development and lower levels of ki-67 in els female hippocampus compared with controls at p12 (p = 0.006), p16 (p < 0.001), and p21 (p = 0.001), but not later time points. for dcx levels in female hippocampus, a significant effect of age (f(7,63) = 93.407, p < 0.001 ; eta = 0.912), no main effect of treatment (f(1,63) = 0.804 ; p = 0.373 ; eta = 0.013) but a significant treatmentage interaction (f(7,63) = 3.240, p = 0.005 ; eta = 0.265) were found. similar to males, there was a decrease in expression of dcx over development with lower dcx levels in els hippocampus at p12 (p = 0.033) and p16 (p = 0.009). thus, els does impact the expression of markers of neurogenesis over development, however, all significant effects are restricted to early developmental time points (prior to p38). here, the effects of els on the development of spatial memory in a mouse model were tested. specifically, testing was carried out to investigate if sex differences exist in risk for the development of disturbances in spatial memory, and if effects were present, to identify when they emerge, and how long they persisted. els, in the form of maternal bedding restriction, led to early and persistent impairments in male performance on the nop task. in females, els was associated with impaired performance during the pre - adolescent period, but these effects resolved prior to young adulthood. the current results suggest that els may have sex and developmentally selective effect on cognitive functioning, with effects in males being early emerging and more persistent than those observed in female mice. in the current study, control reared male mice could perform the nop task as early as 21 days of age (p21) with females showing a trend toward successful performance at p21 and successful performance by p28. these results are consistent with the work of stanton and colleagues, who have found in rats that novel object location abilities develop as early as p21 (jablonski. thus, the task and chosen time points for measuring nop function were appropriate and allowed us to assess the impact of els on this behavior throughout early development. els led to persistent impairments in spatial performance in male mice and early but transient effects in females. the current study leveraged a modified version of the fragmented maternal care paradigm developed by the lab of dr. tallie baram (rice., 2008, bath., 2016). (2015). observed significant effects of els on cognitive outcomes in adult mice on the nop task : impaired performance in adult male mice and no impairments in adult females. the current results confirm the adult effects observed in that report, and significantly extend that work to demonstrate that female cognitive ability is also affected by els. however, the effects of els on female cognitive ability appear to be dependent upon the age of testing. specifically, impaired performance in els female mice was observed immediately post weaning (p21) as well as during pre - adolescent stage (p38), but not at other developmental time points. the current data suggest that female function may in fact be impacted by els, but that the effects in females either resolve, or that females develop an alternative strategy to promote success on this task by early adulthood. however, the mechanisms underlying the persistent impairments in memory function observed in males, or the transient impairments observed in females, are not fully understood. the neural mechanisms supporting nop performance have been investigated across a variety of species including rodents and non - human primates. based upon that literature, multiple nodes within a broader network based on lesion studies, the broader circuit supporting nop function includes the hippocampus (aggleton., 1992, barker and warburton, 2011) and more specifically a reliance upon an intact dentate gyrus (dg) and ca3, but not ca1 (lee., 2005). additional studies have shown that success on the nop task further relies upon an intact fornix (aggleton., 1991, chudasama and muir, 1997, eacott and norman, 2004), anterior thalamus (aggleton., 1991), anterior cingulate cortex (weible., 2009), peri - rhinal cortex (wiig and burwell, 1998, barker and warburton, 2011), and medial prefrontal cortex (rogers., 1992, chudasama and muir, 1997, barker and warburton, 2011). in pharmacological studies, nop performance has also been shown to be dependent upon cholinergic signaling. regional modulation of cholinergic activity in the frontal cortex or hippocampus (dunnett., 1990) or septum (torres., 1994, steckler., 1995) have been shown to contribute to impairments in nop performance. in the previous report by naninck and colleagues, the author argue that els - associated changes in dentate gyrus (dg) neurogenesis may mediate the effects of els on cognitive functioning in the novel object placement task. such effects could be consistent with prior work demonstrating involvement of hippocampal as well as para - hippocampal regions in object - location memory tasks (wiig and burwell, 1998, liu and bilkey, 2001, lee., 2005, parron., 2006, bachevalier and nemanic, 2008, assini., 2009). the current study did not directly assess rates of neurogenesis in dg, but instead included proxy measures of gene expression for markers of cell proliferation and differentiation in whole hippocampus. in all groups, a significant decrease in the expression of markers of cell proliferation and differentiation were found across early postnatal development, consistent with decreasing rates of neurogenesis over the early postnatal period. els led to a more rapid developmental decline in expression of these markers in both male and female mice. however, the majority of the effects of els on ki-67 and dcx expression dissipated by p21, the start of behavioral testing. during the adolescent and adult period, no effects of treatment or treatmentsex interactions were observed for gene expression, despite ongoing impairments in behavioral performance in els reared males, but intact performance in els reared female mice. ki-67 and dcx expression change over development and across the life - cycle of the cell, with ki-67 being expressed during the process of cell division and dcx being expressed during the process of cellular differentiation. in our previous work, declines in dcx gene expression were mirrored by a decrease in density of dcx - immunoreactive cells, suggesting that gene expression can serve as a proxy measure for quantification of cell number. based upon these data, markers of cell proliferation and differentiation do not appear to predict impairments in performance on this task. it should be noted, that in the work of naninck, the authors quantified rates of neurogenesis in the same animals that had performed the nop task, but in the current study, measures of ki-67 and dcx were collected from behaviorally nave animals. further, in the work by naninck, the authors observed elevations in rates of neurogenesis in both sexes, but sex selective effects on survival, which were correlated with performance. it is possible that gene expression levels for markers of proliferation and differentiation in the whole hippocampus, collected here, may not provide adequate sensitivity to detect changes in region specific (e.g. dg) rates of neurogenesis or differences in rates of survival of newly born cells. therefore, more intensive study would be required to fully resolve this issue. in the current report, els led to significant and persistent impairments in the nop task in male mice, but transient effects on nop performance in els reared females. multiple possible factors could contribute to deficits in nop performance, including stress associated changes in object interest (e.g. neophobia), elevations in anxiety - like behavior, stress effects on locomotion and object exploration, or stress associated changes in spatial memory processing. here, extensive analysis of rodent behavior was carried out across different phases of the task to identify whether deficits in performance were related to altered cognitive ability or some other variable, such as elevated anxiety or differences in object exploration. in the current analysis of the data, observed deficits in performance do not appear to be due to stress associated changes in locomotor function, object interest, or willingness to approach the objects. thus, the observed deficits are likely due to effects on processing of spatial information. in the current study, a 25-min interval was employed between the t1 exploration and t2 recognition phases of the task. this interval was chosen, as rodents show greater difficultly with task performance over extended delays, especially at younger ages (westbrook., 2014). by using a 25-min delay, this allowed for a task that was both sufficiently difficult and accessible to young animals. such studies may provide additional insights into disruption in spatial memory function in control and els reared male and female animals and identify further deficits in memory consolidation at these young ages. a principle goal of much of the work investigating els effects on cognitive development in animal models, is to provide model systems of early adversity that phenocopy traits associated with pathological outcomes in humans, and to then leverage those models to understand the neurobiological substrates of dysfunction. here, els in the form of maternal bedding restriction resulted in persistent impairments in spatial skills in male mice, but only transient effects in female animals. this work is consistent with prior work assessing cognitive function following rearing in a bedding restriction stress paradigm (naninck., 2015), and suggests replicability of this model across labs. in addition, this model may provide a useful tool for understanding the possible neurobiological substrates of risk, and possibly resilience, for cognitive deficits following early adversity, as well as a tool to better understand developmental changes in behavioral deficits and their relation to changes in symptom expression in human exposed to early adversity. however, it should be noted that previous work in other model systems of early life stress (including data from multiple strains of mice and rats) have yielded somewhat conflicting results. differences in outcome could be the consequence of differences in the timing of stress induction, the mode and severity of the stressor, the species used, or the timing and form of behavioral testing. specifically, in models of prenatal stress, some have observed selective impairments in female balbc mouse offspring (wang., 2011) while others found a male bias in impairments in various species of rats (mueller and bale, 2007, salomon., 2011, schulz., 2011, wang., 2016) using the nop or morris water maze tasks in adolescence or adulthood. still other studies using either mice and rats as model systems, failed to find any sex differences (benoit., 2015, nazeri., 2015) or showed els to be associated with improvements in cognitive performance (zuena., 2008, barbie - shoshani., 2016). studies investigating the effects of early postnatal stress manipulations on cognitive outcomes have been less numerous and have similarly resulted in disparate findings, that appear to depend upon the specific timing and form of stress used. for example, rats receiving low licking and grooming early in life showed improvements in spatial memory performance when tested as adults (barha., 2007). however, maternal separation or maternal deprivation, which involve greater handling of subjects, were associated with female selective effects, with effects being observed in adolescence and adulthood (wang., 2011, 2013). given that behavioral outcomes may be highly dependent upon the form, timing, and severity of stress, as well as the age at testing, a greater understanding of how manipulation of these variables can drive disparate outcomes will be important in understanding risk factors for negative outcomes and the importance of the timing of these events on brain and behavioral development. furthermore, in the context of translational research, care should be taken when considering the timing, duration, and method of stress, as well as the timing of testing of behavioral performance when considering models systems approaches to understanding pathology. here, a comprehensive developmental approach was used to assess the impact of els, in the form of maternal bedding restriction, on the development of spatial abilities in a mouse model. the current results significantly add to our understanding of the effects els on the development of spatial learning in this model, provide greater insights into sex selective disruptions in function, and provide clarity with regard to the timing of emergence of these effects. more work will be required to understand the neurobiological underpinnings of these effects, and the factors that confer risk and resilience in male and females. | disruptions in early life care, including neglect, extreme poverty, and trauma, influence neural development and increase the risk for and severity of pathology. significant sex disparities have been identified for affective pathology, with females having an increased risk of developing anxiety and depressive disorder. however, the effects of early life stress (els) on cognitive development have not been as well characterized, especially in reference to sex specific impacts of els on cognitive abilities over development. in mice, fragmented maternal care resulting from maternal bedding restriction, was used to induce els. the development of spatial abilities were tracked using a novel object placement (nop) task at several different ages across early development (p21, p28, p38, p50, and p75). male mice exposed to els showed significant impairments in the nop task compared with control reared mice at all ages tested. in female mice, els led to impaired nop performance immediately following weaning (p21) and during peri - adolescence (p38), but these effects did not persist into early adulthood. prior work has implicated impaired hippocampus neurogenesis as a possible mediator of negative outcomes in els males. in the hippocampus of behaviorally nave animals there was a significant decrease in expression of ki-67 (proliferative marker) and doublecortin (dcx - immature cell marker) as mice aged, and a more rapid developmental decline in these markers in els reared mice. however, the effect of els dissipated by p28 and no main effect of sex were observed. together these results indicate that els impacts the development of spatial abilities in both male and female mice and that these effects are more profound and lasting in males. |
primary cardiac tumors are quite rare, especially in the pediatric age group,13 and their atypical presentations often prevent a timely diagnosis.4 most primary cardiac tumors in the pediatric age group are benign ; autopsy studies in children have reported incidence rates ranging from 0.027% to 0.08%.4 one echocardiography database has reported an incidence of 0.17%, which suggests that one or two new primary cardiac tumors will be detected for every 1000 first - time pediatric echocardiograms.4 fibromas are generally reported as the second most common primary cardiac tumor in the pediatric age group.3, 5 however, in a more recent review of boston children s hospital database from 1980 to 2005, fibromas were the third most common tumor. to date, no distinct genetic inheritance or familial predisposition has been associated with cardiac fibromas. these neoplasms are often intramural and involve the left ventricular free wall or the interventricular septum. less frequently, they can be multiple and invade the right ventricular free wall, the atrial septum, or its free wall. although rare, intracavitary fibromas have also been reported.4, 6 it is very rare for primary intracardiac tumors to occur in the supravalvular pulmonic or aortic positions.7 a case of a seven - year - old boy with an intraluminal pulmonary artery fibroma was reported in the pediatric cardiology journal (21 : 480482 2000) from southwest texas methodist hospital. we herein present the case of a girl with an intraluminal fibroma in her ascending aorta. we present a 23-month - old girl admitted for the evaluation of a cardiac murmur with a history of one episode of syncope two months before admission. she had no positive history for syncope and no positive family history for congenital heart diseases or any kind of cardiac tumors. on physical examination, her growth and development were within normal limits (height = 85 cm, weight = 11 kg)., there was a grade 3/6 systolic ejection murmur at the second right inter - costal space with radiation to the neck. transthoracic echocardiography demonstrated mild left ventricular hypertrophy and a large intraluminal mass in the ascending aorta, but its borders were not obvious. doppler interrogation of the ascending aorta showed a turbulent, high - velocity ante grade flow with a 66 mmhg peak pressure gradient and a 46 mmhg mean pressure gradient. by color mapping, turbulency began at the level of the aortic valve. in order to confirm the transthoracic echocardiography findings, transesophageal echocardiography was performed, which confirmed the presence of a large intraluminal mass in the ascending aorta. the mass was elongated and occupied about 60% of the ascending aorta s area (3 cm 1 cm 1 cm). the turbulent antegrade flow around the margins of the mass was obvious and there was no more adherence. the distal end of the mass juxtaposed the initiation of the transverse aorta (figure 1). the high likelihood of the mass being tumoral and its hazardous location precluded catheterization, and the child underwent surgery for mass resection. given the position of the mass, cannulation via the femoral artery, inferior vena cava, and superior vena cava was done. with cerebral protection during cardiopulmonary bypass, the arteriotomy of the ascending aorta was preformed. the mass was found to be entirely within the ascending aorta with firm adherence to the anterior leaflet of the bicuspid aortic valve (figure 2). the tumor was excised subtotaly because it could not be detached from the aortic valve leaflet. the arteriotomy was closed, and the child was weaned from the cardiopulmonary bypass machine without any difficulty. repeat transthoracic echocardiograms following the operation did not reveal any evidence of residual supravalvular aortic stenosis and there was no aortic insufficiency. in gross pathology description, the mass was solid, firm, and creamy with a nodular appearance (3 1 1 cm). microscopic sections showed a benign neoplasm composed of fascicles of fibroblasts with variable amounts of collagen and a scanty number of lymphocytes arranged in a focally myxomatous stroma without any evidence of malignancy such as increased mitotic figures or areas of necrosis (figures 3a & 3b). the most common tumors in newborns and infants are rhabdomyomas, fibromas, and intrapericardial teratomas ; whereas in older children and adolescents, myxomas, rhabdomyomas, and fibromas are prominent. rhabdomyomas constitute 45% to 80% of all primary cardiac tumors in the pediatric age group. these tumors can be diagnosed in the prenatal period but are most frequently diagnosed in the newborn infant. although fibromas have recently been reported in utero and in patients younger than 1 month of age, they are found much less commonly than rhabdomyomas in this age group. in a recent review at boston children s hospital, fibromas were the second most common tumors (17%) in patients diagnosed between 1 month and 1 year of age. these primary tumors are rarely seen in older children, adolescents, or young adults.4 no known sex predilection is recognized, although the rarity of different benign cardiac tumors prevents an accurate determination of a male - to - female ratio.6 on two - dimensional echocardiography, cardiac fibromas are seen as a single, bright, intramural, echogenic mass. ct scanning is often performed and might provide clues regarding tissue characterization, with central calcification suggestive of a cardiac fibroma.6 this case has two interesting aspects : presenting symptom of the tumor and its location. our patient was previously healthy with a normal development, and she sought medical attention after a syncopal event and a murmur was noted on the auscultation of the heart. fibromas are predominantly intramural tumors, and extensive intramural fibromas can encroach and obliterate the intracavitary space. a similar case has been previously reported with an intraluminal fibroma in the proximal main pulmonary artery.8 although benign, fibromas may become life - threatening by causing arrhythmias or obstruction to blood flow.8, 9 acquired supravalvular aortic stenosis and syncope due to the tumor were the presenting features of our patient. although complete resection is preferable, our patient s tumor was not amenable to complete resection due to its firm adherence to the anterior cusp of her bicuspid aortic valve. syncope can be the presenting symptom of the tumors of the intraluminal ascending aorta as was the case in the patient described here with an unusual location of fibroma. transesophageal echocardiography is useful for the evaluation of anatomic details and can be of assistance for surgical planning. | primary cardiac tumors are quite rare, especially in the pediatric age group, and their atypical presentations often prevent a timely diagnosis. most primary cardiac tumors in the pediatric age group are benign. fibromas are generally reported as the second most common primary cardiac tumors in the pediatric age group. these neoplasms are often intramural and involve the left ventricular free wall or the interventricular septum. although benign, fibromas may become life - threatening by causing arrhythmias or obstruction to the blood flow. a case of supravalvular intraluminal ascending aorta fibroma in a 23-month - old girl, presenting with syncope, is described here ; the location is rare and the presentation atypical for this type of tumor. transesophageal echocardiography helped us to evaluate the anatomic details of the tumor and plan surgery. |
breast cancer is the most common cancer among women globally and one of the main concerns of societies, especially for women. according to the american cancer society report, breast cancer is diagnosed in about 1.3 million women annually worldwide and around 465,000 will die from the disease. present data is showing an increase in the annual incidence rates of the disease and is ranked as the first recorded malignancies among iranian women since 1999. it is also ranked as the second cause of death in females of all cancer mortalities. results of recent studies in iran indicated that the age of onset of breast cancer is about 10 years earlier than the developed countries. of the available cancer control measures for breast cancer, only screening has the potential for a rapid and major effect, though this will be restricted to a reduction in mortality rather than a reduction in incidence. establishing population - based screening programs, including periodical mammography, breast examination (bpx) by a trained medical staff and monthly breast self - examination (bse) is useful in detecting breast cancer at an early stage. bse+bpx as simple non - invasive tests would be a suitable option for countries in economic transition, where incidence rates are on the rise and limited resources do not permit screening by mammography. moreover, some studies have shown that annual mammography does not result in a reduction in breast cancer specific mortality for women aged 40 - 59 years compared with physical examination alone or the usual care in the community. it attempts to clarify whether bpx combined with bse would reduce the cumulative incidence of advanced stage (stage 3 or worse) and mortality from breast cancer in a population of yazd. the present work is the first report of a multicenter community based intervention evaluating the effect of bse+bpx on the reduction of morbidity and mortality of breast cancer. there are three distinctive phases in this trial with 10 years duration : phase - i included pilot of questionnaire (3 months) along with gathering the baseline data with 9 months duration, phase - ii comprised of active intervention phase with 4 rounds of annual screening of bpx+bse and phase - iii involves follow up with 5 years duration. following project approval by the deputy for research affairs and the ethics committee of shahid sadoughi university of medical sciences, a pilot study for validating project questionnaire begun in november 2008. subsequently, three months later, it was finalized and approved by the scientific committee of the project. data from previous studies were used to calculate the required sample size. considering 30% reduction in breast cancer mortality at a significance level of 0.05 (one - side) and a power of 0.9, a total of 12,660 women of 35 - 64 years age group (6,330 in the case group and 6,330 in the control group) were selected from four areas of yazd. the required sample of each group was selected by the mode of multistage selection. at first, two health centers from middle socio - economic (se) areas and two health centers from high se areas of the city were selected in the form of stratified random sampling. then, from each se area, one was randomly assigned as the intervention group and other as the control group. appropriate to the number of women in the age group of 35 - 64 years living in each area, the allocated size was divided into clusters (urban blocks). finally, the required number in each cluster was selected by determining the head cluster to complete the sample size (table 1). the exclusion criteria were having a personal history of breast cancer or detection of the disease at the beginning of the study, residing in the city of yazd for less than five years, suffering from severe illness expecting survival of less than 10 years, and disaccord to attend the designated center for annual evaluation. stages of sample selection of eligible women age 35 - 64 years allocated to study groups tools of enquiry were a pre - tested pre - coded questionnaire, physical examination and paraclinical procedures such as mammography and fine needle aspiration. firstly, all individuals were visited at their homes and debriefed on the importance of breast cancer and the goal of the project. after obtaining written informed consent, secondly, the participants in the case group were requested to attend a designated center to fill - in specific questionnaire followed by breast physical examination combined with training on self - examination of breast by skilled female general physicians. the flow diagram in figure 1 represents the follow up steps of the participants in the case group. shows the follow up flow diagram of intervention group attending health centers morbidity and mortality data of the individuals in the control group were enquired yearly by phone and adjusted with the active registry systems of cancer and death events available at the deputy for health affairs of the university. main outcome measures of the study are a comparison of cumulative incidence of advanced (stage 3 or worse) breast cancer, the survival rate of breast cancer and mortality rate from breast cancer among the study groups. evaluation and monitoring of the project were done weekly and monthly by the selected supervisors of the provincial deputy for health affairs, quarterly by the national supervisor of the program and by random control telephony of 5% of the filled questionnaire by the executive team of the program. using the spss software, descriptive statistics such as frequencies, percent, mean (sd), tests of chi - square and student t - test with 95% confidence level were analyzed. personal identifying information of a total 12,602 individuals in both groups (6,302 in experiment and 6,300 in control groups) was analyzed. comparison of socio - demographic and socio - economic factors such as age, age at marriage, family size, number of live births, occupation, education level, total family income and marital status of the participants showed no significant difference between the groups (p<0.05). the majority of those were householders (90% experiments, 91% controls), lower education level (62.9% experiments, 67.4% of controls) and married (94.3% experiment, 94.1% control). the response rate of individuals from both groups, during the phase of primary data collection at their homes, was 100% and the response rate of 84.5% was noted for the intervention group visiting the assigned health centers to undertake breast physical examination. the efforts for controlling breast cancer at the level of primary prevention are limited to a few risk factors with marginal effectiveness. consequently, the main attention and activity for the control of breast cancer have to be concentrated on secondary prevention level applying screening tests. developing countries are showing an increase in the incidence of breast cancer, but they face inadequate resources to implement screening with high - cost equipment such as mammography. since mammography is the only known effective screening test in reducing mortality from breast cancer, it requires expensive technology, highly trained radiologist, and radiographers, which is scarce in most developing countries. breast physical examination (bpx) has been recommended as an alternative to mammography in these settings. in an unscreened population in indonesia, bpx is known to be almost as effective as mammography in detecting prevalent breast cancers. furthermore, in women under the age of 50, there is little evidence of a benefit at least in the first 10 years after screening is initiated, or if a benefit exists it is less in older women. miller. also showed that annual mammography does not result in a reduction in breast cancer specific mortality for women aged 40 - 59 years compared with physical examination alone or with the usual care in the community. in this context, some commentators in the developed countries have strongly emphasized on the rationale in which screening by mammography should be urgently reassessed by policy makers. a greater benefit of early detection could be obtained in communities where most cancers that present clinically are larger and a higher proportion are node positive. in a study in boston (usa), 80% first abnormally sign or symptom of cancer was reported by self - detected breast and only 6% abnormalities detected by clinical breast examination. in a study done in india, a similar study was carried out in yazd in which 92% of women heard about breast self - examination, but only 17.4% performed it monthly and only 4.7% with good performance. data from the first year of our study showed that the response rate from participants in both groups with respect to data collection at their homes was 100% and for the participants in the intervention group visiting the assigned centers for the first breast physical examination was an acceptable level of 84.5%. these indicate that our study settings, design, and methodological manner were at a favorable level. on the one hand it is known that iran has a high number of mortality with an increasing incidence rate of breast cancer. additionally, marginal practice by iranian women results in late attendance by health centers. on the other hand, availing some evidence suggests that a benefit might be derived from a program that incorporated both annual bpx and bse. consequently, yazd province was selected as a national site for the community based trial of breast cancer. it is anticipated that the obtained results and experiences from this trial would generalize to other parts of the country. our study limitations were migration of few participants and loss of contact due to change in phone numbers that resulted in limited serial follow up and unavailability of national identification number. further details of the results obtained from annual follow - ups and related outcomes will be presented in the next reports. our data show that difference of socio - demographic and socio - economic factors between the groups were not statistically significant. in addition, with response rates of 100% in collecting the primary information at the homes of the participants as well as 84.5% for the experimental group in visiting the assigned centers for the first breast physical examination, it is fair to claim that our study settings, design, and methodological manner were at a favorable level. | there is some evidence to suggest that a benefit might be derived from a program that incorporated both annual physical examination of the breast (bpx) and the teaching of breast self - examination (bse). current investigation presents the profile of a multicenter community based intervention for evaluating the effect of bse+bpx on the reduction of morbidity and mortality due to breast cancer amongst women residing in urban areas of yazd (iran) from 2008 to 2018. there were three distinctive phases in this trial with 10 years duration : pilot phase with the duration of 1 year, active intervention phase with 4 rounds of annual screening of bpx+bse and follow up phase with 5 years duration. tools of enquiry included a pre - tested questionnaire, repeated annual physical examination of the breast and more importantly mammography, sonography, and fine needle aspiration (fna). data were analyzed using descriptive statistics such as frequencies, percent, mean (sd), tests of chi - square and student t - test with 95% confidence level. comparison of socio - demographic and socio - economic factors such as age, age at marriage, family size, number of live births, occupation, education level, total family income and marital status showed that no significant difference was seen between the groups (p>0.05). a response rate of 84.5% was seen by participants of the experiment group visiting the health centers for the first bpx. our results showed that except for the education and marital status, the difference in other main demographic and socio - economic factors between the groups were not significant, and the response rate of individuals in the experiment group was at an acceptable level. |
overweight or obesity among american children has reached epidemic proportions. over the past 25 years, childhood overweight or obesity has nearly quadrupled in the united states, affecting almost 17% of children and adolescents aged 219 in 2007 - 2008 [2, 3 ]. childhood overweight or obesity is defined as having a body mass index (bmi) greater or equal to the age- and sex - specific 95th percentile of the 2000 centre for disease control growth charts. overweight - obese youth are increasingly suffering from comorbid conditions once considered limited to adults. despite considerable efforts to halt or reverse the growing rates of childhood overweight or obesity paediatric overweight or obesity has been implicated in a myriad of serious health concerns. short - term consequences of overweight - obese status include chronic orthopedic and psychological disorders, nonalcoholic fatty liver diseases, metabolic syndrome, as well as a host of cardiovascular diseases such as hypertension and type ii diabetes mellitus [5, 6 ]. long - term consequences have proven the persistence of childhood obesity into adulthood, with an estimated 50% of obese adolescents becoming obese adults. this persistence into adulthood incites a cascade of cardiovascular risk factors and other chronic morbidities, dramatically increasing the risk for premature mortality. the economic burden of overweight and obesity on the american healthcare system is expected to intensify with persevering rates of childhood overweight and obesity. increased rates of overweight and obesity among children have been attributed to an increase in sedentary pursuits and a decrease in physical activity. furthermore, children who participate in higher levels of physical activity are less likely to display risk factors for cardiovascular disease and more likely to have positive outcomes in weight regulation [913 ]. motivating children to participate in physical activity may also preclude the persistence of childhood obesity into adulthood [14, 15 ]. promoting optimal physical activity levels among children can reduce the overall incidence and prevalence of overweight and obesity [16, 17 ]. further research is required to better explore and understand the relationship between physical activity and obesity prevention. with growing rates of childhood overweight and obesity, it becomes increasingly more important to promote healthy eating as poor dietary behaviors are a known risk factor for the development of obesity [19, 20 ]. in addition, nutritional deficits and poor eating habits that develop in youth have been implicated with long - term health, growth, and developmental issues. obesity often results from an energy imbalance, with a greater energy intake to expenditure, with overweight or obese youth less likely to compensate for excess energy intake throughout the day than normal - weight children. exploring strategies that reduce overweight and obesity by targeting physical activity and healthy eating is a necessary endeavour. the united states department of agriculture 's dietary guidelines for americans from the age of 2 years old focuses on balancing caloric intake with physical activity. the message purported is to decrease the amount of calories consumed and increase calories expended through physical activity. to achieve this, the surgeon general suggests the following for a healthy lifestyle : an increase in consumption of fruits, vegetables, whole grains, and lean proteins ; reduction of sodas and juices with added sugars ; increase amounts of water consumed ; limit dairy products to low fat or nonfat ; be more physically active, including limiting screen time to a maximum of 2 hours per day. in addition, the centers for disease control and prevention recommends that children and adolescents partake in 60 minutes of physical activity a day. this should consist of aerobic activity (making up the bulk of the 60 minutes), muscle strengthening activities (a minimum of 3 days a week), and bone strengthening activities (a minimum of 3 days a week). efforts to mitigate the formidable effects and prevalence of childhood overweight or obesity have been aimed at reducing sedentary behavior and poor nutrition ; indeed, compared to the rest of the world, early adolescents in the united states exhibit the worst rates of physical activity and the least healthy diets. while several factors have been blamed for this disparity in healthy behavior, including preference for indoor pastimes, low energy levels, time constraints, unsafe neighborhoods, a lack of motivation, insufficient resources and poor social support, it is screen - based activities that seem to garner the most public criticism. while both watching television and playing video games have been accused of increased sedentariness among youth and growing rates of childhood overweight or obesity, video game playing has shown the strongest positive correlation, with the duration of screen time forecasting weight status [29, 30 ]. the united states houses the highest percentage of youth under 18 years of age using the internet, with approximately 93% of teens (aged 1217 years old) going online. while only 15% of households do not have a home computer 83% of american youth have access to at least 1 video game console in their bedroom. thus, as a possible forward - thinking strategy, researchers can look at video gaming as a means of promoting physical activity and healthy nutrition among at - risk children, replacing passive screen time with active screen time. exergaming (video games that are a form of exercise) can be used to motivate direct physical activity in combating overweight and obesity among children. similarly, interactive educational video gaming can aid in developing self - care abilities and healthy behavioral skill building. the goal of this paper is to enlighten researchers to the possible benefits of active and educational video games targeting diet and physical activity in children. our objective is to review the current literature on the role of video games (development and use) in the prevention of childhood overweight and obesity and provide a summary of findings that can be used to spur future research. we conducted a systematic review of the literature utilizing the bibliographic databases embase and pubmed in december 2010. the following search terms (mesh headings for pubmed and keywords for embase) were used : obesity, overweight, physical activity, fitness, exercise, energy expenditure, heart rate, energy metabolism, nutrition, bmi, diet, video gam, exergam, active video gam, active computer gam, new generation computer gam, exertainment, active gam, and computer gam. search terms were determined by examining the previous literature in the area. the search was limited to articles written in the english - language and published between 1998 and 2011. articles focusing on a 0- to 18-year - old population were included (preschool child, school child, and adolescent in embase). two reviewers (s. guy and a. r.- leewing) hand searched references present in the included articles with a specific focus on journal articles discussing the use of video games to combat obesity. we retrieved 181 studies in total, 87 from embase and 94 from pubmed. further examination eliminated 4 references not classified as journal articles (2 were conference abstracts, 1 comment, and 1 forum document). each article was reviewed by 2 reviewers (s. guy and a. r.- leewing). articles were excluded if they only described the negative effects of screen time (watching television, playing video games, surfing the internet) on physical activity levels. articles that only described obesity interventions in general without specific reference to video game interventions were also excluded. of the 124 articles, 18 pertained to interventions utilizing video games and/or computer games. upon hand searching references within retrieved articles, 18 journal articles were included (figure 1). a previous literature review was eliminated ; however, it was examined for potentially useful references. systematic reviews [26, 3335 ] discovered during hand searching were used to inform study choice and discussion material. the results are organized into sections based on the type of game (table 1). all studies in this section focused on exploring physical activity and on determining the amount of physical activity expended during active video game play. (2008) conducted a randomized controlled trial with a 12-week home - based dance dance revolution (ddr) (konami digital entertainment, redwood city, ca) intervention. the comparison group received ddr to use as often as they desired, while the multiplayer group played ddr with other children once a week in a 60-minute class at a sports center. a total of 29 children with a low fitness level aged 912 years from 4 primary schools were recruited. (2008) measured aerobic fitness, physical and sedentary activity through self - report, anthropometry, body composition, playing time through self - report, and perceived competence in sport. results showed that the multiplayer group played the game more (901 min) than the comparison group (376 min) ; however, this was nonsignificant. epstein. (2007) examined the reinforcing value and activity levels of active dance and bicycle games in 18 overweight and 17 nonoverweight children aged 812 years. there were 3 conditions for the dance game (dancing with music, dancing with a video, and ddr) and 3 conditions for the bicycle game (bicycle alone, bicycle with video, and freekstyle) (electronic arts, redwood city, ca). findings show ddr to be more reinforcing than dancing alone or dancing while watching the video ; however, no difference was found across the 3 bicycle conditions. graf. (2009) compared energy expenditure rates in children playing active video games in relation to treadmill walking. the sample comprised of 23 healthy children ranging in age from 10 to 13 years old. participants were measured for energy expenditure, heart rate, step rate and perceived exertion watching television, playing ddr at 2 skill levels, playing wii bowling and boxing (nintendo, redmond, wa), and walking at 2.6, 4.2, and 5.7 km / h. energy expenditure while playing the active video games or walking increased 2- to 3-fold in comparison with watching television. wii bowling and beginner level ddr elicited a 2-fold increase in energy expenditure compared to watching television. a study conducted in the united kingdom compared energy expenditure during sedentary and active gaming. eleven participants aged 1315 years played 4 computer games for 15 minutes each : sedentary xbox 360 (microsoft, redmond, wa), wii sports bowling, tennis, and boxing. (2007) found that energy expenditure was 51% greater during active gaming and highest during wii tennis. further expanded on the energy expenditure in upper limb movement in comparison to total - body movement while engaged in sedentary and active video gaming. youths between 1117 years old played 3 active wii games and one sedentary video game (xbox 360). energy expenditure, heart rate, and nondominant upper limb activity were significantly greater during boxing in comparison to tennis or bowling. (2009) measured oxygen consumption, energy expenditure, and perceived exertion during a comparison of stationary cycling with and without a video game. children participating in this study (n = 20) were at risk for overweight and between the ages of 714 years old. participants rode a stationary bicycle (cateye gamebike, usa) for 20 minutes. testing session 1 consisted of riding the bicycle, while in session 2 the bicycle controlled the speed in the game cars (pixar entertainment inc.). energy expenditure was significantly higher (4.4 1.2 k cal min) when cycling in conjunction with the video game than riding the bicycle on its own. a comparison of energy expenditure during sedentary video gaming and television viewing, active video game play, and treadmill walking was undertaken by lanningham - foster. energy expenditure was calculated while participants watched television seated, played a traditional video game seated (disney 's extreme skate adventure activision, los angeles, ca), watched television while walking on a treadmill at 1.5 mile per hour, and played active video games (nicktoons movin ', thq, calabasas hills, ca ; ddr ultramix 2). active video games resulted in a larger increase in energy expenditure than playing the traditional video game seated. energy expenditure increased by 382 181 kj / hour above levels of energy expended during rest. obese children had significantly greater increases in energy expenditure in response to active video games. 2007) examined energy expenditure and physical activity associated with playing active and sedentary video games using playstation 2 (sony corporation, tokyo, japan) eyetoy games. each participant (n = 21) completed the study protocol. this involved resting while seated, playing a sedentary video game, and playing active video games (eyetoy knockout, homerun, groove, antigrav, and dance uk). significant increases in energy expenditure and heart rate were found during active game playing. step counts increased from 122 to 1288 steps during active video games in comparison to sedentary game play. the energy expended during active video game play was comparable to light or moderate exercise. a recent publication by maddison. (2009) provides an overview of their egame study (a 2-arm parallel randomized controlled trial) and includes the plan for phase 3 which is their future direction. the phase 3 objective is to determine the effects of an active video game intervention (sony eyetoy) over 6 months on bmi, body composition, waist circumference, cardiorespiratory fitness, and physical activity levels in 330 overweight children. all australian participants ranging in age from 10 to 14 years will be randomized to either an active video game upgrade package or to a control group (no intervention). phase 1 contained focus groups with children, and phase 2 was a laboratory study (described above). a 6-month pretest, posttest trial with a noncomparative design was conducted by madsen. obese children (n = 30) aged 918 years old were provided with ddr and motivated biweekly with a semistructured telephone interview for 24 weeks. the goal was for children to play 30 minutes a day for 5 days a week. findings show that few children used ddr regularly with a lack of association between ddr use and change in bmi. children reported that group play, competition, and greater variety would increase their motivation to play. maloney. (2008) [45, 46 ] examined physical activity and enjoyment with a controlled group comparison design of an intervention home - based ddr play and control group. a total of 60 children between the ages of 7 - 8 years old participated in a 28-week study. the authors collected self - reported screen time, ddr use, accelerometry, pedometry, body composition, blood pressure and pulse, anthropometry, game satisfaction, and assessment of participation support. participants played ddr for 89 min per day (mean) and used the game the most in week 1. absence of other video games and parent participation was associated with this high level of usage in week 1. by the end of the study, mcdougall and duncan (2008) reported a mixed - methods, noncomparative study, where british children (n = 12) aged 811 years old engaged in school lunch - time active video game play for 1 week. children played on average for 24 minutes per day with game play resulting in 10% to 11% of the recommended steps allocated daily and 11 minutes of sustained moderate - to - vigorous physical activity per day. mellecker and mcmanus (2008) examined energy expenditure and cardiovascular responses in children during seated (10-pin bowling computer game) and active gaming (xavix bowling and j - mat) (shiseido, tokyo, japan). a total of 18 children aged 6 to 12 years participated in a 25-minute gaming protocol : 5 minutes of seated, 5 minutes of seated bowling, 5 minutes of xavix bowling, 5 minutes of seated rest, and 5 minutes of xavix j - mat. in each game format, energy expenditure was significantly higher than resting (39% for seated bowling, 98% for xavix bowling, 451% for xavix j - mat). (2009) tested the feasibility of a newly developed walking media station they had created. twenty - nine healthy children between the ages of 6 and 13 years old tested the media station in a laboratory and home setting. each participant completed the following protocol : rest, playing a computer bowling game seated, walking, and walking while playing a computer bowling game. murphy. (2009) randomized 35 (17 female, 18 male) overweight children with endothelial dysfunction, between the ages of 7 and 12, to 12 weeks of exercise using ddr or to a nonexercising delayed treatment control group. the exercising intervention group saw significant improvements in flow - mediated dilation, exercise time on a graded test, mean arterial pressure, weight, and peak vo2 compared to the control group. during the study 13 intervention participants achieved normal endothelial function. a randomized controlled trial conducted by ni mhurchu. (2008) evaluated the effect of home - based active video game play on the physical activity levels of 20 children aged 10 to 14 years in new zealand. the intervention group (n = 10) received an upgrade package for their sony playstation 2 console, including a sony eyetoy camera, eyetoy active games, and dance mat. the participants and families were instructed to substitute usual video game play with active video games. the control group received the same upgrade package at the end of the study without an intervention. the authors used the following measurements to evaluate physical activity : accelerometry, self - reported activity, physical activity questionnaire, established activity compendium, anthropometry, and body composition. results showed that physical activity was significantly higher in the intervention group than the control at both followups. no significant group differences were found between time spent in moderate or vigorous physical activities. the intervention group showed weight loss (mean = 0.13 kg) and a reduction in waist circumference (mean = 1.4 cm) at the end of 12 weeks. the physiological cost, relative reinforcing value, and satisfaction of playing wii sports bowling compared with wii punchout ! (a traditional sedentary video game) was the subject of investigation for penko and barkley (2010). a sample of 11 lean and 13 overweight or obese 8 to 12 year olds participated in 4, 10-minute activity sessions : resting, treadmill walking, sedentary video game play (wii punchout !), and active video game play (wii sports boxing). participants performed a computer task designed to assess relative reinforcing value. results showed that average heart rate, oxygen consumption, and liking were significantly greater for wii than all other conditions. ridley and olds (2001) examined energy cost and observed the behavior of children playing video games and games in an australian game centre. the authors measured the energy cost of 10 elementary school children (5 females and 5 males aged 1012, with a mean of 12.5 years) under 5 experimental conditions which were each 5 minutes in duration : seated in front of a sedentary video game and playing 4 games daytona (a simulated driving game), final furlong (a simulated horse - racing game), air hockey (table hockey), and mini dunxx (a minibasketball shooting game). gross energy cost ranged from 7.6 to 2.5 ml kg min. (2010) conducted a study in hong kong examining preferences and physical activity levels during interactive electronic games. seventy overweight and nonoverweight children aged 9 to 12 participated in 2, 60-minute recreation sessions. session 1 involved playing either an active (xavix bowling) or an online bowling game. session 2 was a choice of an active (aerostep, shiseido co. of japan) or an online electronic running game. participants chose to play the games during 94% of their session time with time split between active (52%) and online (48%) gaming. participants who were male (n = 35) and nonoverweight (n = 50) expended relatively more energy during active games than females and overweight children. a within - subjects design of a comparison between cardiovascular response and energy expended among television watching, sedentary gaming, and active video game play was carried out by straker and abbott (2007). measures of interest include heart rate, energy expenditure, oxygen uptake, and ventilation. twenty healthy 912-year - old children, who had previous experience with playing electronic games, were recruited. energy expenditure and heart rate increased from resting during active video gaming (sony eyetoy) by 224% and 59%, respectively. the exertion levels observed during active video game play were equivalent to moderate intensity activity. (2006) determined the difference between the submaximal energy cost of movement and cardiorespiratory measures for overweight and nonoverweight children playing ddr. each group of children (n = 22) completed 12 minutes of ddr and a maximal treadmill walking test. there was no difference in heart rate and energy costs associated with ddr between overweight and nonoverweight groups. heart rate intensity levels, but not oxygen consumption reserve, were sufficient for developing and maintaining cardiorespiratory fitness. these games are either stand - alone or embedded within a larger intervention study containing elements such as face - to - face group education sessions. (2011) [5759 ] targeting healthier food choices (fruit and vegetable consumption), body composition, and physical activity were evaluated in a 2-group randomized controlled trial. participants (n = 133) aged 10 to 12 years participated in either the intervention group, which played escape from diab and nanoswarm : attack from inner space in sequence, or the control group, which played diet and physical activity knowledge - based games on popular websites. the authors found that playing the video games in the intervention increased fruit consumption by 0.67 servings per day but did not increase water intake, moderate - to - vigorous physical activity, or body composition. the fun, food, and fitness project is a randomized 2-arm parallel randomized controlled trial designed to prevent obesity. this 12-week intervention, taking place at a summer day camp and at home, was designed to motivate the participants (n = 35, 8 years old, african - american females) to eat 5 servings of fruit and vegetables, drink 5 glasses of water, and engage in 30 minutes of physical activity per day. participants in the intervention group attended a special 4-week summer day camp and received an 8-week home internet intervention featuring a computer game with comic strip characters who overcome barriers to goals with regard to physical activity. the control group attended a summer camp with usual activities followed by a monthly home internet intervention without the game. squire 's quest [61, 62 ] is a multimedia game that aims to increase preferences for fruit, juice, and vegetable consumption among children. in a 2-group a population of 1578 4th grade students were recruited to participate in the study. the intervention group received 10 sessions twice a week with a duration of 25 minutes of squire 's quest. findings indicate an increase of fruit and vegetables servings per day (1.0) in the intervention group. the mypyramid blast - off game educates children about the food pyramid and physical activity. in a pretest, posttest study moore. a total of 126, 4th and 5th grade washington, dc students took part in an intervention of 6 classes taught over 3 months aimed at increasing knowledge about nutrition and physical activity. both schools received the educations and activity content. while 1 school received a more didactic presentation on playing the blast - off game, the other school required students to use individual computers to evaluate their diets in small groups. results of this study show an increase in nutrition knowledge of the control group. in both groups activity time was increased and systolic blood pressure decreased. (2008) evaluated the use of 2 interactive games a video game and a board game that were interconnected in relation to theme, character, and foods in a nutrition education program for obese children in brazil. two hundred children aged 810 years took part in this study with each taking part in weekly 30-minute game sessions over a 4-month period. both games were based on the food pyramid and promoted the learning of nutritional concepts. pempek and calvert (2009) reported a cross - sectional study examining how marketing games (advergames) affect the consumption of snack type. participants (30 low - income 3rd- and 4th - grade african - american children) were randomly assigned to 1 of 3 conditions of which they would play 2 levels of each game : a healthier advergame, a less healthy advergame, or the control group. the classic arcade game pac - man (namco, tokyo, japan) was used as a prototype for 2 versions created. in the healthier game option, 10 points were awarded for each nutritious snack eaten and penalized the same amount of points for every less nutritious snack. while in the less healthy option children were rewarded 10 points for every less nutritious snack and penalized for every healthier snack eaten. in the intervention groups children were asked to choose a snack after they played the game. in the children playing the healthier version of the game selected and ate significantly more healthy snacks. metakenkoh is an internet - based adventure game that targets physical activity promotion and healthier food choices. d. r. southard and b. h. southard (2006) present preliminary results of a 4-week randomized controlled trial of 63 children aged 911 years. intervention group participants played a game and wore a pedometer (with the pedometer controlling game performance)., underweight and normal weight children in the intervention group showed an increase in activity. in comparison, a decrease in physical activity was observed in the control group. the overweight and at - risk participants in both groups showed a slight increase in activity levels. in the boy scout 5-a - day badge study, 473 boy scouts aged 10 to 14 years old participated in a 2-group randomized controlled trial for 9 weeks. this program was aimed at increasing fruit and vegetable consumption that included knowledge games focusing on diet and contained interactive comic characters that underwent challenges to eating more fruits and vegetables. while the game can not be evaluated by its own, the study saw an increase of 0.83 servings per day of fruit and vegetable and a 1.24 increase in fruit and vegetable items available at home in the intervention group. (2001) conducted a 2-group randomized controlled trial in 1876, 3rd to 5th graders (16 schools) evaluating 4 nutritional teaching computer games aimed at increasing nutritional knowledge and improving eating habits. the schools taking part (16) were randomized into 2 groups : an intervention group, which received nutritional learning games ; and a control group, where a teacher provided the nutritional information. (categorization of food), guess who (food contents), granny smith (food choices), and the restaurant (nutritional balance) for 1 hour twice a week for 5 weeks. researchers have utilized a number of commercial active video games or new - generation video games in an effort to quantify their impact on children 's physical activity levels. the games included in the reviewed papers are aerostep ; dance dance revolution ; wii sports ; freekstyle ; nicktoons movin ' ; eyetoy games such as knockout, xavix bowling ; xavix j - mat. educational video games, either as a stand - alone or part of a larger intervention, are predominantly focused on dietary and nutrition issues. games included in this paper include escape from diab, guess who, granny smith, nanoswarm, squire 's quest, mypyramid blast - off game, the restaurant, an altered version of pac - man, metakenkoh, and store. assumptions are often made about the value of video games and their potential to improve lifestyle and dietary habits. although video games may have benefit, it is equally important to have an evidence - based approach to developing and evaluating these games as they are in fact an intervention. appropriate strategies to evaluate the games and deliver them in ways that are reproducible are important from a knowledge translation perspective. we found a lot of heterogeneity in terms of the outcomes also, both the metrics that were used and the methods of measurement. it may be time to consider some guidance for those who design and use these games in research to have common metrics that can be compared across different games. this will, in the future, allow us to provide more relevant and interpretable comparisons. evidence is indisputable about the need for an ecological, multilevel approach in childhood obesity interventions. educating both the parent and child, given the complex interactions between social determinants in a family setting, is essential to the success of an intervention. it is unreasonable to expect a total and significant behaviour change or outcome based on modifying nutrition knowledge alone. while education is undoubtedly important for motivating behaviour change, other factors such as home environment, parent income, and parental education level have been implicated in obesity. although a parent 's knowledge of healthful eating and their socioeconomic status may play a role in obesity, it is necessary to note these 2 factors may not be the main driving forces. a recent report of national health and nutrition examination survey data showed that (a) childhood obesity prevalence decreased as the education level of the head of the household increased ; however, this trend was not consistent across race and ethnicity and (b) the majority of children and adolescents who are obese are not from low - income households. this suggests that a lack of financial resources may not be a main barrier to healthful eating. further research is needed looking at the role an educated child can play in transforming the outlook of the family as parents. while additional research is needed to understand and determine the most effective avenues to promote healthy eating, it is well supported that educating children in healthy eating not only provides immediate benefit but fosters long - term health habits as well. thus, while active and serious gaming poses an excellent opportunity for increased physical activity and nutritional knowledge, it is not a sole solution to the obesity epidemic among children. looking at integrating avg and educational gaming in classroom settings or in collaboration with community or national level programs such as the expanded food and nutrition education program (efnep) is a potential step forward to using gaming as a tool for combating obesity. research has proven that avg use can elicit light to moderate physical activity among youth. while we are not advocating that a child only plays video games to get their recommended daily physical activity, we are proposing that physical activity as a result of avg engagement can contribute toward daily recommendations of physical activity. gaming companies invest significant resources into determining the multifactors that influence choice and duration of game use. so the question becomes, how can we sustain use of health promoting games in children ? one avenue of exploration is the involvement of the family unit parents and siblings. emphasizing the social aspects of gaming, including competition and feelings of camaraderie, may be an effective means of promoting game use. a review conducted by biddiss and irwin (2010) found fun to be the primary reason for participation in physical activity. conducting needs assessments to determine what game factors constitute fun may not only encourage physical activity while gaming but additionally promote user sustainability. given the fact that a child 's attention is already captured by video gaming, why not develop games targeted at obesity reduction that use active new generation style games to increase children 's knowledge and self - care ability ? the time that children already spend playing video games can be simultaneously used to promote physical activity and health behavior education. the climate in north america lends itself to periodic episodes of limited outdoor play thus making safe, indoor play an easier option. capitalizing on the novelty of active video gaming can encourage physical activity and simultaneous learning, a strategy which again may promote sustained use of the game. engaging participants in game development is a strategy employed by many corporations. by examining product development and marketing strategies using these strategies in a pure research setting may prove advantages when developing an appropriate and effective avg for overweight and obese children. self - initiation and choice are key factors in motivating physical activity among children. intrinsic motivators such as enjoyment, mastery, and achievement drive initiation and long - term continuation with behaviors. biddiss and erwin (2010) suggest five strategies on how to sustain video game play in children. first, avgs must provide positive feedback and be accessible through low cost and ease of use. second, early exposure to active gaming, as opposed to passive or sedentary gaming, may encourage greater acceptance of avgs ; this strategy may allude to a future need for games that appeal to a wide range of ages and interests. third, acceptance and motivation to play avgs may increase when game play is perceived as a personal choice rather than a prescribed treatment therapy. and finally, short- and long - term reinforcement (i.e., enjoyment, goal - achievement, and skills development, resp. additional research on video gaming and motivation is required to explore goal setting and achievement as well as factors that initiate game play among children. also, intensities and durations of physical activity over extended periods (> 12 months) must be analyzed. a propensity toward short - term home - based studies has curtailed research in long - term adherence and efficacy of avg use among children (> 7 months). additional long - term research is required to determine the role of game diversification, the importance of story or plot development in avgs, and the potential benefits of group play. while these strategies have been successful for short - term game play, their role in long - term adherence and efficacy is yet to be determined. given the prevalence of past studies and media attention towards the negative effects of video gaming (e.g., increased screen time), it is important to consider the potential barriers that may impede avg use. research into overcoming potential barriers would be advantageous to the future of health gaming. an interesting avenue for future research is the potential of avg play as an entry way into organized sports. psychological factors accompanying avg play may include increases to self - efficacy, self - competency, and self - empowerment. these qualities may be translated to an increase in confidence and attitudes towards organized sports, ultimately leading to a greater likelihood of the child participating in organized sports activities. as children learn and become more familiar with the rules and play of sports activities, they may become more inclined to participate in physical activity. an additional area to question and possibly incorporate into the research is what value do people put on their health ? the premise is that people will want to play games that improved their self - health and quality of life. however, we do not really know whether people play games, because it is a game. knowing this will help us design games that clearly state that their value has more subtle or hidden if people value their health, they may be more likely to engage in healthful behaviours in a sustainable way rather than sporadically. would knowing that the game is aimed at promoting healthy behaviour encourage or impede the use of the intervention by children ? clearly with the environmental constraints in both emerging economies and developed economies, where access to green space and exercise facilities is limited, consideration has to be given to other avenues for exercise, which makes a good case for healthful games. interdisciplinary research teams would prove invaluable to the development of health video games ; individuals with specialized knowledge in one field may collaborate with an expert in another to ensure a tailored intervention. the stigmatization of video gaming, with implications of increased sedentary screen time and decrease in physical activity, is slowly being eroded by the advancement of active video games. the potential of active video gaming or exergaming in the fight against childhood obesity is evidenced through the studies referenced in this paper. however it is also important to consider interactive educational video games that aid in self - management and skill building as an equally valuable tool to combat childhood obesity. in a society so dependent on technology, using that dependency to create a more healthful existence becomes an easy choice. the popularity of games such as farmville (zynga inc.) and the realms of second life (linden research inc.) suggest a new avenue of games in obesity prevention and self - care : social gaming. rather than fight the enormous role (given how many hours children spend playing them) gaming has in children and adolescent 's lives, we should embrace the opportunity to influence health behaviour through this avenue and use an evidence - based approach to do this in a thoughtful way | increasing epidemic proportions of overweight children in the united states presents formidable challenges for education and healthcare. given the popularity and pervasiveness of video gaming culture in north american children, the perfect opportunity arises to investigate the potential of video games to promote healthful behaviour. our objective was to systematically review the literature for possible benefits of active and educational video games targeting diet and physical activity in children. a review of english - language journal articles from 1998 to 2011 using embase and pubmed was conducted. thirty - four studies concerned with children, video games, physical, and/or nutritional outcomes were included. results of these studies that showed some benefit (increased physical activity and nutritional knowledge as a result of gaming) demonstrate the possibility of video games to combat childhood obesity looking beyond the stigma attached to gaming. |
since first described by quain in 1847 (1), hundreds of abdominal aortic stenosis have been reported with many variations in anatomy. though, the complete absence of infrarenal aorta, seen in presenting case, is extremely rare. this represents the first case of complete segmental occlusion of infrarenal aorta in the old patient. a 52-yr - old woman presented to the clinic with one - year history of bilateral claudication. blood pressures were measured at 130/80 mmhg in the forearms and at 100/70 mmhg in the thighs, with ankle - brachial indexes of 0.77. serologic tests including c3, c4, venereal disease research laboratory test (vdrl), and immunoglobulin g were unremarkable except mild elevated erythrocyte sedimentation rate (esr) of 22 mm / hr and low tittered anti - nuclear antibody of 1:80 with speckled pattern. the abdominal aorta was reconstituted by multiple collaterals, such as marginal artery of drummond, the anastomosis between superior mesenteric and inferior mesenteric artery, bilateral epigastric, lumbar, and abdominal superficial arteries. for her knee joint arthritis, she was started with methylprednisolon of 8 mg and non - steroid anti - inflammatory drug. after negative results of pathergy test and human lymphocyte antigen b 51, we ruled out the behcet 's disease and she tapered the medications to stop successfully. she wanted the antihypertensive drug for mild hypertension and was taken amlodipine at 5 mg daily. one year later, computer tomographic angiography showed patent bypass graft and decreased collateral vessels (fig. the etiology of abdominal aortic stenosis is poorly understood. the young age of most of the patients suggests that the malformation may be congenital. maycock first proposed an unequal fusion of embryonic aorta as a congenital mechanism (3). in first month of fetus, the paired primitive dorsal aortas fuse to form the abdominal aorta. incomplete fusion due to infection or inflammation may lead of kinking of the aorta resulting to permanent constriction. congenital rubella has been suggested for such vascular anomaly (4, 5). in alagille syndrome which is congenital pulmonary artery stenosis transmitted in an autosomal dominant inheritance, deletion of the jagged 1 gene has been suggested to relate with abdominal aortic coarctation (6). acquired etiologies were also suggested in fibromuscular dysplasia or takayasu 's arthritis (7, 8). in our patients, several inflammatory presentations including the knee joint arthritis, recurrent oral ulcers, and elevation of esr have suggested the possibility of behcet 's disease. we ruled out the behcet 's disease after the negative study of pathergy test. considering the complete absence of infrarenal aorta without any inflammatory reaction, in review of cases for abdominal aortic stenosis, coarctation, hypoplasia, and middle aortic syndrome were used diversely. the term " coarctation " implies a congenital anomaly, and " hypoplasia " is to longer segment of coarctation. middle aortic syndrome is the clinico - anotomical entity involving long segmental narrowing of the abdominal aorta and various visceral arteries irrespective of its etiology. we described this case by " hypoplasia ", because 4 cm of infrarenal aorta was absent. presenting symptoms of abdominal aortic hypoplasia are related to the extent of the stenosis, and the collateral system. in case involving renal artery, patients typically present with uncontrolled hypertension. infrarenal aortic hypoplasia can give rise to claudication when the collateral circulation is not sufficient. an abdominal aortogram via angiography, ultrasound, computed tomography, or magnetic resolution imaging can be used to diagnose and to determine the location and extent of disease. in case of renal artery stenosis claudication and aneurysmal dilatation also need surgical correction. in this case, as the subrenal aortic stump was too short, graft was interposed between the subdiaphragmatic thoracic aorta to the iliac bifurcation. careful physical examination for inspection of dilated superficial arteries and auscultation of bruits can provide a clue for rare causes of ileofemoral insufficiency such as abdominal aortic stenosis. when suspected, an angiography is mandatory to diagnose, and surgical intervention is necessary to improve severe claudication. | we describe a case of infrarenal aortic hypoplasia in a 52-yr - old woman who presented with claudication. computed tomographic angiography revealed an abrupt absence of the infrarenal aorta, with collateral flow reconstituting the iliofemoral systems. after a polytetrafluoroethylene graft was interposed between the aortic stump and the iliac bifurcation, the patient 's claudication resolved. |
every time we discuss about immunization program in adult and in elderly people, we should consider two important changes that happened in our current society : the increasing ageing of population and a different old age compared with the past. it has been estimated that since 2005 to 2030 world population over 65 will double (from 500 million to 1 billion). in 2030 elderly people will be 40% of all population and in italy in the next 10 years elderly people will be the double compared with 0 - 14 population. elderly people showed some social and demographic characteristics in the past decades : in the end of working life few years remained to live ; the end or working life coincided with exclusion of every role in social or recreational activities ; in the most of cases after - work activities were domestic (particularly for women) ; younger generations took care of elderly, usually inside the same home. today we are witness to a new paradigm : usually a 65 years old person does n't feel old ; usually life expectancy at the time of retirement is long ; most of retired people are included in social and recreational activities ; elderly people claim quality of life ; over 80 people are increasing in number and usually they live with comorbidity and they represent a high cost for each health service. every year, influenza is responsible of 40,000 deaths in europe, most of them in elderly people and with chronic diseases. the economic burden to manage these preventable diseases with adult immunization, other than year - of - life lost, is more than 10 billion dollars every year. in spite of availability of effective and safe vaccines for these diseases, they are underused. which are the reasons to immunize adult people with vaccines ? at least one of these issues is a good reason to immunize them : because they did n't have immunization in childhood ; because at present time new vaccines are available ; because human immune system gets old and acquired immunity can decline ; because elderly people and people with chronic diseases have more susceptibility to diseases preventable with vaccines (influenza, pneumococcal pneumonia). the lists of immunizations for adults and high risks subjects are reported in tables i and ii. vaccination schedule for adults broken down age group. recommended when there are other risk factors (chronic disease, occupational, behavior). adult immunizations for high - risk groups and categories. recommended when there are other risk factors (chronic disease, occupational, behavior). tetanus, diphteria and pertussis (tdp) immunization is strongly recommended because in italy the most of cases of tetanus are in adults who did n't receive any other dose after childhood. about pertussis in italy, as in other countries, the immunization is very spread in childhood population, but the immunity related to vaccine declined in the next 6 years and so young adults, adults and elderly become susceptible to disease another time. we recommend that every time patients request tetanus immunization or when we suggest tetanus immunization, we can propose the booster with tdp immunization. the high degree of antigenic shift and drift in circulating influenza strains leads to the periodic introduction of strains with high susceptibility in the population, resulting in pandemic spread of disease as in 2009. influenza vaccine is effective and safe to prevent influenza and complications and to decrease related mortality in high - risk group (patients with chronic diseases, older people and those in contact with high - risk persons). furthermore, it is important to consider that 2014 - 2015 season quadrivalent influenza vaccine was available to cover incidental mismatching in co - circulating virus b - strains. streptococcus pneumoniae is a widespread airway transmittable bacterium causing severe diseases as pneumonia and meningitis. pneumococcal infections can be the complications of other respiratory infections as influenza and they usually occur in wintertime. everyone can take a pneumococcal disease but over 65 and patients with chronic diseases have higher mortality rate. overall pneumococcal infections offer the growing rate of antibiotic resistance : immunization is one of the main weapon of prevention against these diseases. one - third of people that had a previous infection with varicella zoster virus, will develop a clinical herpes zoster. the herpes zoster vaccine is recommended in adults over 60 years and older, regardless of their history of herpes zoster. in current competences all general practitioners (gps) must integrate in their daily practice the immunization policies of the national health service. regional and local health services organize the mandatory immunization acting in accordance with the national immunization plan. gps ' duty is to orient, counsel and give recommendations on vaccines characterizing and selecting population groups at high - risk. we can suggest immunization in adults according to age - group (young adult, adult or old people) (tab i) or according to risk - group (patients with chronic disease, immunocompromised conditions, pregnancy) (tab. iii) or for employment categories (health care professionals and social care professionals) or for lifestyle (international travelers, history of drug abuse or sexual transmitted diseases). it 's possible, in this way, to have agenda about age groups or about risk categories. the mission of our college works in the following directions : education to vaccines and immunization policy ; immunizations belong to gps ' daily practice and their scientific knowledge ; to offer tools of education and information ; to invite pharmaceutical industries make information service to primary care doctors ; to provide gps innovating tools to value own practice, understand the weakness and criticism and supply solving to improve immunizations in adults and in elderly people. millegpg (mille general practice governance) is an informatic tool able to have an active interaction with the most spread electronic patient data record (millewin) in italy. this application has been developed in collaboration with italian college of general practitioners (simg). millegpg provides gps with a series of " dashboards " by which they can check several performance indicators. more than 200 performance indicators (epidemiological, ongoing and outcome) are embedded millegpg encompassing three main domains (clinical audit, appropriateness and risk management. all indicators have been conceived according to international clinical guidelines during several meetings involving gps and specialists.. when gp wants to analyze the cohorts of patients with chronic disease (for example patients with chronic obstructive pulmonary disease or with diabetes or with heart failure) in the domain " risk management & prevention ", it 's possible to have the list of patients under 65 years old that should receive influenza vaccine. the other can be recall or in a proactive way (every time patient comes in office for a prescription, the nurse or the doctor receive an alert on the patient record) or call the patient by mail or e - mail. this model can be replicated for other immunizations and in this way each gp (or group) can act a real governance of immunizations. gp should adopt a systematic approach to immunization programs that includes educating patients and office staff using reliable sources of information, standing protocols during patient encounters and all practice management resources. recall and reminder systems have resulted in increases of up to 20 percent in rate of vaccination. synergic policies of education, information and professional tools can improve the competences and behaviours for benefit to patients and society. | summaryvaccine - preventable disease significantly contributes to the morbidity and mortality of adults worldwide. the rates of vaccination against influenza, pneumococcal disease and tetanus in adults and in high - risk group of people are far from the optimal coverage as suggested by minister of health. general practitioners (gps) can contribute to increase immunization in adults and in elderly people because these age groups attend frequently the surgery of their family doctors for reasons related to their chronic diseases. the gps, on their side, can proactively involve patients through informatics tools that supply lists of specific patients and electronic alerts in patient records. |
substantial coronal and radicular tooth structure is lost as a result of endodontic therapeutic procedures. hence, endodontically treated teeth are known to present increased susceptibility for mechanical and biological failures than intact vital teeth. the higher risk of fracture is also attributed to other factors such as effect of endodontic irrigants, age changes in dentin, and bacteria - dentine interaction. in - vivo investigations have indicated compromised tactile sense to perceive the functional overload in root canal treated teeth. the amount of remaining tooth structure is the most critical factor for the fracture resistance of endodontically treated teeth. the rehabilitation of root canal treated tooth is complete only with the resumption of its shape and full function. the choice of restorative techniques for these teeth is mainly influenced by the extent of tooth structure destruction, type, and location of the tooth in the dental arch. the endodontic post is indicated for teeth with the substantial amount of coronal tooth structure destruction. the indiscriminate use of post is discouraged by the researchers due to its associated multiple risks. frequently, clinician encounters a tooth with large central defect with normal height and peripheral dentin wall. the predicament of a restorative dentist to utilize the endodontic post in this clinical situation will be addressed by evaluating the fracture strength of the tooth with varying dentin wall thickness. though the influence of remaining tooth structure height on fracture resistance is evaluated by many researchers, studies on the influence of remaining dentin wall thickness on fracture resistance are few. the objective of this in - vitro study was to investigate the effect of coronal dentin wall thickness on the fracture strength of root canal treated teeth. it was also aimed to evaluate the influence of endodontic post on fracture resistance at various dentin wall widths. recently extracted 50 human mandibular premolars were used for the study with the mean root length of 15.83 1.79 mm. the teeth included in the study were intact, caries - free teeth without previous endodontic treatment. the teeth were divided into five groups of ten each (n = 10) depending on remaining coronal dentin wall thickness and post endodontic treatment [figure 1 ]. flowchart for sample distribution the remaining teeth samples were root canal treated ; the canal cleaning and shaping was accomplished with stainless steel k - files. the root canals were enlarged up to 40 master file and obturated with gutta - percha with cold lateral condensation technique. the procedure of remaining group preparation is described below : group 2a : the teeth samples were root canal treated as explained earlier. the central dentin core was removed to keep the 2.5 mm surrounding dentin wall thickness with 6 mm height. the standardization of dentin wall thickness was done with a digital caliper by measuring at three points in each four dentinal walls. the post space was prepared with the sequential use of gates - glidden, peeso, and calibrated reamer. a minimum of 5 mm gutta - percha apical seal was maintained during post space preparation. the cementation of the epoxy resin fiber post (easy post, dentsply international, york, usa) was done by using self - adhesive resin luting cement (breeze, pentron clinical, orange, ca, usa) following the manufacturer 's instruction. the access cavity and central defect were restored with posterior composite restorative material (filtek p90, 3mespe, st. the remaining dentin wall thickness was maintained at 2.5 mm as described in group 2a. the tooth was restored without endodontic post with posterior composite.group 3a : the teeth samples were prepared similar to group 2a. the remaining dentin wall thickness for this group the teeth were restored with an epoxy fiber post and posterior composite core.group 3b : the tooth samples were similar to group 3a with remaining dentin wall thickness of 1.5 mm. the central dentin core was removed to keep the 2.5 mm surrounding dentin wall thickness with 6 mm height. the standardization of dentin wall thickness was done with a digital caliper by measuring at three points in each four dentinal walls. the post space was prepared with the sequential use of gates - glidden, peeso, and calibrated reamer. a minimum of 5 mm gutta - percha apical seal was maintained during post space preparation. the cementation of the epoxy resin fiber post (easy post, dentsply international, york, usa) was done by using self - adhesive resin luting cement (breeze, pentron clinical, orange, ca, usa) following the manufacturer 's instruction. the access cavity and central defect were restored with posterior composite restorative material (filtek p90, 3mespe, st. the remaining dentin wall thickness was maintained at 2.5 mm as described in group 2a. group 3b : the tooth samples were similar to group 3a with remaining dentin wall thickness of 1.5 mm. the root surface area of all samples was covered with two layers of adhesive tape. the teeth were implanted vertically inside the identical polymethyl methacrylate acrylic mounting block with the help of twist drill (inside the canal) oriented in dental surveyor [figure 2 ]. teeth were removed from the mounting block while acrylic was in a rubbery stage of polymerization. the samples were prepared with torpedo diamond bur for circumferential chamfer finish line of 0.75 mm. the analogous nickel - chromium metal coping were fabricated and were cemented on the teeth with type i glass - ionomer cement (medicem, promedica dental material gmbh, neumunster, germany). composite picture for sample preparation the adhesive tape which was applied before implanting them in acrylic block was removed from the root surface. the load was applied on the buccal cusp at 30 angulation with crosshead speed of 2 mm / min until the failure [figure 3 ]. control sample testing on universal testing machine the data obtained were statistically analyzed using spss 19 (ibm corporation, armonk, new york, usa) for one - way anova and post - hoc tukey honestly significant difference (hsd), to find a statistically significant difference between the tested groups at 0.05 significance level. recently extracted 50 human mandibular premolars were used for the study with the mean root length of 15.83 1.79 mm. the teeth included in the study were intact, caries - free teeth without previous endodontic treatment. the teeth were divided into five groups of ten each (n = 10) depending on remaining coronal dentin wall thickness and post endodontic treatment [figure 1 ]. flowchart for sample distribution the remaining teeth samples were root canal treated ; the canal cleaning and shaping was accomplished with stainless steel k - files. the root canals were enlarged up to 40 master file and obturated with gutta - percha with cold lateral condensation technique. the procedure of remaining group preparation is described below : group 2a : the teeth samples were root canal treated as explained earlier. the central dentin core was removed to keep the 2.5 mm surrounding dentin wall thickness with 6 mm height. the standardization of dentin wall thickness was done with a digital caliper by measuring at three points in each four dentinal walls. the post space was prepared with the sequential use of gates - glidden, peeso, and calibrated reamer. a minimum of 5 mm gutta - percha apical seal was maintained during post space preparation. the cementation of the epoxy resin fiber post (easy post, dentsply international, york, usa) was done by using self - adhesive resin luting cement (breeze, pentron clinical, orange, ca, usa) following the manufacturer 's instruction. the access cavity and central defect were restored with posterior composite restorative material (filtek p90, 3mespe, st. the remaining dentin wall thickness was maintained at 2.5 mm as described in group 2a. the tooth was restored without endodontic post with posterior composite.group 3a : the teeth samples were prepared similar to group 2a. the remaining dentin wall thickness for this group the teeth were restored with an epoxy fiber post and posterior composite core.group 3b : the tooth samples were similar to group 3a with remaining dentin wall thickness of 1.5 mm. the central dentin core was removed to keep the 2.5 mm surrounding dentin wall thickness with 6 mm height. the standardization of dentin wall thickness was done with a digital caliper by measuring at three points in each four dentinal walls. the post space was prepared with the sequential use of gates - glidden, peeso, and calibrated reamer. a minimum of 5 mm gutta - percha apical seal was maintained during post space preparation. the cementation of the epoxy resin fiber post (easy post, dentsply international, york, usa) was done by using self - adhesive resin luting cement (breeze, pentron clinical, orange, ca, usa) following the manufacturer 's instruction. the access cavity and central defect were restored with posterior composite restorative material (filtek p90, 3mespe, st. the remaining dentin wall thickness was maintained at 2.5 mm as described in group 2a. group 3b : the tooth samples were similar to group 3a with remaining dentin wall thickness of 1.5 mm. the root surface area of all samples was covered with two layers of adhesive tape. the teeth were implanted vertically inside the identical polymethyl methacrylate acrylic mounting block with the help of twist drill (inside the canal) oriented in dental surveyor [figure 2 ]. teeth were removed from the mounting block while acrylic was in a rubbery stage of polymerization. the samples were prepared with torpedo diamond bur for circumferential chamfer finish line of 0.75 mm. the analogous nickel - chromium metal coping were fabricated and were cemented on the teeth with type i glass - ionomer cement (medicem, promedica dental material gmbh, neumunster, germany). composite picture for sample preparation the adhesive tape which was applied before implanting them in acrylic block was removed from the root surface. the load was applied on the buccal cusp at 30 angulation with crosshead speed of 2 mm / min until the failure [figure 3 ]. the data obtained were statistically analyzed using spss 19 (ibm corporation, armonk, new york, usa) for one - way anova and post - hoc tukey honestly significant difference (hsd), to find a statistically significant difference between the tested groups at 0.05 significance level. table 1 illustrates the mean and maximum compressive force values at which teeth failed for all the groups. it was followed by the group 2a with 28.97 mpa and group 2b at 27.70 mpa. group 3a had an average fracture strength at 23.39 mpa compared with 16.38 mpa for group 3b. there was statistically significant difference between the force required to fracture among groups 1, 2, and 3 with p = 0.000 and f = 16.307. the mean and maximum compressive fracture strength values for all groups and one - way anova test the tukey hsd multiple comparison tests [table 2 ] revealed no significant difference in fracture strength between the groups 1 and 2a with p = 0.994. the comparative fracture strength of groups 1 and 2b with a p = at 0.824 also showed no significant difference. the result shows that statistically significant difference between the groups 1 and 3a (p = 0.016) and 3b (p = 0.000). the relative compressive fracture strength between group 2a and groups 3a, 3b was significant with the p values of 0.045, 0.000, respectively. the groups 2a and 2b showed no statistically significant difference in maximum compressive load with p = 0.514. on the contrary, there was a statistically significant difference (p = 0.001) was noticed between the groups 3a and 3b. a tukey hsd comparisons of the significance level among groups hence, it can be inferred from the results that the tooth samples with 2.5 mm remaining dentin thickness are considerably similar to the control group in their mean compressive fracture strength. the tooth samples with remaining 1.5 mm dentin thickness (3a, 3b) had less fracture strength compared to the control and groups 2a, 2b. the endodontically treated teeth with significant loss of coronal tooth structure often require endodontic post to retain the core material before crown fabrication. hence, the restorative dentist should take due care and make an informed decision while incorporating post in the treatment plan. the study included teeth samples with different dentin wall thickness with or without the post to analyze their influence on fracture resistance. the results of the study illustrate the control group (group 1) showed the highest compressive fracture strength (29.75 mpa). it was followed by groups 2a (28.97 mpa), 2b (27.70 mpa), and least fracture strength 16.38 mpa was observed in group 3b. as reported by al - wahadni., the intact tooth was more fracture resistant than the endodontically treated teeth. the researchers found the biomechanical stress distribution in post core restored teeth was distinctly dissimilar to an intact tooth. the endodontic post restored tooth flexes as a single unit while intact natural teeth exhibit compressive force on one side and tensile stress on the opposite side. the difference between the flexural strength of post and remaining tooth structure leads to fracture of the tooth. the important observation of the study was that there was no significant statistical difference in fracture resistance between the control (group 1) and groups 2a, 2b with p value of 0.994, 0.824, respectively. with the presence of sound coronal dentin more than 2.5 mm, the researchers strongly suggest the self - supported coronal dentin improves the fracture resistance by favorable stress transmission to the root. though the contribution of coronal dentin toward fracture resistance is controversial, the majority of researchers advocate the preservation of coronal tooth structure during endodontic - post restoration. they suggest that the retained coronal dentin also provides the irregular contact surface with the core, helps in increasing retention and antirotational feature of dowel and core. the previous researchers were of the opinion that bridging by collagen fibers and water content in sound dentin walls also contributes to improved fracture resistance. the study results revealed the group 3a mean fracture strength significantly less than the group 2a and control group. the mean compressive fracture strength of the samples with 1.5 mm remaining dentin wall thickness without post (group 3b) was at 16.38 mpa. the fracture resistance was significantly affected due to the remaining dentin wall thickness in group 3a. according to tjan., sound dentin around the post is critical for a better prognosis of endodontically treated tooth than the cervical metal collar. they reported that teeth with 2 - 3 mm retained dentin thickness were less prone to fracture under horizontal impact force. stokes also emphasized on the sound dentin preservation around the post for the favorable distribution of the functional load. it is suggested the fragile dentin wall lead to a significant elastic modulus difference between dentin wall and restorative material, predisposing it to fracture. henry, conducted a photoelastic study and concluded that there is less concentration at the cervical finish line with sound coronal dentin. the results showed a statistically significant improvement in the fracture strength from group 3b (16.38 mpa) to group 3a (23.39 mpa) with post inclusion. the result endorses the observation by manning. regarding the need for post and full coverage crown for concluded that the filling materials alone are insufficient to restore the fracture resistance in the tooth with greater cervical preparation and thin remaining dentin wall thickness. patel and gutteridge reported the fracture resistance was not significantly different between teeth without coronal dentin and teeth with partially retained dentin in a lingual or buccal wall. hence, the presence of 360 coronal tooth structure enhances the fracture resistance of post restored teeth. though the study was performed on sound extracted natural teeth, change in moisture content, presence of invisible microcracks, functional age changes, morphological changes of pulp and dentin are difficult to standardize. within the limitation of the study, we conclude the remaining tooth coronal tooth structure width contribute significantly toward the fracture strength of endodontically treated teeth. the endodontic post in a root canal treated tooth with 2.5 mm remaining dentin wall thickness does not contribute significantly toward the fracture strength. endodontic post significantly reinforces root canal treated teeth with remaining dentin thickness < 1.5 mm. | background : remaining dentin wall thickness may influence the fracture resistance of tooth.aims:to investigate the effect of various coronal dentin wall widths on the fracture strength of root canal treated teeth.materials and methods : fifty recently extracted single canal mandibular premolars were used for the study. ten unrestored teeth were used as control (group 1) ; remaining teeth were root canal treated and divided into four groups (n = 10). the groups 2a, 2b and 3a, 3b were having 2.5 mm, 1.5 mm remaining dentin with and without post, respectively. the samples fracture resistance was tested under the universal testing machine. the data were analyzed with one - way anova and post - hoc tukey test for comparative evaluation.results:the mean fracture strength observed in group 1 was (29.75 mpa) followed by group 2a (28.97 mpa), group 2b (27.70 mpa), group 3a (23.39 mpa), and group 3b (16.38 mpa). there was no statistically significant difference between control and groups 2a and 2b with p > 0.05. the post contributed significantly for fracture resistance in group 3a.conclusion:the endodontic post is not required in root canal treated teeth > 2.5 mm coronal dentin wall width while the post is essential for a tooth with < 1.5 mm dentin wall width to improve fracture resistance. |
in refractory temporal lobe epilepsy (tle), it is important to be able to determine which hemisphere is dominant and hosts the majority of the language areas. when a patient is evaluated as a potential candidate for resective surgery, language mapping should be able to indicate which hemisphere is dominant and precisely identify where the language areas are situated within the brain. while the general principle of mapping language for tle in children might be the same as for adults, many challenges are encountered in the mapping process because children are not small adults but differ from adults in many aspects. to understand some of the differences between children and adults in tle features, a brief overview of tle then, a brief summary of language development and lateralization differences between normal children and children with epilepsy is provided. the semiology of temporal lobe originating seizures is not as well characterized in children compared to adults and is dependent on age. for example, infants have a predominance of behavioral arrests, they also tend to have more prominent convulsive activity than adults, and their seizures appear clinically generalized. in younger patients, the automatisms are first discrete and mostly orofacial, but the complexity of hand automatisms increases with age. after the age of 3 years, tonic or myoclonic spasms decrease, as do other motor phenomena, which might have been reminiscent of frontal lobe seizures, and the overall semiology becomes closer to that observed in adults [1, 2 ]. mesial temporal sclerosis (mts) is the most common adult etiology, while in children it is relatively rare. in the pediatric population, when mts is present, it is often accompanied by a neocortical pathology (dual pathology) [36 ], and curative surgery therefore necessitates a temporal lobectomy instead of a selective amygdalohippocampectomy to maximize the chances of being seizure - free. other pathologies, such as focal malformation of cortical development, tumors (such as gangliogliomas and dysembryoplastic neuroepithelial tumors), are frequent and also necessitate a neocortical resection. because language is not only affected by interindividual differences but also variably modified by epilepsy, exact language mapping is required before any neocortical resection to minimize postoperative neurological deficit. epilepsy surgery has been shown to lead to better cognitive development [8, 9 ] if the epileptogenic zone can be completely resected, but it also carries a higher risk of some language deficit (up to 50% in a series). language is progressively acquired over the years, and its development might be affected by seizures themselves, age at the onset of seizures, seizure severity, and the underlying pathology. surgery also has a different impact depending on the age at which it is performed. the general population, independent of handedness, has a 1018% chance of having a right or bilateral dominant hemisphere (5% in a right - handed population and 22% in a left - handed population [1114 ]) the majority of them are left handed. by contrast, among the epilepsy population, 77% have an atypical pattern for language (right or bilateral) as determined by the wada test or functional mri (fmri) [1517 ]. language might be displaced to the contralateral hemisphere or be reorganized within the same hemisphere, either a different location in that hemisphere, or compensated with additional areas recruited [14, 15, 1825 ]. it has also been shown that more children with epilepsy have an atypical language network than adults with epilepsy ; whether these findings are correlated with the age of onset of the seizures is a matter of debate. some studies have shown a difference in language pattern with early - onset seizures [17, 19, 26 ], while other studies have not been able to show that correlation [16, 2730 ]. in addition, the percentage of atypical language seems even higher when the epilepsy is probably symptomatic (formerly known as cryptogenic, i.e., : no lesion detected on the mri). however, even if language could be influenced by seizures and might be shifted to the contralateral side in some patients, it could also remain in the normal anatomical location on the left even when the seizures are arising from that area, which is important to remember when considering surgery to treat epilepsy. while it was initially thought that language lateralization was acquired later in life, recent functional magnetic resonance imaging (fmri) and magnetoencephalography (meg) studies have shown signs of lateralization in infants, with the left frontal region being implicated in the discrimination of speech sounds, for example, [3235 ]. however, there seems to be an increase in lateralization with age as shown by fmri and meg studies in normal children [36, 37 ], which could explain the better potential outcome in language reorganization following surgery before the age of 5 - 6 years [3840 ]. for example, verb - generation and story - processing tasks demonstrate more changes in lateralization over time than word - picture matching tasks [36, 4143 ]. all of the tools to assess language were developed in adults and then adapted for pediatric populations. while new technologies allow us to move away from invasive techniques, these technologies still carry challenges when applied to children. when considering all these methods, there are general constraints based on the age of the patient, which will be the same for all. currently, language assessment is not performed in infants and toddlers, except in specific research settings ; therefore, the following will focus on children who have developed enough language skills to communicate and be tested by visual and/or oral questions. because all of the techniques require cooperation of the subject, it is important to keep age - specific abilities in mind when developing the tests. in addition, antiepileptic drugs as well as cognitive delay resulting from epilepsy might affect attention span. cognitive psychologists have made us quite aware of the timeline of language development, which should be considered when developing tests of language function. development of phonological, semantic, grammatical, and pragmatic components of language during childhood influences the design of the studies. to obtain satisfactory results and map the language accurately, it is essential to use tasks that are appropriate for the age of the patient. in addition, processes occurring during brain development (such as the formation of synaptic contacts and myelination) also affect most of the imaging and mapping techniques and may thus influence the results. the last point to consider when interpreting the results of a patient compared to a study is that many imaging studies have a relatively small number of subjects, and the subject population is heterogeneous. for the same type of study, a pediatric study should have a larger number of subjects than an adult study because there is significantly more variability among children due to development, yielding an even more heterogeneous population. the various mapping techniques are described below, beginning with neuropsychological assessment, moving to more invasive tests, such as cortical stimulation, sodium amobarbital (wada) test, and nuclear medicine (single - photon emission computed tomography spect and positron emission tomography pet), then to contemporary tools, which are being used increasingly more in the clinic (fmri and meg), and finally to newer tools that are currently being assessed, such as functional near - infrared spectroscopy (fnirs) and diffusion tensor imaging (dti). in the context of epilepsy, clinical language assessment begins with a neuropsychological assessment that helps to determine lateralization and guides the decision as to whether more in - depth assessment is needed (i.e., if language is thought to be on the left and the surgery is a right temporal lobectomy, it is not generally necessary to investigate further). the neuropsychological assessment includes a battery of standardized, age - appropriate tests for language and memory, among other cognitive domains, such as attention, visual - spatial skills, motor skills, and executive functions, interpreted in the context of developmental milestones, academic skills, psychosocial functioning, and so forth. this global assessment helps determine assets and deficits and detect whether the pattern is consistent with dysfunction in a specific region of the brain or with a known neurological syndrome. as part of the neuropsychological test battery, fused dichotic word listening tests (fdwlts) have proven to be cost - effective, noninvasive methods for identifying language dominance as left, right, or bilateral in adults, children, and adolescents [4547 ]. during this behavioral test, different words are presented to both ears simultaneously, and the subject reports which one they heard. the number of correct answers for each ear is counted, and the value indicates a right or left ear advantage. the rationale behind this test is that contralateral projections from the ear to the brain are stronger than ipsilateral projections. it is well known that ongoing seizures in refractory epilepsy can affect development therefore by reducing the number of seizures, surgery does help in ameliorating learning and general development deficits. however, studies show that temporal lobectomy of the dominant hemisphere, even in children, leads to some degree of postoperative deficit in language. a study of 24 children with complex partial seizures, aged 5.815.7 years, showed a preoperative left - language dominance in 65% of subjects, with an estimated language delay of 1.73.5 years. postoperatively, these same children had an increase in language delay in all areas except for receptive syntax. while language lateralization is important, it is not the only factor when considering surgery. the traditional notion of two areas well demarcated anatomically within the frontal and temporal lobes broca and wernicke has been replaced by the knowledge of a language network that has some interindividual variability. when further information beyond laterality alone is required, imaging or further investigations with more language specificity are undertaken. the wada test consists of an injection of intracarotid sodium amobarbital to freeze half of the brain to lateralize function. the procedure consists of a dose of 40125 mg (depending on the body weight) of sodium amobarbital into the internal carotid through a femoral catheter. once the patient has returned to a normal baseline after anesthesia, the side where the seizure focus is present is tested first followed by the opposite side. the contralateral injection is typically performed 3045 minutes after the first using the same procedure as for the first. hemiplegia is first examined, and then language is tested either by a pediatric epileptologist or neuropsychologist. this is subject to variation from one centre to the next and is sometimes even performed on different days. the sodium amobarbital test requires full cooperation of the child, who has been subjected to a stressful situation because the injection also causes transient hemiplegia. while the situation can be explained and tolerated by older children (teenagers) [52, 53 ] or adults, it is extremely difficult in younger children. nonetheless, successful sodium amobarbital testing has been reported in children as young as 2 years old [53, 54 ]. in a study that tested 22 patients between the ages of 5 and 12 years (median 10), language lateralization was clearly identified in 50% of patients ; ten children had left hemispheric language while one had a right hemispheric dominance. furthermore, the percentage of successful sodium amobarbital procedures was higher among children with higher iqs (100% over iq 70, 57% of iq < 70). a later study from the same group reported a 62.5% success rate in 42 children ; 7.5% failed because of inconclusive results from the test (intact language after both injections, or a mix of intact language after one injection and then noncooperation), and 30% failed because of inadequate cooperation. similarly, schevon reported a successful sodium amobarbital test in 57% of patients younger than 10 years but as high as 93% in patients older than 10 years of age. however, the information obtained during the sodium amobarbital test is whether language is impaired after injecting sodium amobarbital into a particular side and therefore determines the dominant side. it might help to detect some bilateral patterns and predict language deficit after a proposed surgery ; however, a cortical map of the language network can not be created based on this procedure. another useful function of the sodium amobarbital test, which will not be discussed here, is to assess memory, which can be performed in the same setting. nuclear imaging, such as single - photon emission computed tomography (spect) and positron emission tomography (pet), has been used in the past to map eloquent cortex, including language areas. however, even though studies have shown good correlation between nuclear imaging and intraoperative language mapping or sodium amobarbital tests, such imaging exposes children to radiation. furthermore, these imaging methods are limited by the lack of spatial and temporal resolution [5759 ]. the subtraction of the ictal and interictal spect then registering to the mri) and pet are still used to find the epileptogenic zone, but, currently, they are not as commonly used to map language [60, 61 ]. cortical stimulation to map eloquent cortex was described by penfield in the early 1950s. however, because the procedure required a cooperative patient in the operating room, until the advent of implanted subdural or depth electrodes, it was only amenable to older children or adults. leaving subdural electrodes in place allows the use of cortical stimulation in the perioperative period. it also allows distribution of the language tests and mapping on different days, which is more suitable to children 's shorter attention spans. however, because it is invasive and carries some surgical risks, subdural electrodes, grids, or depth electrodes would not only be implanted for language mapping, but might also be used when invasive recording is otherwise necessary to determinate the epileptogenic zone. of note, only a restrictive part of the brain is exposed and, therefore, available for testing. another drawback of this method is the fact that, especially with strips and grids, only the gyri are recorded and the activity in the depth of the sulci is not. the main difference between cortical stimulation mapping (and the sodium amobarbital test) and the other mapping techniques is the fact that stimulation directly interferes with the language task under examination. therefore, cortical stimulation identifies areas that are critical to language instead of highlighting active areas that are part of the language network for a given task but might not be essential (such as seen with nuclear medicine or fmri). even though perioperative testing made stimulation more amenable to children and can now be performed by the bedside without restricting the child 's movements significantly, it has again proved to be more difficult in children than in adults [54, 6365 ]. standard stimulation protocols used in adults had to be modified to obtain some response from the stimulation in children. protocol using rectangular biphasic pulses of current on two adjacent contacts, starting the stimulation at 1 ma with a 0.3 ms pulse of alternating polarity and a train duration of 35 seconds. the stimulation intensity is then increased by steps of 1 ma and pulse duration by steps of 0.10.2 ms until after - discharges are seen, a seizure occurs, or there is a physiological response (speech arrest, motor, or sensory response). the various protocols use frequencies in the 2050 hz range, an intensity between 1 and 20 ma, a pulse train 325 seconds in duration, and a pulse width between 0.14 and 0.2 ms [54, 6668 ]. while intensities as low as 24 ma in adults generally evoke a response, in children, those intensities might have to be as high as 16 - 17 ma to be effective [67, 6971 ]. the possible reasons for the difficulties encountered include incomplete myelination and the greater proportion of small fibers. the chronaxie, which is the pulse duration needed for stimulation to evoke a response, is directly affected by the myelin deposition, and increasing myelination leads to a decrease in time of chronaxie. schevon. found that 10.2 years was a cut - off age for successfully mapping cortex. he studied children with both the sodium amobarbital test and cortical stimulations and found that, before the age of 10, 19% of children had positive cortical stimulation versus 87% for children older than 10. while a positive response is in general synonymous with critical language areas, the tasks administered during the stimulation are important, and, for example, expressive tasks show a better correlation with the sodium amobarbital test than receptive tasks. however, within the expressive tasks, the generation of sentences might generate a larger perisylvian network of expressive and receptive language [73, 74 ] than a verb - generation task. various studies have used cortical stimulation to study intrahemispheric reorganization of language in epileptic patients. for example, kadis. demonstrated anterior reorganization of language in expressive language in the left frontal lobe. the basics of fmri will not be discussed here because they are explained in another chapter of this issue (wang.functional magnetic resonance imaging for language mapping in tle). once again, pediatric fmri studies are more challenging than most adult fmri studies. first, the cognitive level of the child is dependent on his age ; therefore, the battery of tests should be age dependent. in addition, it is more difficult for a child than an adult to stay perfectly still in an mri during acquisition of the task. yuan showed a difference in age and gender in the motion of the head during acquisition ; younger (5- to 9-year - old) male children moved the most. all groups moved less when engaged visually instead of just with an auditory stimuli (picture - word matching versus syntactic prosody, story processing, and verb generation). another study showed a similar trend and demonstrated better results in children with normal developmental milestones as well as older children. in addition, because children have smaller heads, the head coil should be adjusted for younger age groups. this modification is especially important for younger patients who, in addition to a small head have a shorter neck, would have their head in the lower quadrant of the coil if using an adult coil. in addition, the thickness of the skull changes with age, which influences the quality of the image. to adjust for all ages would require an institution to have different head size coils and to avoid surface coils that increase heterogeneity due to the thinner skull (signal is enhanced in thinner skulls of younger compared to older people) [77, 78 ]. thinner skulls also produce increased physiological noise due to the increased heart and breathing frequency in children. first, the addition of videos for viewing at the beginning of the acquisition, during the anatomical mri as well as in between runs, would likely reduce motion artifact. second, the buttons subjects push to answer questions should be adapted to smaller children 's hands. third, the child should be brought into the room and the magnet before the task without being rushed to acclimatize to the environment ; he / she should be fully prepared for the task and the environment before beginning the session. despite the above - mentioned problems, fmri is starting to replace sodium amobarbital tests and cortical stimulation as a clinical tool for language mapping in some comprehensive epilepsy centres because it is less invasive, has a good spatial resolution (13 mm), and shows a good correlation with more invasive techniques [15, 72, 8082 ]. fmri can be used to investigate lateralization of language (laterality indexes are calculated based on the number of voxels activated on each side) as well as to show a more precise localization of language areas. as previously stated, there are various types of tasks that can be used, some active (verb generation, semantic decision, sentence completion, etc.) and some involving passive listening. the activations can be analyzed by contrasting any of these tasks with a resting state, during which the patient is instructed to do nothing, or between any of the conditions. the language map (pattern and lateralization) resulting from the analysis should differ depending on which tasks have been studied and contrasted. language protocols have to be developed especially for children and according to their age groups. in addition, delay in language development is common among epileptic children, which should be taken into account and tested before putting the child in the scanner. fmri shows the entire network of areas involved in the specific task (figures 1 and 2). because it is important to be as sensitive as possible to decrease the risk of postoperative deficit, paradigms capturing a wider network are usually preferred in pediatric populations (i.e., verb generation). while some studies have shown the possibility of performing 3 - 4 language tasks during image acquisition, so many tasks might be difficult in a clinical setting where the available time to train the child on the task and to remove him / her from the magnet in between tasks is less. however, one must be aware that only one language task might not be enough. the authors illustrated with two case reports that hemispheric dissociation in language function is possible, which can only be detected when administering different types of language tasks (i.e., vowel identification tasks and beep story). the design of the study must be carefully developed to adequately capture the language network. verb - generation tasks, in which the subject is asked to generate a maximum number of verbs related to a noun that is being presented (i.e., horse : jump, ride, etc.), seem to have a good reliability in determining hemispheric dominance [41, 82, 85 ]. story - processing tasks, in which the subject listens to a story with the instructions of listening carefully enough to be able to answer questions on each story after the mri, seem to produce wider bilateral activation and show some asymmetries in pathological subjects [86, 87 ]. another task commonly used in settings is a picture - word matching, in which the subject sees one or a couple of images and must decide whether it matches the name that is presented orally. in younger children, a passive language (listening) task can also be used for patients between the ages of 2 and 4 years. however, it is difficult to control whether the subject is actually listening to the story as opposed to dreaming or sleeping. in each task, different designs for each age group must be developed to provide an appropriate level of difficulty for the patient. all tasks should include resting states or control tasks, such as button press, sensory test, or a finger - tapping task, to compare the task data to. the fmri signal uses the hemodynamic response and the changes in oxyhemoglobin and deoxyhemoglobin to determine which areas of the brain are activated. because cerebral blood flow varies across ages, should it be corrected for age when doing a group study ? does the immaturity of the brain affect the bold signal ? the grey / white matter ratio also varies greatly until the age of 7 years then continues to change over time. the major fiber pathways are in place by the age of 3, but the average density of neurons and synapses changes until the age of 16, when it stabilizes until the late 60s. at the age of 7, the average synaptic density of the frontal lobe is approximately 1.4-fold greater than that of an adult [77, 90 ]. do these developmental variations provide a stable enough environment to use the same methods of analysis when investigating a pediatric population ? while most pediatric studies that have analyzed correlations with other modalities have found a good correlation, which indicates that fmri seems to yield valid data even in children, those questions have to be considered. the pediatric brain differs from an adult one, so group studies should not be normalized to an adult atlas, such as the talairach atlas [88, 91, 92 ]. this problem does not occur in a clinical setting in which the images of the patient are directly coregistered to his / her own anatomical mri. careful analysis of the data is of paramount importance, keeping in mind that misused statistics can show anything. one should be sure of how the analysis has been performed and of the statistical value of the results before interpreting the data. similar to other functional imaging modalities, fmri can be used to assess verbal and non - verbal memory. while in adults, memory mapping by fmri is sometimes used as a clinical tool, it is usually part of a research protocol in the pediatric population. because the hippocampus represents only a small area, the motion artifact and the difficulty of the tasks (without real - time feedback to know if the child is actually doing the task), it is difficult to have a valid study in a child. while it is not as evident as using meg, some studies have attempted to use fmri to detect interictal spikes (spike - triggered fmri) [94, 95 ]. the creation of superconducting quantum interference devices (squids) in the late 1960s / early 1970s allowed a different method of recording of brain electrical activity. meg captures neuronal activity by recording the net current of the flow of ions, leading to an intracellular electrical current generating a magnetic flux. repetitive events generate event - related potentials leading to evoked magnetic fields, which can then be recorded by the 248 channels positioned around the head. because magnetic fields are less deformed than electrical fields on the scalp, the spatial resolution of the meg (24 mm) is better than the eeg, and the images can be coregistered with an anatomical mri to visualize them. in addition, because meg measures neuronal activity, its temporal resolution is excellent (10 sec). in epileptic patients, meg allows the recording of interictal spikes, sometimes seizures, and eloquent cortex. breier. demonstrated a good correlation between an meg study and sodium amobarbital test in the pediatric population such results have been reproduced in adults too ; papanicolaou. showed no differences in language pattern with age, but other studies on normal subjects comparing meg to fmri for receptive language mapping showed significant differences in language patterns between the two methods, and another study showed good concordance between meg and fmri lateralization in normal teenagers for picture verb - generation, but only 75% concordance for the word verb generation task, and even lower when looking at precise localization (voxel overlap 50%). in patients requiring surgery near language - related eloquent brain areas (mostly for tumors), a german group combined meg and fmri and showed a good congruence between the two modalities (96%) ; however for some patient language activation was only seen in one of the modality. meg is still in its infancy when considering language mapping in epileptic patients, and more studies are required to see how it can be used in presurgical planning. the national institute of neurological disorders and stroke is currently recruiting patients for a large trial (nct00706160). near - infrared spectroscopy is a noninvasive technique used to measure hemodynamic changes using the different light absorption spectra of oxyhemoglobin (hbo) and deoxyhemoglobin (hbr). it necessitates a light - emitting source (at two different wavelengths, usually between 680 and 1000 nm), a detector, and a dispersive element. this method is also called optical tomography because it uses an exogenous optical tracer to extrapolate the blood flow, blood volume, or oxygenation of a specific region of the brain. for example, it can be used in conjunction with a bolus injection of indocyanine green to measure cerebral blood flow. it is also used to detect changes produced by neuronal activity (similarly to fmri). when there is regional activation during a task, there is an initial dip with a reduction of hbo and an increase of hbr, then a large increase of hbo and a decrease of hbr (focal arterial blood flow). because of the shallow penetration of photons (35 cm below the scalp), the advantage of fnirs is that it does not restrict the child to a small space such as an mri scanner does, and because the device is directly on the head of the child, motion artifacts are not a problem. the other advantage of fnirs is that the child can actually speak during the task ; therefore, his understanding and involvement in the task are actively monitored. fnirs seems to be more sensitive to bilateral speech pattern, which is sometimes more difficult to analyze with fmri, as shown by benke., when there is a dissociation between frontal and temporal activations [104, 106, 107 ]. diffusion tensor imaging is a technique that enables tracing of neuronal tracts in the brain. because of the tubular nature of neurons, water can move freely in the direction of the axis but is restricted transversally by the membrane. when applying various field gradients to the brain in the mr scanner, the difference between the diffusivity in the two axes can be represented by a tensor and subsequently mapped to the brain by coregistering it with an anatomical image to obtain mean diffusivity and fractional anisotropy maps. these maps can aid in the understanding of functional connectivity of the brain by displaying fibers connecting two regions [108, 109 ]. there are currently no pediatric studies investigating the use of dti and fmri together for language mapping. however, in adults, a few groups have tried to predict language lateralization by studying the arcuate fasciculus, inferior longitudinal fasciculus, or uncinate fasciculus and their asymmetry reflected by the anisotropy value, the association between anisotropic or mean diffusivity values and language deficits in patients with tle, or finally with dti and cortical mapping to assess colocalization of language areas in the anterior and posterior part of the arcuate fasciculus. a number of studies have shown that increased mean diffusivity and decreased fractional anisotropy, interpreted as structural compromise of the white matter tracts, are associated with language deficits in patients with epilepsy [111114 ]. transcranial magnetic stimulation (tms) is a noninvasive technique, which uses focal magnetic field generated by a rapidly changing current within a conducting coil. the coil can be applied to the scalp, so the magnetic field has a direct effect on the brain by depolarizing or hyperpolarizing neurons. experiments have shown that tms can induce a transient change in behavior by interfering in a manner similar to cortical stimulation. there are several methods for delivering the magnetic field, including single - pulse, paired pulse, and repetitive pulses. a first study on language and tms was performed in adults by pascual - leone., who was able to induce speech arrest when stimulating the perisylvian cortex with 10-s trains of repetitive tms (rtms) applied at rates of 825 hz. variations in the frequency of the stimulation were tried, and speech was also disrupted at 4 hz in another study. the same group showed a good correlation between sodium amobarbital test and tms in 12/16 epilepsy patients, while other studies showed better even better correlations [115, 118 ]. while some studies have been performed in children and one review article described the safety of tms in children (including a potential for increased risk of seizures in children younger than 5 years), there are no language studies in this group of patients. some motor studies have shown that it is feasible ; however, recommendations state that tms should be avoided in young children. language mapping in children requires a specialized multidisciplinary team with specific protocols designed from a developmental perspective. however, an increasing number of noninvasive techniques have been shown to be reliable and are being implemented clinically in preoperative investigation for epileptic children. | temporal lobe epilepsy (tle) in children is a slightly different entity than tle in adults not only because of its semiology and pathology but also because of the different approach to surgical treatment. presurgical investigations for eloquent cortex, especially language, must take these differences into account. most diagnostic tests were created for adults, and many of the assessment tools need to be adapted for children because they are not just small adults. this paper will highlight the specific challenges and solutions in mapping language in a pediatric population with tle. |
diabetes mellitus (dm) which is characterized by hyperglycemia is a common metabolic disorder that happens due to insulin deficiency and insulin resistance (1). it is associated with central nervous system (cns) and peripheral nervous system (pns) neurological complication (2). dm in long - term causes a vast range of peripheral neuronal deficits including reduced motor nerve conduction velocity, axonal shrinkage, impaired nerve regeneration, and deficient axonal transport (3). stz - induced diabetes is a well - defined experimental model for dm because the nerve damages are similar to the nerve degeneration that occurs in human diabetic neuropathy (4). it has been suggested that diabetes is one of the primary risk factors in the creation of senile dementia of the alzheimer 's type (sdat) (5). although the complete mechanisms through which diabetes could mediate these effects are not fully understood, it seems that hyperglycemia, oxidative stress and subsequent free radicals generation, lead to increased cell membrane damage (lipid peroxidation) and initiates death signaling pathways) 6, 7). portulaca oleracea l. (portulacaceae) which is referred to as purslane (khorfeh in persian) has been given the term global panacea and is listed by the world health organization as one of the most used medicinal plants. purslane is a rich source of antioxidants (vitamins a, c, and e, -carotene, and glutathione) and omega-3 fatty acids (8). other bioactives found in purslane are coumarins, alkaloids, saponins, polyphenols and anthocyanin (10). the aqueous extract of purslane hasn't shown any cytotoxicity or genotoxicity, and has been certified safe for daily consumption as a vegetable (11). a wide range of biological effects related to purslane has been reported including being neuroprotective (12, 13), effective in cognition improvement (14), being anxiolytic (15, 16), analgesic (17), antidiabetic (18, 19) and having antioxidant effects (8 - 10). although there are many different aspects in brain 's structure, neuroendocrine regulation and neurotransmitter systems of males and females (20, 21), most of the studies have been focused on the neurobehavioral changes which take place in male animals following diabetes. on the other hand, incidence of diabetes increases after menopause (22). therefore, due to lack of enough knowledge concerning the effects of purslane on neurobehavioral changes and hyperglycemia that happen in female diabetic animals, the present study was designed and conducted. ovx rats not only are well - defined models to assess post - menopausal events (23, 24), but also they do n't get influenced by the effects of ovarian steroids which may alter the behavior of rats during the estrus cycle. all the other chemicals were of analytical grade and were obtained from commercial sources. preparation of aqueous extract purslane which was cultured in khouzestan, ramin agriculture and natural resources university, was obtained as a gift. 1500 ml of distilled water was added to 300 grams of dried purslane powder in a sealed glass container and it was set aside for about 72 h. the filtrated extract was concentrated in a rotary evaporator under reduced pressure at 55 c and dried in a bath of warm water. thirty virgin female wistar rats (weighing 190 - 200 g, 12 weeks of age) were obtained from the faculty of veterinary medicine of shahid chamran university (ahvaz, iran). they were maintained under standard laboratory conditions with 12 h light / dark cycle and free access to standard laboratory rat food and tap water. the used protocol and animal ethics were approved by the research council of the faculty of veterinary medicine of shahid chamran university of ahvaz. ovx rats were randomly divided into three groups (10 in each group) : normal control (control), diabetic (dia) and aeop treated diabetic (dia+aeop) groups. following confirmation of diabetes (blood sugar>300 mg / dl), dia+aeop group were treated daily by oral gavage of 300 mg / kg of aeop dissolved in 4 ml of saline (25) for 35 days. both control and dia groups were treated similarly with the same procedure and same volume of the solvent of the extract (saline). anesthesia was induced by ketamine - xylazine mixture (100 mg / kg-10 mg / kg, respectively) to remove both of their ovaries. the dorsal mid lumbar area was shaved and swabbed with surgical scrub, iodine and alcohol. a 1 - 2 cm dorsal midline incision was made halfway between the caudal edge of their ribcage and base of the tail at both right and left sides. ovaries and oviducts were exteriorized, clamped and removed. with 10 days of recovery, injection of freshly prepared stz (60 mg / kg, dissolved in 0.1 m citrate buffer, ph 4.5). five days after stz injection, animals with blood glucose greater than 300 mg / dl were used for the study. glucose measurement glucose was measured by a glucometer from a drop of blood taken from their tail. glucose was measured twice : once 5 days after injection of stz to assess the induction of diabetes and again at the last stage of the study to assess effects of aeop on diabetic hyperglycemia. morris water maze (mwm) the spatial learning and memory ability of the rats were evaluated using mwm (26). briey, the system included a circular pool (height : 50 cm, diameter : 150 cm) which was lled with water (22 1 c) to a depth of 25 cm and a video tracking device captured the swimming pathway by a video - tracking software (borj sanat co, tehran, iran). a hidden square platform (10 10 cm) was placed in the center of the southwest quadrant, submerged 1 cm below the surface of the water. the maze was geographically divided into four quadrants ; northeast (ne), northwest (nw), southeast (se) and southwest (sw), and four starting positions including north (n), south (s), east (e) and west (w) ; which were equally spaced around the perimeter of the pool. in the training trials, each rat was trained in the maze for four consecutive days, which was repeated four times per day and the swim path of every rat was recorded for 90 seconds. swim speed, the escape latency and the distance traveled to reach the hidden platform were recorded by the software. in the probe trial which took place 24 h after the last training trial, spatial memory of the rats was evaluated in the maze without the platform. at this point, the activity of the rats was recorded for 60 s to compute the total distance traveled, the percentage of distance traveled in the target quadrant and the percentage of time spent in the target quadrant as a measure of retention and memory performance of the rats. elevated plus maze (epm) the epm was designed according to pellow and file (27). the epm is comprised of two open arms) 50 10 cm (and two closed arms (50 10 40 cm), which are connected by a common central square 10 10 cm. rats were placed in the central square, facing an open arm and were allowed to explore the apparatus for 5 minutes. the maze was cleaned with ethanol (70%) after each test. a video tracking device captured every rat 's pathway by a video - tracking software (borj sanat co, tehran, iran) and measured : a) total time spent in open arms, b) total time spent in closed arms, c) number of entries into open arms and d) number of entries into closed arms. finally, the following parameters were calculated : 1) percentage of time spent in the open arms relative to the total time spent in the maze 2) percentage of entries into open arms relative to the total number of entries into any arm. forced swimming test (fst) the fst was performed according to porsolt. (28). a vertical plastic cylinder (50 cm in height, 20 cm in diameter) was filled with water (23 - 25 c) to a depth of 30 cm. each animal was placed in the cylinder for 6 min, to measure immobility - floating and swimming behaviors during the last 4 min. immobility - floating was considered as the rat being passively in the water and only making slight movements to keep its head above the water line and swimming was considered as making active swimming motions, more than necessary to merely keep the head above the water (i.e., moving around in the cylinder). tail pinch stressor (tps) rats were subjected to a mild tail pinch according to the method which is described by gomez. cm) which one side of them was made of wire - mesh and they were allowed to acclimatize for 15 min. a softwood biting block was put in each chamber. the rat 's tail was put through a hole in the center of the wire - mesh wall and at the distance of 8 cm from its tip a plastic clothes clip was put on it for 5 min. the duration of non - functional masticatory activity (nfma) displayed during tail pinch exposure was measured by an observer. vigorous gnawingbiting of the wooden block and/or audible experimental bruxism (chattering or scraping of the teeth) were considered to be nfma. duration was defined as the total time in which nfma was displayed during the observation period which was 5 min ; although a prerequisite was that rats had to exhibit a minimum of 5 consecutive seconds of nfma for it to be considered valid. the reason that 5 s was decided upon as an appropriate portion of time, was to prevent the scoring any intermittent masticatory activity which was not significant. data were analyzed by the software spss, version 16 (spss ; chicago, il, usa). repeated measures and one way analysis of variance (anova) followed by tukey test were used to assess treatment differences among the groups. fasting blood sugar (fbs) the results of measuring the fbs following 12 h of fasting are demonstrated in (figure 1). diabetes induction significantly increased fbs (p0.05, figure 4). data on the stz - induced diabetic rats both treated and not treated (dia) with purslane (dia+aeop, 300 mg / kg) and the control indicating the total distance traveled in the probe trial of mwm. each bar is represented as mean sem and is analyzed by anova followed by tukey test. : indicates the significant difference vs. control (: p0.05), but the number of total arm entries were significantly lower in dia group (p0.05). data on the stz - induced diabetic rats both treated and not treated (dia) with purslane (dia+aeop, 300 mg / kg) and the control indicating immobility and swimming behavior assessed by fst. each bar is represented as mean sem and is analyzed by anova followed by tukey test. n = 10 total time of stress - induced nfma was severely greater in the dia group compared to the control (p0.001, figure 7). data one the stz - induced diabetic rats both treated and not treated (dia) with purslane (dia+aeop, 300 mg / kg) and the control indicating nfma in tps. each bar is represented as mean sem and is analyzed by anova followed by tukey test. : indicates the significant difference vs. control group (: p0.001). the aim of the present study was to assess psychobiological effects of aeop in diabetes induced ovx rats, as a model of postmenopausal women. aeop not only ameliorated hyperglycemia, but also improved the diabetes related deficits in mwm, as well as motor deficit in epm and nfma in tps. prolonged hyperglycemia, the most important cause of most diabetes complications, is thought to lead to cognitive impairments in diabetic patients (31, 32). therefore, amelioration of diabetes related neurobehavioral impairment by aeop may be partly related to its ability to attenuate hyperglycemia. the mwm is a commonly used paradigm for experimenting on learning abilities and memory of rodents (33). increased escape latency and distance traveled to reach the hidden platform in dia group could be related to spatial learning impairment, while reduction of swim speed as well as the total distance traveled in this group are indications of motor impairment. this finding in the diabetic group is in accordance with the decrease in total arm entries in epm. since swim speed was significantly lower in diabetic animals, interpretation of learning and memory should be with caution. however, the distance traveled to reach the hidden platform may be more decisive in this context. deficits in spatial cognitive tasks have been reported in dm animal models, such as the stz - diabetic rat in the mwm (35). the hippocampus and cerebral cortex of mammals which play a pivotal role in a diverse set of cognitive functions are very sensitive to the oxidative damage that occurs in stz - diabetic animals (36). enhancement of lipid peroxidation in these regions leads to a significant motor and memory deficit in behavioral functions of diabetic animals (37). in summary, chronic oral administration of aeop did enhance the memory consolidation and the capability to recall stored information and spatial memory in diabetic ovx rats. many plant ingredients, including polyphenols, flavonoids and various plant extracts, have antioxidant effects (38). there are various antioxidants in purslane including anthocyanins, antioxidant vitamins (vit a, c and e), melatonin, flavonoids and etc. the epm is widely used to assess anxiety - related behaviors in rodents (40). the percentages of open arm entries and time spent in the open arms which have been approved as a measure of anxiety (41), decreased significantly in dia group. both ovariectomy, via estrogen depletion (23, 24), and diabetes (42) increases anxiety - like behavior in rodents. aeop not only reversed the diabetes related decrease of locomotor activity but also increased the percentage of time spent in the open arms and the percentage of open arm entries. the anxiety - like behavior of aeop - treated group not only was lower than diabetic rats, but also was lower than the control group. this may be related to higher anxiety of ovx rats compared to intact ones (23, 24) or to high anxiolytic effects of aeop. the later assumption is supported by the results of the previous studies which have indicated the anxiolytic effect of aeop and ethanol extract of purslane on normal mice (15, 16). however, we did not find any report that indicates the anxiolytic effect of aeop on diabetic or ovx models. since both the naturally occurring and synthetic flavones have been shown to bind to gabaa benzodiazepine receptors with high affinity and to exert anxiolytic - like effects in rodents, they have been suggested as ligands for these binding sites (43). furthermore flavonoids naturally are the ligands of benzodiazepine receptors and have anxiolytic effects (44). fst is frequently used to assess antidepressant - like activities in rodents (45). in the present study, we utilized fst to evaluate the effects of aeop on the depressive behavior of diabetic ovx rats. in some studies immobility however in our study there were no differences in the depressive behavior of normal and diabetic rats. tail pinch is used to stimulate stress - related behavioral activities in rats (29, 30). the use of a peripheral stressor which is unavoidable for the animal, like tail pinch, produces a series of behaviors including chewing and gnawing / biting (29, 30). the total time of stress - induced nfma which was severely increased in the dia group, was improved by aeop - treatment. these responses attenuate a variety of stress - related changes in rats, such as increase in the activity of hypothalamo - pituitary - adrenal axis and catecholamine utilization in cns (49). in this context, central dopamine is traditionally considered related to the etiopathogenesis of stereotyped behavior and bruxism (50). rutledge. (51) showed that diabetes alter the sensitivity of dopaminergic system, which leads to modulation of nociception in stz - diabetic rats. these changes may be related to the mechanism of increase of nfma in diabetic rats. purslane has many valuable compounds, it is rich in antioxidants (8 - 10), and also has neuroprotective (12, 13) and antidiabetic effects (17, 18). thus, the positive effects of aeop observed in this study may be attributed to these properties. in the present study we showed anti - stress, antidiabetic and spatial memory improving effects of aeop in diabetic ovx rats, as well as its anxiolytic effect on both diabetic ovx and normal ovx rats for the first time. | diabetes mellitus is one of the most common causes of neuropathy. although antioxidant and antidiabetic effects of the aqueous extract of purslane (portulaca oleracea) (aeop) have been demonstrated before by other researchers, we did not find any study that assessed the psychobiological effects of aeop in diabetes induced animals. thirty ovariectomized (ovx) female wistar rats were randomly divided into 3 groups of control, dia and dia+aeop. the latter group was orally treated by 300 mg / kg of aeop for 35 days. dia and dia+aeop groups were made diabetic by ip injection of 60 mg / kg of streptozotocin (stz). the psychobiological effects of aeop were assessed by morris water maze (mwm), elevated plus maze (epm), forced swimming test (fst) and tail pinch stressor (tps). aeop significantly decreased hyperglycemia (p0.05). diabetes significantly increased their non - functional masticatory activity in tps (p0.001) while it was improved in dia+aeop group. we showed that aeop has significant anxiolytic effects and it can improve spatial cognitive performance, locomotor deficit and stress in diabetic ovx rats. |
carbon nanotubes have been extensively studied because of their interesting physical properties and potential applications. motivated by this success, scientists have been exploring nanotubes and nanostructures made of different materials. in particular, theoretical studies have also been carried out to investigate the electronic, optical and elastic properties of bc2n nanotubes using the first - principles and tight - binding methods, respectively [3 - 6 ]. besides the elastic and electronic properties, theoretical and experimental research on phonon properties of bc2n nanotubes is also useful in understanding the properties of the nanotubes. furthermore, symmetry properties of nanotubes have profound implications on their physical properties, such as photogalvanic effects in boron nitride nanotubes. studies on the symmetry properties of carbon nanotubes predicted the raman- and infrared - active vibrations in the single - walled carbon nanotubes, which are consistent with the experimental data and theoretical calculations. a similar work was carried out by alon on boron nitride nanotubes, and the results were later confirmed by first - principles calculations. and the symmetry of bc2n nanotube was reported. the purpose of this study is to extend the symmetry analysis to bc2n nanotubes and to determine their line groups. the number of raman- and infrared (ir)-active vibrations of the bc2n nanotubes is determined accordingly. similar to carbon or boron nitride nanotubes, a single - walled bc2n nanotube can be completely specified by the chiral vector which is given in terms of a pair of integers (nm). however, compared to a carbon and boron nitride nanotubes, different bc2n nanotubes can be obtained by rolling up a bc2n different directions, as shown in fig. 1a, because of the anisotropic geometry of the bc2n sheet. if we follow the notations for carbon nanotubes, at least two types of zigzag bc2n nanotubes and two types of armchair nanotubes can be obtained. for convenience, we refer the two zigzag nanotubes obtained by rolling up the bc2n the a1 and the a2 directions as zz-1 and zz-2, respectively, and two armchair nanotubes obtained by rolling up the bc2n the r1 and r2 directions as ac-1 and ac-2, respectively. the corresponding transactional lattice vectors along the tube axes are ta1, ta2, tr1, and tr2, respectively, as shown in fig. it is noted that ta2 is parallel to r2, tr1 to b1, and tr2 to a2. we first consider the achiral carbon nanotubes with the rotation axis of order n, i.e., zigzag (n, 0) or armchair (nn). the nonsymmorphic line - group describing such achiral carbon nanotubes can be decomposed in the following way : (1) where is the 1d translation group with the primitive translation tz = |tz|, and e is the identity operation. the screw axis involves the smallest nonprimitive translation and rotation. the corresponding bc2n sheet of the zigzag (n, 0) bc2n nanotubes (zz-1) (fig. in this case, thednhanddndpoint groups reduce tocnvdue to the lack of horizontal symmetry axis / plane, ands2nvanishes for the lack of the screw axis. 2,(3) to determine the symmetries at the point of the 12 n (nis the number of unit cells in the tube andn = nfor zz-1 bc2n nanotubes) of phonons inzz-1 bc2n nanotubes and the number of raman- or ir - active modes, we have to associate them with the irreducible representations (irrep s) ofcnv. here, two cases need to be considered. 2d projections of zigzag bc2n nanotubes (zz-1).zis a glide plane nis odd (orn = 2 m + 1, m is an integer) the character table of c(2m+1)v possesses m + 2 irrep s, i.e.,(4) the 12 n phonon modes transform according to the following irreps:(5) stands for the reducible representation of the atom positions inside the unit cell. the prefactor of 4 in reflects the four equivalent and disjoint sublattices made by the four atoms in the zz-1 bc2n nanotubes. is the vector representation. of these modes, the ones that transform according to (the tensor representation) or are raman- or ir - active, respectively. out of the 12 n modes, four have vanishing frequencies, which transform as and corresponding to the three translational degrees of freedom giving rise to null vibrations of zero frequencies, and one rotational degree about the tube s own axis, respectively.(7)(8) nis even (orn = 2m, mis an integer) the character table of c2mv possesses m + 3 irrep s, i.e.,(9) the 12 n phonon modes transform according to the following irreps:(10) is the vector representation. of these modes, the ones that transform according to (the tensor representation) or are raman- or ir - active, respectively. out of the 12 n modes, four (which transform as and) have vanishing frequencies.(12)(13) the numbers of raman- and ir- active modes are 30 and 18, respectively, for zz-1 bc2n nanotubes irrespective n. the armchair (n, n) bc2n nanotubes (ac-1) (fig. thednhanddndpoint groups reduce tocnhowing to the lack ofc2axes ands2nvanishes for the lack of the screw axis.(14) the point group of the line group is readily obtained from eq. (at the point) of the 12 n (n = n) phonons in ac-1 bc2n nanotubes and the number of raman- or ir - active modes, two cases need consideration, by associating them with the irrep s of cnh. 2d projections of armchair bc2n nanotubes (ac-1).zis a glide plane nis odd (n = 2 m + 1) the character table of c(2m+1)h possesses 4 m + 2 irrep s, i.e.,(16) the 12 n phonon modes transform according to the following irreps:(17) and is the vector representation. of these modes, the ones that transform according to (the tensor representation) or are raman- or ir - active, respectively. out of the 12 n modes, four (which transform as and) have vanishing frequencies.(19)(20) the character table of c2mh possesses 4 m irrep s, i.e.,(21) the 12 n phonon modes transform according to the following irreps:(22) is the vector representation. of these modes, the ones that transform according to (the tensor representation) or are raman- or ir - active, respectively. out of the 12 n modes, four (which transform as and) have vanishing frequencies.(24)(25) the numbers of raman- and ir- active modes are 19 and 10, respectively, for ac-1 bc2n nanotubes in irrespective of n. the numbers of raman- and ir- active phonon modes for zz-1 bc2n nanotubes are almost twice as for ac-1 bc2n nanotubes, which is similar to boron nitride nanotubes. the nonsymmorphic line group describing the () -chiral carbon nanotubes can be decomposed as follows:(26) where ; wheredris the greatest common divisor of and ; dis the greatest common divisor of and ; sn / dand snare the screw - axis operations with the orders ofn / dandn, respectively. 26,(27) where and are the rotations embedded in sn / dand sn, respectively. for chiral (nm) bc2n nanotubes, the point group dn reduces to cn due for the lack of c2 axes. here,, where dr is the greatest common divisor of and ; d is the greatest common divisor of and. 4a and b, respectively, for zz-2 and ac-2 bc2n nanotubes, n = 4n. the point group corresponding to the two models is expressed as:(28) the character table of cn has n irrep s, i.e.,(29) the 12 n phonon modes transform according to the following irreps:(30) where and. of these modes, the ones that transform according to and/or are raman- and/or ir- active, respectively. out of the 24 n modes, four (which transform as and) have vanishing frequencies.(31)(32) experimentally, only several raman / ir - active modes can be observed. the e2 g mode around 1580 cm is related to the stretching mode of c c bond. the experimental raman spectra between 100 and 300 cm should be attributed to e1 g and a1 g modes. nis odd (orn = 2 m + 1, m is an integer) the character table of c(2m+1)v possesses m + 2 irrep s, i.e.,(4) the 12 n phonon modes transform according to the following irreps:(5) stands for the reducible representation of the atom positions inside the unit cell. the prefactor of 4 in reflects the four equivalent and disjoint sublattices made by the four atoms in the zz-1 bc2n nanotubes. is the vector representation. of these modes, the ones that transform according to (the tensor representation) or are raman- or ir - active, respectively. out of the 12 n modes, four have vanishing frequencies, which transform as and corresponding to the three translational degrees of freedom giving rise to null vibrations of zero frequencies, and one rotational degree about the tube s own axis, respectively.(7)(8) nis even (orn = 2m, mis an integer) the character table of c2mv possesses m + 3 irrep s, i.e.,(9) the 12 n phonon modes transform according to the following irreps:(10) is the vector representation. of these modes, the ones that transform according to (the tensor representation) or are raman- or ir - active, respectively. out of the 12 n modes, four (which transform as and) have vanishing frequencies.(12)(13) the numbers of raman- and ir- active modes are 30 and 18, respectively, for zz-1 bc2n nanotubes irrespective n. the armchair (n, n) bc2n nanotubes (ac-1) (fig. thednhanddndpoint groups reduce tocnhowing to the lack ofc2axes ands2nvanishes for the lack of the screw axis.(14) the point group of the line group is readily obtained from eq. (at the point) of the 12 n (n = n) phonons in ac-1 bc2n nanotubes and the number of raman- or ir - active modes, two cases need consideration, by associating them with the irrep s of cnh. nis odd (n = 2 m + 1) the character table of c(2m+1)h possesses 4 m + 2 irrep s, i.e.,(16) the 12 n phonon modes transform according to the following irreps:(17) and is the vector representation. of these modes, the ones that transform according to (the tensor representation) or are raman- or ir - active, respectively. out of the 12 n modes, four (which transform as and) have vanishing frequencies.(19)(20) the character table of c2mh possesses 4 m irrep s, i.e.,(21) the 12 n phonon modes transform according to the following irreps:(22) is the vector representation. of these modes, the ones that transform according to (the tensor representation) or are raman- or ir - active, respectively. out of the 12 n modes, four (which transform as and) have vanishing frequencies.(24)(25) the numbers of raman- and ir- active modes are 19 and 10, respectively, for ac-1 bc2n nanotubes in irrespective of n. the numbers of raman- and ir- active phonon modes for zz-1 bc2n nanotubes are almost twice as for ac-1 bc2n nanotubes, which is similar to boron nitride nanotubes. the nonsymmorphic line group describing the () -chiral carbon nanotubes can be decomposed as follows:(26) where ; wheredris the greatest common divisor of and ; dis the greatest common divisor of and ; sn / dand snare the screw - axis operations with the orders ofn / dandn, respectively. 26,(27) where and are the rotations embedded in sn / dand sn, respectively. for chiral (nm) bc2n nanotubes, the point group dn reduces to cn due for the lack of c2 axes. here,, where dr is the greatest common divisor of and ; d is the greatest common divisor of and. 4a and b, respectively, for zz-2 and ac-2 bc2n nanotubes, n = 4n. the point group corresponding to the two models is expressed as:(28) the character table of cn has n irrep s, i.e.,(29) the 12 n phonon modes transform according to the following irreps:(30) where and. of these modes, the ones that transform according to and/or are raman- and/or ir- active, respectively. out of the 24 n modes, four (which transform as and) have vanishing frequencies.(31)(32) experimentally, only several raman / ir - active modes can be observed. the e2 g mode around 1580 cm is related to the stretching mode of c c bond. the experimental raman spectra between 100 and 300 cm should be attributed to e1 g and a1 g modes. in summary, the symmetry properties of bc2n nanotubes were discussed based on line group. all bc2n nanotubes possess nonsymmorphic line groups, just like carbon nanotubes and boron nitride nanotubes. contrary to carbon and boron nitride nanotubes, armchair and zigzag bc2n nanotubes belong to different line groups, depending on the index n (even or odd) and the vector chosen. by utilizing the symmetries of the factor groups of the line groups, it was found that all zz-1 bc2n nanotubes have 30 raman- and 18 ir- active phonon modes ; all ac-1 bc2n nanotubes have 19 raman- and 10 ir - active phonon modes ; all zz-2, ac-2, and other chiral bc2n nanotubes have 33 raman- and 21 ir - active phonon modes. it is noticed that the numbers of raman- and ir- active phonon modes in zz-1 bc2n nanotubes are almost twice as in ac-1 bc2n nanotubes, but which is almost the same as those in chiral, zz-2, and ac-2 bc2n nanotubes. the situation in bc2n nanotubes is different from that in carbon or boron nitride nanotubes. | the symmetry properties of the single - walled bc2n nanotubes were investigated. all the bc2n nanotubes possess nonsymmorphic line groups. in contrast with the carbon and boron nitride nanotubes, armchair and zigzag bc2n nanotubes belong to different line groups, depending on the index n (even or odd) and the vector chosen. the number of raman- active phonon modes is almost twice that of the infrared - active phonon modes for all kinds of bc2n nanotubes. |
the online version of this article (doi:10.1007/s00239 - 009 - 9239 - 0) contains supplementary material, which is available to authorized users. alpha-2-fucosyltransferases (2fts) are enzymes required for the biosynthesis of the terminal glycan motif fuc2gal-r found in abh and lewis histo - blood group antigens. the biologic functions of such carbohydrate motifs are not completely clear yet, but their main expression at the surface of epithelial cells that constitute doors of entry for pathogens, as well as on soluble mucins present in these epithelia, suggests that they might have functions related to interactions with microorganisms, pathogenic or not (marionneau. consistent with this view, helicobacter pylori strains, campilobacter pilori, uropathogenic strains of escherichia coli, lactobacilli strains, and several strains of caliciviruses are known to attach to 2fucosylated glycan structures (boren. 1993 ; le pendu. 2006 ; ruiz - palacios. 2003 additional cellular functions such as involvement in the development of the olfactory system, angiogenesis, interaction between dendritic cells and the vascular endothelium, and regulation of apoptosis (garcia - vallejo. 2006)recently have been suggested. like other glycosyltransferases, 2fts are type ii membrane proteins anchored in the golgi apparatus. they present a short intracytoplasmic tail and a transmembrane domain in n - terminal location, followed by a stem region and the catalytic domain, which can be subdivided into two subdomains, the n- and c - terminal subdomains. in mammalian genomes, three 2 ft genes are located in tandem and designated as fut1, fut2, and sec1 (oriol. 2000). the coding sequence of each of the three genes is comprised within a single exon, and it has been suggested that this monoexonic structure results from an l1-retrotransposition event that occurred within the 2 ft mammalian ancestor gene (saunier. their tandem localization and earlier sequence comparisons using only primates suggested that the fut1, fut2, and sec1 genes originated from two successive duplications. the first one would have given rise to fut1 and to the ancestor of both fut2 and sec1, whilst the second duplication event would have generated fut2 and sec1. gene duplication is generally considered important for adaptation because it allows advantageous mutations in one of the duplicates to promote a new role without impairing the original function exerted by the other duplicate (ohno 1970). however, theoretical studies suggest that one of the duplicates has a high probability to become silenced rapidly (walsh 1995), which would be consistent with the silencing of sec1 in catharrinians (apoil. in addition, it has been proposed that functional diversification is a rare event because gene conversion tends to homogenize the variation between duplicated genes (walsh 1987). for that reason, gene conversion is considered the main mechanism of concerted evolution of gene families. previous studies have not detected gene conversion in primates because each of the fut1, fut2, and sec1 genes appeared as separated clusters (apoil. 2000) ; however, very recently the occurrence of gene conversion between fut2 and sec1 was reported in a sec1-fut2-sec1 human allele (soejima. 2008). here, with the aim to improve our understanding of the evolution and the biologic role of the 2fts gene family, we addressed two main questions. first, what are the evolutionary relations between fut1, fut2, and sec1 when looking at a broader phylogenetic context ? complete coding sequences of 2fts fut1, fut2, and sec1 genes were retrieved from genbank and aligned with clustal w (thompson. 1994), followed by visual inspection (see table 1 for a list of species used, abbreviations, and genbank accession numbers).table 1list of the coding sequences of the 2fts genes, fut1, fut2 and sec1, retrieved from genbank and used in this studyspecies namecommon namegenegenbank accession no.abbreviationhomo sapienshumanfut2u17894human_fut2fut1m35531human_fut1sec1u17895human_sec1pantroglodytescommon chimpanzeefut2af080604chimpanzee_fut2fut1af080603chimpanzee_fut1sec1ab006612chimpanzee_sec1pongo pygmaeusorangutanfut2ab015636orangutan_fut2sec1ab006610orangutan_sec1gorillagorillagorillafut2af080606gorilla_fut2fut1af080605gorilla_fut1hylobateslarlar gibbonfut2af136648gibbon_fut2h. agilisagile gibbonsec1ab006609gibbon_sec1chlorocebusaethiopssabaeusgreen monkeyfut2d87934green_monkey_fut2fut1d87932green_monkey_fut1sec1d87933green_monkey_sec1macaca fasciculariscynomolgussec1af080608cynomolgus_sec1bostauruscowfut2x99620cow_fut2fut1nm_177499cow_fut1sec1af187851cow_sec1musmusculusmousefut2af064792mouse_fut2fut1u90553mouse_fut1sec1y09882mouse_sec1rattusnorvegicusratfut2ab006138rat_fut2fut1ab015637rat_fut1sec1af131239rat_sec1susscrofapigfut2u70881pig_fut2fut1u70883pig_fut1sec1u70882pig_sec1oryctolagus cuniculusrabbitfut2x91269rabbit_fut2fut1x80226rabbit_fut1sec1x80225rabbit_sec1xenopustropicalisfrogfut1nm_001004772frog_fut1monodelphis domesticaopossumfut2-likexm_001362239opossum_fut2_like list of the coding sequences of the 2fts genes, fut1, fut2 and sec1, retrieved from genbank and used in this study phylogenetic relations between mammalian fut1, fut2, and sec1 genes were analyzed using the entire catalytic domain, corresponding to nucleotide positions 235 to 1083, 184 to 1026, and 193 to 1033 of the human fut1, fut2, and sec1 sequences, respectively. only the catalytic domains of these enzymes were used because the three genes are highly divergent for the transmembrane and stem regions, which could not be aligned with confidence. the optimal model of sequence evolution was estimated using the modeltest web server (posada 2006). this model was then used to estimate a maximum likelihood (ml) tree using phyml (guindon and gascuel 2003). 2006a, b) was used to detect possible phylogenetic incongruences, such as those due to gene conversion. ml trees were estimated for each of the segments identified by gard and compared using the shimodaira hasegawa (sh) (shimodaira and hassegawa 1999) test implemented in paup (swofford 2000). in addition, the program geneconv (sawyer 1989) was used to confirm the conversion events inferred by visual inspection of the phylogenetic trees recovered for each fragment. geneconv looks for aligned segments in which pairs of sequences are similar enough to be suggestive of past gene conversion. the programs finds and ranks the highest - scoring fragments globally for the entire alignment (global fragments), and if specified, also for each sequence pair (pairwise fragments). p values are obtained by permutation (in this case 10,000) ; however, whereas global p - values compare each fragment with all possible fragments for the entire alignment, pairwise p - values compare each fragment with the maximum that might have been expected for that sequence pair in the absence of gene conversion. global fragments have p - values that are multiple comparison corrected for all possible sequence pairs, whereas pairwise fragments have a built - in multiple comparison the program also distinguishes between inner fragments, i.e., gene - conversion events between ancestors of two sequences in the alignment, and outer fragments, i.e., evidence of past - gene conversion events that may have originated from outside of the alignment. a mismatch penalty was allowed (gscale = 1) ; therefore, conversion fragments did not have to be identical. ml trees were also obtained with phyml from the amino acid sequences for each of the segments identified by gard using the best - fit model suggested by prottest (abascal. phylogenetic analysis of the nucleotide sequences corresponding to the catalytic domain (supplementary data) yielded evolutionary relations somewhat different from those previously published based on full protein sequences (apoil. for example, rabbit, rat, and mouse fut2 sequences clustered, with high support, with their corresponding sec1 sequences. this was also the case for the pig fut2 and sec1 sequences, but here the pair was embedded inside the main fut2 group. gard analyses indicated 2 highly supported recombination break points at positions 720 and 912 (fig. 1). the resulting fragments were named segment a (nucleotide position 184720) ; segment b (nucleotide position 721912) ; and segment c (9131026 human fut2 nucleotide positions). according to sh test, the resulting phylogenetic trees for each segment were significantly different (p 1.0207479273154191274rabbit_fut2 ; rabbit_sec10.0465>1.018488169839353913pig_fut2 ; gibbon_sec10.04650.14306208551344195371693pig_fut2 ; pig_sec10.04650.2627920892972239724212cow_fut2 ; human_sec10.04650.12367208426219127171604cow_fut2 ; gibbon_sec10.04650.11739208551344195371703pairwise sim p 465bonferroni - corrected ka (blast - like) p where ka p is not corrected for multiple pairwise comparisons. bc p is ka p 465number of polymorphic sites in the fragmentnumber of mismatches within the fragmenttotal number of mismatches between two sequencespenalty per mismatch for the two sequencesfig. 3amino acid alignment of the catalytic domain of the 2fts proteins of a the n - terminal region and b the c - terminal region. the homologous regions between fut1, fut2, and sec1 that may have resulted by gene conversion are highlighted in light grey. amino acid substitutions shared between fut1 and fut2, but not shared with sec1, are highlighted in black. closed square = stop codon ; dashes = alignment gaps ; dots = identity with the consensus sequence ; and question marks = consensus sequence represents nonconsensual amino acids. the consensus sequence is based on the amino acid sequences of the 2fts proteins of the mammals presented list of global inner fragments (484 polymorphisms and 849 aligned bases) obtained with geneconv where inner fragments are runs of matching sites with penalties only fragments with sim p 0.05 are listed corrected for multiple comparisons bonferroni - corrected ka (blast - like) p, where ka p is not corrected for multiple pairwise comparison. bc p is ka p 465 number of polymorphic sites in the fragment number of mismatches within the fragment total number of mismatches between two sequences penalty per mismatch for the two sequences additional pairwise fragments obtained by geneconv with bc pairwise simpval < 0.05 or listed global fragments with significantly better bc simpval (3 pairwise fragments considered per pair) bonferroni - corrected ka (blast - like) p where ka p is not corrected for multiple pairwise comparisons. bc p is ka p 465 number of polymorphic sites in the fragment number of mismatches within the fragment total number of mismatches between two sequences penalty per mismatch for the two sequences amino acid alignment of the catalytic domain of the 2fts proteins of a the n - terminal region and b the c - terminal region. the homologous regions between fut1, fut2, and sec1 that may have resulted by gene conversion are highlighted in light grey. amino acid substitutions shared between fut1 and fut2, but not shared with sec1, are highlighted in black. closed square = stop codon ; dashes = alignment gaps ; dots = identity with the consensus sequence ; and question marks = consensus sequence represents nonconsensual amino acids. the consensus sequence is based on the amino acid sequences of the 2fts proteins of the mammals presented ml trees were estimated at the peptide level for each of the a, b, and c segments of the catalytic domain (amino acids 62342 of the human fut2 enzyme). the trees obtained for each of the segments (data not shown) were consistent with the ones obtained at the nucleotide level. 3), we noted that the number of sites that are identical between fut1 and fut2, but that differ in sec1, is greater in the c - terminal (which includes both b and c segments from amino acid position 237342) than in the n - terminal subdomain (positions 62236), with the exception of the sus and oryctolagus sequences that suffered gene conversion. our results confirm the evolutionary scenario for the origin of 2 ft genes previously reported for primates (apoil., two duplication events could explain the emergence of these genes : an ancestral duplication event originated fut1 and the ancestor of fut2 and sec1, and the ancestor of fut2 and sec1 duplicated and originated the fut2 and sec1 genes. the idea that in mammals gene conversion between 2 ft genes is rare (apoil. 2000) is severely challenged by our results. when only primates are considered, gene conversion is not apparent because fut1, fut2, and sec1 cluster by gene, creating three independent clusters (apoil. 2000) ; however, gene conversion still could be detected with statistical methods as those implemented in geneconv. indeed, when we include other 2 ft mammal genes and use gard and geneconv, we can readily see that gene conversion between fut2 and sec1 has been common. an inspection of the trees, with distinct clades formed by conspecific fut2 and sec1 sequences, and the different segments detected by geneconv, suggests that multiple independent events of gene conversion occurred in the evolution of the 2 ft gene family in mammals. the conversion events have different lengths and can span the three different segments previously detected with the gard software. note that the two phylogenetic break points detected maximize the phylogenetic disagreement and not the exact limits of the conversion events. in our analyses, multiple gene - conversion fragments involving primates were detected by geneconv in segment a (30 of 52). given that all of the sec1 and fut2 primate sequences form a single group in the tree, although defined by a very short branch with a small bootstrap value, all of these fragments might be parsimoniously explained by a single gene - conversion event in the ancestor of primates. evolution after this conversion event, with accumulation of specific mutations in each gene, would explain why they cluster by gene. the clustering of all the other mammals by species and not by gene suggests an ongoing gene - conversion process between fut2 and sec1 within species. in addition, the position of the opossum sequence in segment c within the nonprimate fut2 clade was not expected, although the use of a small segment (120 nt) and the low bootstrap values suggest that this particular result may not be reliable. indeed, the fact that the three genes are located within < 80 kb in the same chromosome prompts gene conversion. in addition, the gene - conversion events may be related to the biologic role of the 2fts. the sec1 gene is inactivated in many primate species, both in old world and new world lineages, by a premature stop codon (apoil. this gene has also been shown to be inactivated in pig and mouse (iwamori and domino 2004), and we recently observed similar evidences in rabbits, where although some sec1 alleles show residual enzyme activity, most are inactive (guillon. 2009). in these species, however, no premature stop codon was observed. altogether, these observations suggest that sec1 is either a pseudogene or that it is on the way to pseudogenisation. at variance, both fut1 and fut2 are active in all mammalian species tested so far (oriol. the fact that the proportion of sites identical between fut1 and fut2, but different in sec1, is higher in the c - terminal subdomain than in the n - terminal domain, and the fact that for most of the species, gene conversion is limited to the n - terminal, suggests that the enzymes must maintain the ancestral characteristics for this particular region (this part of the enzymes most likely resembles the ancestral enzyme that gave rise to this protein family), probably because they require some structural identity to preserve their functionality. nevertheless, based on comparisons with many other glycosyltransferases, some predictions have been made (breton. according to the models, the c - terminal region would correspond to the nucleotide binding domain, whereas the n - terminal part would correspond to the acceptor - binding domain. the latter is generally more variable than the former becasue it should accommodate a number of acceptor substrates much larger than the number of donor substrates. in the case of 2fts, there is a single possible donor substrate, gdp - fuc, whereas the number of acceptor substrates can be quite large. indeed, albeit with different affinities, these enzymes use various acceptor substrates such as gal3glcnac-r, gal4glcnac-r, gal3galnac-r, gal3galnac-r, and gal4glc-r, where r represents the highly variable subjacent chains of glycolipids and of o - linked or n - linked glycan chains of glycoproteins. the redundant nature of sec1 and the different functional constraints on the two regions of the catalytic domain of fut1 and fut2 would explain a higher similarity between these enzymes in the c - terminal part. a comparison of the synonymous and nonsynonymous divergences in both domains indicated that for the three proteins, dn / ds ratios were < 1, suggesting that they are under purifying selection. nevertheless, dn / ds ratios for the c - terminal domain are lower than for the n - terminal domain, consistent with our hypothesis of a higher functional constraint on the c - terminal domain. concerning sec1, dn / ds ratios were also < 1, but they were higher than for either fut1 and fut2, which is at odds with the idea of sec1 being a pseudogene. indeed, if sec1 were a pseudogene, it would be evolving neutrally, and dn / ds should be close to 1. its deviation from neutrality suggests some functional constraints caused by the action of purifying selection. nevertheless, these constraints appear lower than those for fut1 and fut2 (table 1 supplementary material). in humans, both fut1 and fut2 present polymorphisms with null alleles encoding inactive or nearly inactive enzymes, responsible for bombay and the nonsecretor phenotypes, respectively (oriol. likewise, the cell types expressing each enzyme can vary in a species - specific manner. in humans, fut1 null alleles are extremely rare (wagner and flegel 1997), and fut1 is expressed in many cell types, including erythrocytes, the vascular endothelium, some neurons, and epithelial cells (ravn and dabelsteen 2000). in contrast, in humans, fut2 null alleles are almost as frequent as functional alleles, and it has been shown that the gene undergoes balanced selection to maintain both types of alleles at high frequency in various human populations (koda. fut1 has been shown to be involved in some cellular functions, such as adhesion of leukocytes to the vascular endothelium, angiogenesis, and development of the olfactory bulb (amin. 2008 ; moehler. 2008 ; st john. 2006). in contrast, fut2 is mainly expressed in epithelial cells lining the surface of the digestive tract, the upper respiratory tract, and the lower urinary and genital tracts, i.e., in cells in contact with the external environment and potential pathogens (marionneau. 2001). the secretor / nonsecretor polymorphism determined by fut2 has been shown to be associated with sensitivity or resistance to various pathogens, including uropathogenic strains of e. coli, baba - expressing strains of h. pylori, and various strains of norovirus (azevedo. 2008 ; le pendu. the involvement of fut1 in cellular functions and that of fut2 in interactions with pathogens may explain the high frequency of fut2 null alleles in contrast to the rare occurrence of such fut1 alleles. classical studies on the evolution of duplicated genes indicate that the persistence of both duplicates requires their functional differentiation. in the absence of such differentiation, one of the duplicate this is consistent with the inactivation of sec1 in most primate species and with our observation of a limited and most likely ancient gene - conversion event in this lineage. gene conversion involving sec1 after its inactivation may no longer be observed because it would be deleterious to fut2 or fut1. in other species, inactivation of sec1 may be recent or as yet not complete ; therefore, many gene - conversion events between fut2 and sec1 can still be detected. the situation is clearly different in rabbit in which the three genes are involved in gene - conversion events. in such a species, sec1 might have acquired a function distinct from those of the two other 1,2fucosyltransferases genes ; however, that remains to be defined. in conclusion, the gross evolutionary history of 2fts (fut1, fut2, and sec1) seems clear, but the evolution of these genes involved many gene - conversion events that can only be partially characterized and enumerated. it will be difficult to describe the exact phylogenetic relations for each species and gene because these gene conversions differ in position and in length size and because the several histories embedded in the sequences alignment obscures true evolutionary relations. the degree of concerted evolution of the three 1,2fucosyltransferases genes appears to be species - specific, possibly related to the functional differentiation of these genes. below is the link to the electronic supplementary material. supplementary material 1 (doc 113 kb) | the alpha-2-fucosyltransferases (2fts) are enzymes involved in the biosynthesis of 2fucosylated glycan structures. in mammalian genomes, there are three 2 ft genes located in tandem fut1, fut2, and sec1each contained within a single exon. it has been suggested that these genes originated from two successive duplications, with fut1 being generated first and fut2 and sec1 second. despite gene conversion being considered the main mechanism of concerted evolution in gene families, previous studies of primates 2fts failed to detect it, although the occurrence of gene conversion between fut2 and sec1 was recently reported in a human allele. the primary aim of our work was to initiate a broader study on the molecular evolution of mammalian 2fts. sequence comparison leads us to confirm that the three genes appeared by two rounds of duplication. in addition, we were able to detect multiple gene - conversion events at the base of primates and within several nonprimate species involving fut2 and sec1. gene conversion involving fut1 and either fut2 or sec1 was also detected in rabbit. the extent of gene conversion between the 2fts genes appears to be species - specific, possibly related to functional differentiation of these genes. with the exception of rabbits, gene conversion was not observed in the region coding the c - terminal part of the catalytic domain. in this region, the number of amino acids that are identical between fut1 and fut2, but different in sec1, is higher than in other parts of the protein. the biologic meaning of this observation may be related to functional constraints.electronic supplementary materialthe online version of this article (doi:10.1007/s00239 - 009 - 9239 - 0) contains supplementary material, which is available to authorized users. |
testosterone is the main androgen hormone and exerts its physiological effects through a genomic mechanism that is mediated by the androgen receptor. these non - genomic effects are considered to be rapid, in contrast to the genomic effects, and several possible pathways have been identified. the activation of myocardial inflammation signaling enzymes of the mapk family, mediated by the androgen receptor, has been documented, but interestingly this activation is not related to the receptor s transcriptional activity. testosterone and its related androgens has significant anabolic and androgenic effects. since its molecular identification, testosterone has been used as a therapy for hormone replacement, infertility and libido dysfunction, as well as an anti - aging agent. the abuse of androgens for improvement of performance has become a common, yet serious, condition that may have detrimental effects on the athletes. testosterone abuse has been linked to an increase of cardiovascular adverse events such as significant cardiac hypertrophy and myocardial infarction, virilization of women, prostate hyperplasia in men, liver hypertrophy and hepatocellular adenomas or carcinomas [812 ], apoptotic death of myocardial and neuronal cells. on the other hand, recent investigations have shown that testosterone might have anti - ischemic effects and possibly inhibits plaque development. the aim of this study was to examine the effect of testosterone administration at the ultrastructural level of the rat myocardium, as well as to investigate whether testosterone abuse can induce apoptosis. we used high doses of testosterone enanthate for a long period of time mimicking androgen abuse. all procedures for animal use were in accordance with the guidelines of the greek government and the bioethics committee of the medical school of the aristotle university of thessaloniki. we used adult male wistar rats (weighing between 350400 g) separated into 2 groups. group a (experimental) consisted of 6 rats that received 10 mg testosterone enanthate (which equals 7. 5 mg testosterone) intramuscularly for 20 consecutive days, while group b (control group) included 6 rats that were given normal saline via the same route. having completed the administration period, all rats were anaesthetized and sacrificed and their hearts were removed. sections from the hearts were placed in 10% formalin for light microscopy and glutaraldehyde 2.5% for electron microscopy. myocardial tissue samples designated for light microscopy study were dehydrated through a series of increasing ethanol concentrations (25%, 50%, 70%, 80%, 96% and absolute) and finally cleared with xylene. then, the samples were embedded in paraffin wax and sections of 5 m were cut and stained with hematoxylin and eosin. the myocardial tissue samples for electron microscopy were sectioned into small (< 1 cm) pieces and were placed into glutaraldehyde 2.5% for 2 hours and then into osmium tetraoxide 1% for 1 hour. this was followed by staining with uranyl acetate 1% for 16 hours, dehydration with advancing ethanol concentrations and intubation into epon resin. subsequently, ultra - thin sections (600900) were taken and stained with lead citrate (reynolds s stain) and studied using a joel transition electron microscope. we used both qualitative and quantitative analyses which were conducted by 3 of the authors, blinded to the experimental groups. in order to examine the possible activation of apoptosis mouse monoclonal antibodies (ncl - cpp32, novocastra) against caspase-3 (ccp32) were incubated with sections of myocardial tissue after appropriate processing. the paraffin sections were dewaxed, rehydrated and placed in 0.5% hydrogen peroxide / methanol. after washing with tap water, the sections were placed into high temperature and high pressure unmasking solution (0.01 m citrate buffer, ph 6.0) for 1 minute. the sections subsequently were placed into a bath of tap water, washed with tbs buffer and placed in diluted normal serum. then, the sections were incubated with primary (mouse anti - rat - antibody) and secondary biotinylated antibody. the final steps included incubation in abc reagent and in dab, counter - staining with hematoxylin and dehydration. the quantitative evaluation was made from photomicrographs of the myocardial tissue samples from each group by 1 of the authors (v.p.) the area of fibrosis was quantified using imagej software (national institutes of health, bethesda, md, usa), and the fibrosis ratio was calculated by dividing the area of fibrosis by the total myocardial area. the dimensions and areas of mitochondria were measured with the use of imagej and were expressed as mean se for each animal group. the mann - whitney u - test was used for comparisons between the 2 groups. data was analyzed using pasw statistics 18, release version 18.0.0, 2009 (spss, inc., finally, a quantitative measurement of the immunohistochemical staining for caspase-3 was performed using the imagej software. the quantitative evaluation was made from photomicrographs of the myocardial tissue samples from each group by 1 of the authors (v.p.) the area of fibrosis was quantified using imagej software (national institutes of health, bethesda, md, usa), and the fibrosis ratio was calculated by dividing the area of fibrosis by the total myocardial area. the dimensions and areas of mitochondria were measured with the use of imagej and were expressed as mean se for each animal group. the mann - whitney u - test was used for comparisons between the 2 groups. data was analyzed using pasw statistics 18, release version 18.0.0, 2009 (spss, inc. finally, a quantitative measurement of the immunohistochemical staining for caspase-3 was performed using the imagej software. we observed significant myocardial hypertrophy of rats that received testosterone (figure 1), whereas the myocardium of control rats appeared normal, without signs of hypertrophy (figure 2). significant alterations were observed in the myocardial cells and the endothelial cells of the heart capillaries. we found edematous mitochondria in the myocardial cells of the experimental group along with some degree of intracellular edema (figure 3). on the other hand, the mitochondria of the control group appeared to be normal (figure 4). in order to evaluate any differences quantitatively, we measured and analyzed the shape and size characteristics of mitochondria of length exceeding 1.0 m. the maximum length found was 7.96 m. we measured and compared the area (a), the length (l), the width (w) of the equivalent (same area) ellipse w = a/(l) and the elongation (l / w) of 219 mitochondria in the experimental group and 23 mitochondria in the control group. the results presented in table 1, show that the area and the width of the mitochondria were larger for the experimental group, whereas the length and the elongation were larger for the control group. it is indicative that the top 5 values of the area of the mitochondria in the experimental group (9.6830.39 m) were all higher than the highest value of the corresponding area in the control group (7.67 m) ; however, the difference was non - significant (p=0.93), as were the differences in length (p=0.30) and width (p=0.42). on the other hand, the difference in elongation between the 2 groups was statistically significant (p=0.04). there was also disarrangement of the sarcomeres, with disorganization of the myofibrils and the z discus (figure 5). between the capillaries and the myocardial cells we observed collagen fibers, which are an indication of myocardial fibrosis (figures 6, 7). a large number of pinocytoplasmic cysts inside the endothelial cytoplasm was observed (figures 5,8). the appearance of the myofibrils and the mitochondria was normal in the control rats (figure 4). myocardial immunohistochemical staining for caspase-3 was negative for the rats in group b (control group) (figure 9). on the other hand, the myocardial tissue of the rats which received testosterone (group a) exhibited significantly positive staining for caspase-3, which indicates the detection of apoptosis (figures 10, 11). the differences are presented in figure 12, in the form of rgb color histograms, determined by the use of imagej software. to quantify the differences, we calculated the delta - e (e76), as defined by the international commission on illumination (cie), after conversion of the mean rgb values of each image into l, a and b values in the lab color space. the values of e76 between the control and experimental groups were found to be 63.0 and 74.3, for figures 9, 10 and 911, respectively, and only 13.7 between figures 10, 11, which correspond to experimental group images. a value of e76 approximately equal to 2.3 corresponds to a jnd (just noticeable difference). masson trichrome staining showed traces of local collagen fibrils (connective tissue) among the cardiac cells in the control group. figure 13a shows masson s trichrome staining of a representative heart tissue section from a rat of the control group. the amount of collagen fibrils in the experimental group that received testosterone was higher compared to the control group. in sections of cardiac veins, thickening of the muscular tunic with many foamy cells was observed as well as many collagen fibrils in the outer vascular tunica, surrounded by thick adipose tissue (figure 13d). the presence of adipose tissue was evident around the arterioles wall as well. within the adipose tissue we observed significant myocardial hypertrophy of rats that received testosterone (figure 1), whereas the myocardium of control rats appeared normal, without signs of hypertrophy (figure 2). significant alterations were observed in the myocardial cells and the endothelial cells of the heart capillaries. we found edematous mitochondria in the myocardial cells of the experimental group along with some degree of intracellular edema (figure 3). on the other hand, the mitochondria of the control group appeared to be normal (figure 4). in order to evaluate any differences quantitatively, we measured and analyzed the shape and size characteristics of mitochondria of length exceeding 1.0 m. the maximum length found was 7.96 m. we measured and compared the area (a), the length (l), the width (w) of the equivalent (same area) ellipse w = a/(l) and the elongation (l / w) of 219 mitochondria in the experimental group and 23 mitochondria in the control group. the results presented in table 1, show that the area and the width of the mitochondria were larger for the experimental group, whereas the length and the elongation were larger for the control group. it is indicative that the top 5 values of the area of the mitochondria in the experimental group (9.6830.39 m) were all higher than the highest value of the corresponding area in the control group (7.67 m) ; however, the difference was non - significant (p=0.93), as were the differences in length (p=0.30) and width (p=0.42). on the other hand, the difference in elongation between the 2 groups was statistically significant (p=0.04). there was also disarrangement of the sarcomeres, with disorganization of the myofibrils and the z discus (figure 5). between the capillaries and the myocardial cells we observed collagen fibers, which are an indication of myocardial fibrosis (figures 6, 7). a large number of pinocytoplasmic cysts inside the endothelial cytoplasm was observed (figures 5,8). the appearance of the myofibrils and the mitochondria was normal in the control rats (figure 4). myocardial immunohistochemical staining for caspase-3 was negative for the rats in group b (control group) (figure 9). on the other hand, the myocardial tissue of the rats which received testosterone (group a) exhibited significantly positive staining for caspase-3, which indicates the detection of apoptosis (figures 10, 11). the differences are presented in figure 12, in the form of rgb color histograms, determined by the use of imagej software. to quantify the differences, we calculated the delta - e (e76), as defined by the international commission on illumination (cie), after conversion of the mean rgb values of each image into l, a and b values in the lab color space. the values of e76 between the control and experimental groups were found to be 63.0 and 74.3, for figures 9, 10 and 911, respectively, and only 13.7 between figures 10, 11, which correspond to experimental group images. a value of e76 approximately equal to 2.3 corresponds to a jnd (just noticeable difference). masson trichrome staining showed traces of local collagen fibrils (connective tissue) among the cardiac cells in the control group. figure 13a shows masson s trichrome staining of a representative heart tissue section from a rat of the control group. the amount of collagen fibrils in the experimental group that received testosterone was higher compared to the control group. in sections of cardiac veins, thickening of the muscular tunic with many foamy cells was observed as well as many collagen fibrils in the outer vascular tunica, surrounded by thick adipose tissue (figure 13d). the presence of adipose tissue was evident around the arterioles wall as well. within the adipose tissue cardiac arrhythmias, qt dispersion, atrial fibrillation, myocardial infarction, heart failure and atherogenesis have all been linked to androgen abuse by athletes. melchert and welder proposed 4 hypothetical models of anabolic - induced adverse cardiovascular effects : an atherogenic model involving the effects of androgen - anabolic steroids (aass) on lipoprotein concentrations ; a vasospasm model involving the effects of aass on the vascular nitric oxide system ; a direct myocardial injury model involving the effects of aass on myocardial cells. our study shows that the administration of testosterone in high doses exerts toxic effects on the myocardial cells. this probably correlates with the direct myocardial injury model of the effects of androgen - anabolic steroids on the myocardial cells. the morphometric approach showed that the mitochondria of the experimental group were larger and more rounded. the contractile apparatus showed signs of deterioration, with disorganization of the sarcomeres and the z discus. these observations have been reported earlier and have been characterized as typical for early heart failure. another interesting finding of this study, using the masson s trichrome staining, was the presence of collagen fibrils inside the myocardium and particularly between the capillaries and the myocardium cells. this perivascular myocardial fibrosis, together with the hypertrophy that was also induced, may be the cause of myocardial ischemia as well as a substrate for arrhythmias. reported that acute exposure of hearts to testosterone significantly reduces the dp / dt (a measure of cardiac compliance). the myocardial hypertrophy observed in the present study and also in our previous experiments, as well as by other researchers, has a significant role in the testosterone - induced reduction of myocardial compliance. the hypertrophy of the myocardium correlates with the enhanced expression of the androgen receptor after testosterone administration. abnormalities of the circulating levels of other hormones may induce adverse cardiovascular effects. increased levels of the protein hormone leptin have been found to induce cardiac hypertrophy although diastolic dysfunction was not associated with leptin levels. another set of hormones, the thyroid hormones, do have an impact on the myocardial diastolic properties among other various cardiovascular effects. an unusually large number of micropinocytic vesicles was observed in the cytoplasm of the endothelial cells. although it is common for these vesicles to occur in capillary endothelial cells, especially in the striated muscles, the abundance of such vesicles is a sign of heightened pinocytic activity that permits the cell to receive substances through the cell membrane. apoptosis is the programmed cell death and is mediated by 2 pathways : the extrinsic death receptor signaling pathway and the intrinsic mitochondrial control pathway. caspase-3 (cpp 32) is a member of the interleukin-1 beta - converting enzyme (ice) family of mammalian proteases that specifically cleaves substrates at the c - terminal side of aspartic residues. members of this family have been implicated in apoptosis, and caspase-3 acts as a control mediator of programmed cell death in mammalian cells. caspase-3 is synthesized as an inactive 32kd proenzyme and is processed during apoptosis to its active form, which is responsible for the cleavage of poly (adp - ribose) polymerase (parp), actin and sterol regulatory element binding protein (sredp) [3840 ]. in the present study, testosterone overdosing significantly activated apoptosis, as was clearly seen by immunohistochemistry. although it has been reported that apoptosis activated by testosterone enanthate is due to the ester, experimental exposure of myocardial cells to enanthate alone did not activate apoptosis. the abuse of androgen anabolic substances has been causally linked with sudden cardiac death, myocardial infarction, ventricular remodeling and cardiomyopathy. myocardial death without coronary vessel disease or atherosclerosis has also been attributed to the activation of apoptosis by aass. this study showed that testosterone abuse produces significant myocardial hypertrophy and fibrosis as well as of the myofibrils, the mitochondria and the capillaries. the activation of apoptosis was a significant finding that indicates the direct myocardial injury caused by testosterone. as testosterone is now used for a variety of possible indications such as hormone replacement in the elderly or as an antianginal in cardiac patients, more research is required to clarify the exact biochemical route of action as well as the correct dose to prevent adverse effects. | summarybackgroundandrogen abuse is an increasing problem amongst professional and amateur athletes. moreover, testosterone, apart from its widely accepted indications, is used for a variety of other indications such as aging and ischemia. its actions are mainly attributed to a specific genomic mechanism through the androgen receptor, but emerging evidence reveals non - genomic effects as well. the use of androgens has been linked with several adverse effects. the purpose of this study was to examine the effects of testosterone on the morphology and the ultrastructure of the myocardium and to investigate the possible role of apoptosis.material/methodswe used 12 adult male wistar rats, separated into 2 groups. group a consisted of 6 rats that were administered high doses of testosterone enanthate, while group b consisted of 6 male wistar rats that received placebo (normal saline) intramuscularly. after the last day of treatment, all rats were anesthetized and sacrificed, and the hearts were removed and processed for optical and electron microscopy and immunohistochemical detection of caspase-3, an apoptosis marker.resultswe found significant myocardial hypertrophy along with abundant ultrastructural alterations. the immunohistochemical staining of the myocardial cells for caspase-3 was positive in group a (experimental group), which is interpreted as an activation of apoptosis by testosterone treatment.conclusionstestosterone abuse has serious adverse effects, including myocardial hypertrophy, myocardial fibrosis and activation of apoptosis. these findings need to be taken into account whenever androgens are prescribed to improve performance or as hormone therapy. |
several important novel structures evolved in ancestral vertebrates, including a complicated nervous system equipped with integrated sensory systems, bones, and acquired immune systems. importantly, these novel structures probably did not evolve all at once, but via sequential steps. therefore, to understand the early evolutionary history of the vertebrates, we need to reveal the evolutionary sequence of the emergence of these novel structures. this information will have rich implications concerning how the bodies of ancestral vertebrates became more elaborate, and what kinds of adaptation promoted the evolution of the body plan. to infer the evolutionary sequence of vertebrate characteristics, in addition, the genome is another possible chronicle of the early history of vertebrates. it is now widely accepted that vertebrates experienced two rounds of whole genome duplication, which can be traced from conserved genomic segments in chromosomes 1, 2. traces of these genome duplications are also evidenced in the observations that most invertebrate genes have two to four counterparts in vertebrate genomes. in this article, we test the possible relationship of these genome duplications to the evolution of the neural crest. since gans and northcutt (1983) 3 proposed the new head theory, the origin and evolution of the neural crest has been one of the main issues in vertebrate evolutionary biology. the neural crest is a population of cells that differentiates at the boundary between the neural tube and the surface ectoderm. protochordates represented by ascidians and amphioxus, which are the closest relatives of vertebrates, lack comparable cells, such as migratory cells or multipotent cells, at the boundary between the neural tube and the surface ectoderm (but see jeffery. therefore, the neural crest was once regarded as a new cell type that emerged at the origin of the vertebrates. in addition, the neural crest gives rise to several structures that are characteristic of vertebrates, such as cranial sensory organs and the craniofacial skeleton. therefore, gans and northcutt (1983) 3 emphasized that the evolution of the neural crest together with the placode was a key evolutionary event in vertebrate evolution. recent analyses of morphogenetic genes in protochordates have led to a revision of this idea. the expression of amphioxus dll and ascidian pax3/7 homologs in the dorsal midline epidermis abutting the neural tube has led some authors to postulate that the differentiation of the dorsal midline epidermis and dorsal part of the neural tube of protochordates is regulated by machinery comparable to that of the vertebrate neural crest 5, 6. however, the neural crest of vertebrates is quite distinct from its protochordate counterpart in terms of pluripotency and migration. although cells in the amphioxus dorsal midline epidermis migrate, they do so as a cell sheet and do not delaminate from each other. therefore, the evolution of the neural crest may be regarded not as the birth of a new cell population, but as the acquisition of new cell characters, such as migration - delamination and pluripotency 7. to gain insight into how these new cell characters evolved, the genes whose vertebrate homologs are involved in the delamination of the neural crest were analyzed. here, we attempt to reconstruct the evolutionary history of the neural crest by examining the molecular evolution of the genes involved in neural crest development, focusing on whether these genes were involved in neural crest development before or after the genome duplications. when more than two vertebrate paralogs are involved in a certain process of development, it is likely that these genes were involved in the developmental process before the genome duplications occurred. conversely, when only one of the duplicated genes is involved in a certain developmental process, the gene may have acquired its function after the genome duplications occurred. nevertheless, there are some instances in which the primitive function of a gene has been retained by only one of the duplicated genes, such as for the three hedgehog genes of vertebrates (sonic [shh ], desert [dhh ], and indian [ihh ] hedgehog). amphioxus possesses a single ancestral type of the gene (hh) that is expressed in the midline structure endoderm, the notochord, and the floorplate 8. this primitive function of hh has been inherited by shh only, and not by ihh or dhh. therefore, when only one of the duplicated genes is involved in a certain developmental process, the function of the gene in protochordates must be considered. if only one of the duplicated genes possesses a certain role, which is not shared by protochordate homologs, it is more likely that the role was acquired after the genome duplications. since vertebrates experienced two rounds of genome duplication, they should ideally possess four genes corresponding to each protochordate gene. however, the duplicated genes were redundant, and some of them were lost secondarily. when only two paralogs are retained in vertebrates, it is not easy to deduce which round of duplication gave rise to the two genes. moreover, some genes have experienced gene duplications that occurred independently of the genome duplications. for example, it is accepted that teleost fishes experienced a third genome duplication. to avoid the confusion that arises from additional gene duplications, here, we analyzed when genes acquired their functions in neural crest development, and based on this information, we deduced the evolutionary history of the neural crest. we found that many, but not all, of the transcription factors were involved in neural crest development before the genome duplications. in contrast, only some of the paralogs of the cell adhesion molecules and rhob that are involved in neural crest delamination are involved in neural crest development. several transcription factors are known to be involved in neural crest development 9. for most of these transcription factors, are expressed at the neural plate border, i.e., at the lateral edge of the neural plate and the neural crest 10, 11. protochordates have single counterpart genes that are also expressed in the neural plate border 6, 12. therefore, it is likely that pax3/7 acquired a role in neural crest specification before the genome duplications. similarly, of the three mouse msx genes, msx1 and msx2 are expressed in the neural plate border 13 - 17, and their protochordate counterpart also marks the neural plate border 18, 19. molecular phylogenetic studies have indicated that two ascidian zic genes [macho and zicl(n) ] emerged from an ancestral gene independent of the duplication that gave rise to the vertebrate zic genes 21, 22. although gostling and shimeld (2003) 23 did not resolve the molecular evolutionary status of amphioxus zic, their tree is consistent with the idea that genome duplications in early vertebrates gave rise to multiple vertebrate zic genes. two ascidian zic genes are expressed in the notochord, muscle, and neural tube, while a restricted expression pattern along the dorsoventral axis of the neural tube was not observed 21, 22, 24. amphioxus zic is also expressed in the neural tube and somatic mesoderm, and later expression in the neural tube is restricted to the dorsal part 23. the mouse possesses six dlx genes, all of which are expressed in the neural crest or its derivatives 25. although molecular phylogenetic analysis did not resolve the phylogeny of chordate dlx genes, it is consistent with the idea that multiple dlx genes have evolved through the genome duplications of ancestral vertebrates (fig. the six vertebrate dlx genes are located on chromosomes as three pairs, and the three pairs are linked with the hox clusters of vertebrates 26. duplication of the hox cluster of vertebrates is the best - known example of the genome duplications. interestingly, two of these (dlla and dllb) are linked on a chromosome in the tail - to - tail direction as vertebrate homologs 27. our phylogenetic analysis weakly support the close relationship of ciona dlla to vertebrate dlx1, 6, and 7, while dllb is more closely related to dlx2, 3, and 5 of vertebrates. this suggests that the tandem duplication of dlx genes predates the divergence of ascidians and vertebrates ; i.e., the common ancestors of ascidians and vertebrates possessed dlx genes linked in tandem, although the high substitution rate of ciona dlx genes makes the tree topology less convincing (fig. one of the ciona genes, dlxc, and amphioxus dlx are expressed in the neural plate border 5, 27, 28. in the chicken and mouse, the expression of snail and slug in the neural crest have been swapped ; snail, but not slug, is expressed in the mouse pre - migratory neural crest, while slug, but not snail, is expressed in the chicken pre - migratory neural crest 29. this swap can be explained easily if the common ancestors of vertebrates had acquired snail / slug expression before the gene duplication occurred. indeed, in xenopus, both snail and slug are expressed in the neural crest 30. the molecular phylogeny supports the evolution of snail and slug in ancestral vertebrates via gene duplication, and amphioxus and ascidians possess the ancestral type of gene 31. both amphioxus and ascidian snail are expressed in the neural tube, although the expression profiles differ 31 - 32. while amphioxus snail marks the edge of the neural plate that forms the dorsal part of the neural tube 31, the ascidian genes are expressed predominantly in the lateral cells of the neural tube and not in the dorsal part 33,32. three vertebrate paralogs of soxe (sox8, 9, and 10) are expressed in the neural crest. both ascidian and amphioxus possess a single ancestral type of soxe gene 9, 21 (hw, unpublished data). therefore, although soxe expression has not been examined in either ascidians or amphioxus, soxe was likely involved in neural crest specification before the genome duplications. vertebrates possess five ap-2 genes ; three of them, ap-2a, b, and g, are expressed in the neural crest 34. molecular phylogenetic studies support the evolution of four vertebrate genes in the ancestral vertebrates, although we could not resolve the phylogenetic position of ap-2d 35 (fig. ciona has two ap-2 genes, and one of them is not expressed in early embryogenesis, while the other one (ap-2-like2) is expressed in epidermal cells 28. interestingly, careful examination has revealed that the expression of ap-2-like2 is up - regulated in the dorsal midline epidermis (fig. similar up - regulation is also observed in halocynthia ap-2 (fig. 2c). this expression pattern is reminiscent of xenopus ap-2a, which is expressed rather broadly in epidermal cells, and their expression is subsequently up - regulated in the neural crest cells 36. vertebrates have five paralogs of the d group fox genes, of which foxd1 and d2 are relatively closely related 37 molecular phylogenetic studies support the hypothesis that the genome duplications gave rise to foxd1 - 2, d3, d4, and d5 38. in contrast to the genes mentioned above, only one of the paralogs of foxd, foxd3, is involved in neural crest specification 40, 41. ascidian and amphioxus genes are expressed in the endoderm, notochord, and neural tube, and no restricted expression pattern was observed along the dorsoventral axis of the neural tube 38, 39. therefore, foxd3 genes might have become involved in neural crest specification after the genome duplication occurred, although the possibility can not be excluded that foxd became involved in neural crest specification in ancestral vertebrates before the genome duplications, and that function was inherited by one of the duplicated genes, foxd3. delamination of the neural crest depends on the regulated expression of cell adhesion molecules, i.e., cadherins. n - cadherin and cadherin6 are expressed in the pre - migratory neural crest, while others, such as cadherin7 and cadherin11, are expressed in post - migratory neural crest cells 42,43. the ectopic expression of n - cadherin or cadherin7 in the neural tube prevents neural crest delamination without affecting differentiation of the neural crest 44. molecular phylogenetic analyses of cadherin genes have shown that vertebrate cadherin genes can be classified into two families : type i cadherins include e, n-, p-, and r - cadherins, while type ii includes other classic cadherins, such as cadherin6, 7, and 11 45 (fig. type i cadherin is expressed ubiquitously both in ciona and halocynthia 47 (data not shown), whereas type ii cadherin (cicadherinii) is predominantly expressed in the neural tube 48 (fig. 3d). this expression pattern of cicadherinii suggests that in addition to its role in neurulation, type ii cadherin played a role in the formation of neuromeres in the brain of the ancestral chordate. amphioxus possesses two cadherins, whose molecular structures are odd and show similarity to those of non - chordate invertebrates 49, 50. we could not tell when the gene duplication that gave rise to type i and type ii cadherin occurred in the present molecular phylogenetic analysis 50 ; however, multiple copies of type i and type ii genes obviously emerged in ancestral vertebrates (fig. therefore, the evolution of the molecular machinery using these cadherins for neural crest delamination must have occurred after the genome duplications. the rho family of small gtpases is involved in several aspects of regulating cell motility. molecular evolutionary analyses have presented evidence that vertebrates acquired three rho genes that were derived from a single ancestral chordate gene 52 (fig. 3b). halocynthia has a rho gene that is expressed ubiquitously (data not shown). in contrast to the transcription factors involved in neural crest specification, the molecular evolutionary histories of cadherins and rho genes suggest that the genome duplications of vertebrates were essential for the innovation of neural crest cells as migrating cells. it is not certain whether the gene duplications of rho gtpases were essential for the evolution of neural crest delamination, i.e., whether the neural crest requires a specific type of rho gtpase for its control of delamination. the ancestral vertebrate could possibly control the delamination of neural crest cells via the posttranscriptional regulation of the activity of a single rho gene. in the case of cadherins, however, without genome duplication, the ancestral vertebrates could not possess specific repertoires of cadherin molecules for the pre- and post - migratory stages. interestingly, foxd3, which was recruited in neural crest specification possibly after the genome duplications, is involved in regulating cadherin7 expression 53. assuming that the ancestral vertebrates acquired a migratory neural crest before the genome duplications, the delamination of the neural crest must have been regulated using a small set of adhesion molecules. this is not impossible because invertebrates, such as drosophila and sea urchins, regulate several aspects of epithelial - mesenchymal transitions using a small set of cell adhesion molecules. this may result because vertebrates possess more repertoires of cell adhesion molecules, which arose via the genome duplications, and the neural crest may be one of the new cell types that acquired mesenchymal cell properties by using the rich repertoires of cell adhesion molecules. in this sense, it is interesting to determine how the delamination of the lamprey neural crest is regulated. however, it remains to be elucidated whether extant agnathans, such as lampreys and hagfish, experienced the second round of gene duplication 2. if the ancestral vertebrate possessed true migrating neural crest cells before the genome duplication, they must have been controlled by mechanisms distinct from that in present gnathostomes, which utilize multiple sets of cadherins. jeffery. (2004) found that one colonial species of ascidian (ecteinascidia turbinate) possesses migrating cells from the neural tube, and that they differentiate into pigment cells, just like vertebrate neural crest cells 4. as mentioned above, however, the delamination of these cells is unlikely to be regulated by switching several paralogs of cadherin genes. the control of delamination in the ascidian neural crest - like cells is an interesting issue. the mechanism regulating those cells may represent the ancestral machinery of the genetic control of neural crest delamination before the genome duplications. conversely, because the neural crest - like cells begin migration well after neural tube closure, the cells might have acquired migration independently from the vertebrate neural crest. by referring to the molecular evolutionary histories of the genes involved in neural crest cell differentiation and delamination, we reconstructed the hypothetical evolutionary steps of neural crest evolution. most of the transcription factors involved in the neural crest specification have ancestral functions in the neural plate border or neural tube of protochordates. these transcription factors likely acquired roles in neural plate border and neural crest specification before the genome duplications, except for foxd3, which might have been co - opted for neural crest specification after the genome duplications. by contrast, the epithelial - mesenchymal transition of neural crest cells is controlled by genes that evolved after the genome duplications. this suggests that delamination of the neural crest evolved after the genome duplications, although the possibility that primitive neural crest cells control their delamination via a distinct mechanism using a small set of cell adhesion molecules can not be dismissed. the key factor to determine whether the primitive neural crest delaminated prior to the genome duplications may be the lamprey neural crest. the lamprey has true migratory neural crest cells, but it may not have experienced both rounds of genome duplication. the tree is constructed by the quartet maximum likelihood method using treepuzzle 54 based on amino acid sequences of the homeodomain. vertebrate genes can be classified into two groups, and each protochordate gene shows phylogenetic affinity to one of them. this suggests that the tandem duplication of dlx genes predates the divergence of the chordate groups. (a - c) the expression of ciap-2 (a, b) and hrap-2 (c) at neurula stage. embryos in a and c are viewed from dorsal side, while b is the lateral view (anterior to the left). upregulation in the dorsal midline epidermis of ap-2 is observed in both ciona and halocynthia (arrowheads). note that epidermal cells abutting the anterior edge of the neural plate also show upregulation of the ap-2 (arrow). ap-2 from halocynthia roretzi (hrap-2) was isolated from a cdna library of the gastrula stage (acc. no. the tree is constructed by quartet maximum likelihood method using treepuzzle 54 based on the conserved amino acid sequences of the helix - span - helix motif. (a, b) molecular phylogenetic trees of cadherin (a) and rho (b) genes, constructed by quartet maximum likelihood method using treepuzzle 5.0 54. the amino acid sequences of the c - terminus cytoplasmic domain were used for the cadherin gene analysis, while entire amino acid sequences were used for the rho genes. nucleotide sequences of rho genes from branchiostoma belcheri (bbrho) and halocynthia roretzi (hrrho) by following acc. (c, d) the expression of cicadherinii in the neurula (c : lateral view) and tailbud stage embryo (d). cicadherinii is expressed uniformly in the neural plate of neurula embryo (c), while in tailbud stage, it shows more restricted expression in the neural tube (d), suggesting its role in neuromere formation of ascidian cns. | it is now accepted that ancestral vertebrates underwent two rounds of genome duplication. here we test the possible utility of these genome duplication events as a reference time for the evolutionary history of vertebrates, by tracing the molecular evolutionary history of the genes involved in vertebrate neural crest development. for most transcription factors that are involved in neural crest specification, more than two paralogs are involved in that process. these were likely involved in the specification of the neural crest before the genome duplications occurred in ancestral vertebrates, although foxd3 may have acquired that role after the genome duplications. by contrast, the epithelial - mesenchymal transition of neural crest cells is controlled by genes that evolved after the genome duplications, such as cadherin6, cadherin7, cadherin11, and rhob. this suggests that primitive neural crest cells control their delamination by using a small or distinct set of cell adhesion molecules. alternatively, these observations suggest that delamination of the neural crest evolved after the genome duplications. in that case, the neural crest might have evolved in sequential steps ; the specification of the neural crest occurred before the genome duplications, and the neural crest acquired a new cell migration property after the genome duplications. |
recent advances in brain imaging techniques facilitate the investigation of brain activity levels during movement by unrestrained human subjects. imaging studies using single photon emission computed tomography and functional near - infrared spectroscopy (fnirs) have demonstrated that, in the frontal lobe, the primary sensorimotor area and the supplementary motor area (sma) were coactivated during walking in healthy human subjects. for successful execution of bipedal gait, the central nervous system (cns) integrates neural substrates involved in the control of upright posture and stepping movements. concerning cortical mechanisms for the control of locomotion, neurophysiological studies in cats and humans have demonstrated that the (primary) motor cortex modulates ongoing activities in the spinal circuitries via the corticospinal or the pyramidal tract [3, 4 ]. however, studies on the role of the sma in locomotor control are fairly limited. clinical observations have shown that patients with focal lesions involving the sma exhibited mixed signs including disequilibrium and gait abnormalities [5, 6 ]. gurfinkel ' and l'ner and viallet. studied patients with brain lesions and hypothesized that the secondary motor area, particularly the sma, may participate in postural adjustments associated with voluntary movements. thus, our understanding of the functional significance of the sma in the control of bipedal gait in humans remains unclear. in addition, humans commonly exhibit quadrupedalism during infancy, that is, crawling, which then develops into upright bipedalism. experimental evaluations of the functional significance of the sma in postural control during locomotion could include comparative studies of mammalian quadrupedalism and human upright bipedalism or among humans at different developmental stages, such as crawling in infants versus upright bipedalism in adults. however, these intergroup comparisons are applied onto the different cns and musculoskeletal system across the groups ; thus, they hardly extract the main posture - specific differences in different locomotor modes. by contrast, intragroup comparisons of quadrupedal and bipedal gaits are expected to be much more effective because the cns and musculoskeletal system in a single subject can be studied in two modes of locomotion. however, there are currently no previous neuroimaging studies comparing bipedalism and quadrupedalism performed in the same subjects or even those only on quadrupedalism in humans. the aim of the present study was to investigate the functional significance of the sma in the control of locomotor behavior using fnirs by focusing on posture. quadrupedal stance is considered more stable than upright stance, because the center of gravity (cog) is closer to the ground and the base of support bounded by the hands and feet is larger than that by the left and right feet only. based on these biomechanical perspectives, we hypothesized that the activation level of the sma in human adults would correlate with degree of postural instability that accompanies locomotor movements. to test this hypothesis at a coarse - grain level, we asked each subject to perform three locomotor tasks, namely, hand - knee quadrupedal crawling (hkquad task), upright quadrupedalism using bilateral lofstrand crutches (upquad task), and typical upright bipedalism (upbi task) on a treadmill, where the degree of postural instability varied biomechanically, and we measured hemodynamic responses in the sma. ten healthy human adults (five males and five females aged 32.0 7.7 (mean sd) years, range, 2345 years) participated in this study. all the procedures were conducted in accordance with the declaration of helsinki, and they were approved by the ethics committee of chiba - hokusoh hospital, nippon medical school. we used an fnirs system (etg-4000 optical topography system ; hitachi medical co., tokyo, japan) to measure the sma activity, while participants performed locomotor tasks on a treadmill (fitcrew gmjp - t1 - 65 - 12130001 ; greenmaster japan co. ltd., the details of the fnirs system were described in our previous studies [10, 11 ]. in brief, the system emitted near - infrared light (695 and 830 nm) and detected the transmitted light to measure relative changes in oxygenated hemoglobin (oxy - hb) and deoxygenated hemoglobin (deoxy - hb) concentrations. the oxy - hb value was measured every 0.1 s (i.e., sampling rate of 10 hz). eight emission probes and eight detection probes (yielding 24 channels) were positioned with centering on fz according to the international 10/20 system for electroencephalogram electrode placement (figure 1). the interprobe distance was set at 3.0 cm. as reported by okamoto. and in our previous study, the sma was covered by channels 16, 19, 20, and 23. all participants were required to perform three locomotor tasks on the treadmill : hand - knee quadrupedal crawling (hkquad task), upright quadrupedalism using bilateral lofstrand crutches (upquad task), and typical upright bipedalism (upbi task) (figure 2(a)). from biomechanical perspectives, the stability of the stance is proportional to the inverse of the height of the cog, area of the base of support, and weight of the body. thus, we designed the three tasks so that the combinations of the area of the support base (large or small) and the height of the cog above it (high or low) differed. in the hkquad task (figure 2(a), (a)), participants were required to crawl quadrupedally with a dorsal - side - up posture. in this task, the trunk was maintained nearly horizontal (low cog) and supported by all four limbs (large support base). it has been reported that > 96% of humans use hkquad in their infancy and that hand - foot crawling is observed rarely. therefore, we tested hkquad as a relatively natural locomotor mode for human adults. in the upbi task (figure 2(a), (c)), participants were required to walk bipedally with an upright posture. in this task, the trunk was maintained nearly vertical (high cog) and supported only by two legs (small support base). compared with the hkquad task, the posture in this task was considered more unstable because the position of the cog was higher, and the area of the support base was smaller. in addition to the hkquad and upbi tasks, we tested the upquad task as artificial quadrupedalism, where participants were required to walk on their two legs and bilateral lofstrand crutches (figure 2(a), (b)). in this task, the trunk was maintained nearly vertical (high cog), but it was supported by both the legs and a pair of crutches (large support base) (figure 2(b)). the postural instability of this task was considered to be intermediate between that of the other two tasks. compared with the hkquad task, the area of the support base in the upquad task was similar, but the position of the cog was higher, thereby resulting in a more unstable posture, whereas, compared with the upbi task, the position of the cog was similar, but the area of the support base was larger, thereby resulting in a more stable posture. therefore, we were able to rank the tasks in a reasonable manner based on their postural instability, with the upbi task first, the upquad task second, and the hkquad task last. in addition, as shown in figure 2(b), we could distinguish the three tasks based on the two aspects of locomotor control : the orientation of the trunk and the number of stepping limbs that encompassed the support base. therefore, the results could be categorized as one of two cases, as follows. first, if the sma contributes to the control of upright posture, oxy - hb levels may be correlated to postural instability and the differences in hemodynamic responses may be altered between tasks performed in an upright posture and those in a horizontal posture (figure 2(b), left). second, if the sma is involved in stepping movements, the results may be altered between tasks performed with four stepping limbs and those with two stepping limbs (figure 2(b), right). for safety reasons, the treadmill speed in the hkquad and upquad tasks was set at the slowest (0.8 km / h) for all participants. in the upbi task, the heart rate was carefully monitored during practice sessions, and the treadmill speed was set for each participant to counterbalance their heart rate during the performance of the hkquad and upbi tasks. (we monitored heart rates during the practice sessions and set the speed of upbi so that the heat rate during hkquad and the heat rate during the upbi task would be almost the same.) the actual speed of the 10 participants in the upbi task ranged from 1.4 to 3.7 km / h (mean 2.3 km / h). each task comprised five repetitions of locomotion for 30 s each followed by 40 s of rest as a block. during the rest periods between hkquad repetitions, participants were instructed to remain still in the hand - knee position. during the rest periods between upquad repetitions, they were instructed to stand still while supporting themselves with crutches and two legs. during the rest periods between upbi repetitions, the head tilt across the three tasks, a mark was placed in front of participants, and they were instructed to look at it during measurements. body movement artifacts were detected as rapid oxy - hb change (0.2 mmol / l mm during 2 s). when body movement artifacts were observed during measurements, these data were excluded from the analyses. we continued the experiments with each participant until we obtained at least three fair repetitions out of five repetitions of each task. we used oxy - hb concentrations rather than deoxy - hb concentrations because the former are reportedly related to brain activity [1618 ]. first, the data were automatically processed using the integral mode, a command defined by the fnirs system. this command corrects for baseline drift and averages across 3 to 5 repetitions within each task. a first - degree baseline fit was estimated ; the fit was computed between the mean of 5 s period immediately before locomotion and the mean of 3035 s of the rest period. this command is usually recommended for data processing [11, 19, 20 ]. second, we determined the sma waveform by calculating the spatial average across channels 16, 19, 20, and 23 (i.e., the region of interest covering the sma). this process was performed for each task in each participant. finally, we calculated the mean oxy - hb values for three distinct phases during the task : rest, starting, and steady phases (figure 4). the fnirs system measured the oxy - hb value every 0.1 s, and we calculated the temporal mean of a 5 s period for each of the phases. the rest phase oxy - hb value was calculated as the mean of the 5 s period immediately before locomotion. the starting phase oxy - hb value was calculated as the mean of the first 510 s of the locomotion period. the steady phase oxy - hb value was calculated as the mean of 2025 s of the locomotion period. these time periods were selected to consider a delay of several seconds between neural activity and hemodynamic response [21, 22 ]. it is generally accepted that oxy - hb values in the starting phase reflect the cortical activity due to adjustment to the initial movement of the treadmill belt, whereas those in the steady phase reflect the cortical activity related to steady locomotion itself. therefore, to detect significant hemodynamic changes between rest and locomotion, we performed paired t - tests between the rest and the other two phases for all participants according to each locomotor task using spss (ver. the postures during the rest periods differed in the three tasks, as mentioned above (section 2.3) ; thus we did not compare the oxy - hb values across the three tasks. figure 5 shows the grand mean waveforms for the oxy - hb levels in the sma across all participants for the hkquad (a), upquad (b), and upbi (c) tasks. oxy - hb values in the rest, starting, and steady phases for each task are shown in table 1. in the hkquad task, the oxy - hb level progressively decreased from the starting phase until the early rest phase (figure 5(a)). compared with the rest phase, the oxy - hb value in the starting phase was not significantly different, but the value in the steady phase was significantly lower (p upbi > hkquad) did not correlate with the order of the postural instability (upbi > upquad > hkquad). however, qualitative inspection of oxy - hb time course responses showed that, regardless of quantitative differences, oxy - hb response patterns differed between the tasks with an upright posture and the task with a hand - knee crawling posture. these results suggest that the task - dependent patterns of hemodynamic waveforms could at least partially reflect the functional significance of the sma for the control of truncal posture accompanying locomotor movements in humans. to the best of our knowledge, this is the first study to describe hemodynamic responses in the sma during quadrupedal gait in humans. during the hkquad task, oxy - hb value in the steady phase dramatically decreased beyond the level of significance (figure 5(a) and table 1). a decrease in oxy - hb level is often interpreted as indicating deactivation of a brain region [2326 ]. in mammals, the central mechanisms for controlling quadrupedal locomotion largely depend on subcortical structures, and decorticated animals can still walk on a smooth floor without any support. in addition, compared with the quiet stance during rest, crawling involves a hand - knee posture and stepping movements of the arms and legs. overall, our observation of a decrease in oxy - hb level suggests that the blood flow over the sma with a quiet stance could be redistributed to subcortical structures and/or to other cortical areas, such as the primary sensorimotor cortex, during the quadrupedal gait. the current study considered only the sma because of the limited number of nirs probes, but we plan to obtain measurements from the primary sensorimotor cortex as well as the sma in future studies. previous studies using fnirs have shown that oxy - hb level in the sma significantly increased during bipedal walking [2, 22 ]. however, in our study, the upbi task failed to evoke any significant hemodynamic responses in the sma. the first possibility is differences in stepping parameters employed when walking on the treadmill. in our upbi task, the speed was specifically set for each participant to counterbalance the heart rate between the upbi and hkquad tasks, where it ranged from 1.4 to 3.7 km / h. by contrast, in the study by miyai., the cadence was set at 100 steps / min at 1.0 km / h, which should be considerably higher than that in our study. also set the speed higher (3.05.0 km / h) than that used in the upbi task in our study. walking is a rhythmic and stereotyped behavior, which is automatically controlled to a considerable degree at relatively low levels of the nervous system without intervention from higher centers, such as the cerebral cortex. in this study, participants may have become acclimatized to walking on the treadmill after these practice sessions, thereby reducing hemodynamic responses over the sma during the task performance. regardless of the statistical significance, the waveform of oxy - hb responses exhibited a peak - plateau - trough pattern (figure 5(c)). this response pattern is literally observed not only in the sma but also in the prefrontal cortex [28, 29 ] and the medial sensorimotor cortex during bipedal walking by the healthy subjects. this suggests that the peak - plateau - trough pattern might be common features across several cortical regions and that, more importantly, the response pattern observed in the sma during the upbi task in this study could not be a simple noise but be meaningful. in addition, the increased oxy - hb level in the starting phase is generally considered to reflect cortical activity due to adjustments of the posture and movements with increasing treadmill speed, which also applied to our upbi task. thus, it is quite plausible that oxy - hb concentration responded to the upbi task but that it remained below the level of significance. this hypothesis is supported by our data obtained from the upquad task (see below). it is somewhat surprising because walking with a pair of crutches is considered to be more stable than normal walking from a mechanical viewpoint ; therefore, we expected that hemodynamic response in the upquad task would be lower than that in the upbi task. one possible explanation is that because the crutch is an artificial tool, arm movements using the crutch are no longer simple steps ; thus, this may be a tool - use behavior involving bimanual coordination, which was reportedly impaired in monkeys with sma lesions. our preferred interpretation of the response pattern during the upquad task is that the upquad task may be considered as a dual task gait, that is, a combination of usual walking (upbi task) and the putative, bimanual crutch task. using fnirs, mirelman. showed that oxy - hb levels in the prefrontal cortex significantly increased during usual walking while performing a cognitive task, whereas each task alone failed to evoke significant responses. note again that the hemodynamic response pattern (the peak - plateau - trough pattern) in the upquad task (figure 5(b)) was quite similar to that in the upbi task (figure 5(c)). consequently, it seems as if the waveform in the upquad task would be the one, to which the waveform in the upbi task was shifted upward by the facilitatory effect provided by the putative, bimanual crutch task. based on statistical comparisons of the three locomotor tasks, we found no correlations between the quantitative changes in oxy - hb levels in the sma and postural instability. however, the qualitative patterns of the hemodynamic waveforms were different for the tasks with peak - plateau - trough responses (upbi and upquad tasks) compared with the task with a downhill response (hkquad task). mihara. also found the peak - plateau - trough pattern during normal walking in healthy subjects. the consistency of the hemodynamic response pattern observed in the sma among upright locomotor tasks suggests that the sma may contribute to upright posture control during bipedal gait in humans. our interpretations are consistent with clinical observations [5, 6 ], and they support earlier hypotheses regarding the functional role of the sma [7, 8 ]. the limitation of this study is that we could not determine the specific role of the sma in postural control, such as equilibrium and/or weight bearing. to further understand the cortical mechanisms related to upright posture control during locomotion, we will use fnirs in future research to measure the sma activity during walking with the body fully stabilized by a body harness (stepping in the air), walking while being burdened on a tilting ground, and walking in zero - gravity conditions in space. | to understand cortical mechanisms related to truncal posture control during human locomotion, we investigated hemodynamic responses in the supplementary motor area (sma) with quadrupedal and bipedal gaits using functional near - infrared spectroscopy in 10 healthy adults. the subjects performed three locomotor tasks where the degree of postural instability varied biomechanically, namely, hand - knee quadrupedal crawling (hkquad task), upright quadrupedalism using bilateral lofstrand crutches (upquad task), and typical upright bipedalism (upbi task), on a treadmill. we measured the concentration of oxygenated hemoglobin (oxy - hb) during the tasks. the oxy - hb significantly decreased in the sma during the hkquad task, whereas it increased during the upquad task. no significant responses were observed during the upbi task. based on the degree of oxy - hb responses, we ranked these locomotor tasks as upquad > upbi > hkquad. the order of the different tasks did not correspond with postural instability of the tasks. however, qualitative inspection of oxy - hb time courses showed that oxy - hb waveform patterns differed between upright posture tasks (peak - plateau - trough pattern for the upquad and upbi tasks) and horizontal posture task (downhill pattern for the hkquad task). thus, the sma may contribute to the control of truncal posture accompanying locomotor movements in humans. |
a total of 20,653 women delivered in our hospital between january 2002 and december 2010. we did not include the women with known diabetes (n = 204) or those for whom the status for either one of the five risk factors was not known (n = 1674). therefore, 18,775 pregnancies were analyzed. data are routinely entered at birth for all women giving birth in our university hospital by the midwife assisting with the delivery and then checked and collected during maternity stay by a single midwife (i.p.) qualified in data management and storage. a gdm screening was performed early at 15 weeks gestation only for the subjects with a history of gdm or those who had two or more of the following criteria : family history of diabetes ; personal history of glucose intolerance ; pregravid bmi > 27 kg / m ; age > 35 years ; history of a previous pregnancy with gdm, preeclampsia, or malformation ; macrosomia (birth weight > 4 kg) and/or intrauterine fetal death ; current pregnancy with hypertension ; or estimated fetal weight > 90th percentile for gestational age on ultrasound scan. unless gdm had been found in early pregnancy, gdm screening was performed in all women at 2428 weeks gestation or later if it was not possible during this period. in both cases, gdm was diagnosed using a one - step screening and diagnostic test, with a 75-g oral glucose tolerance test (7), and was defined as a fasting plasma glucose value 5.3 mmol / l (the fasting plasma glucose target in the previous french recommendations) (8) and/or a 2-h blood glucose value 7.8 this one - step screening was decided to limit the number lost to follow - up in our population characterized by a widespread geographic origin. screening was precisely prescribed during the hospital routine follow - up visit and then performed out of the hospital. the percentage of women without screening could be considered close to 12.5% (2011 data). all women with diagnosed gdm were referred to a multidisciplinary team including a diabetologist, an obstetrician, a midwife, a dietician, and a nurse educator. these women received individualized dietary advice and instructions on how to perform self - monitoring of blood glucose levels six times a day. they received insulin therapy when fasting, and 2-h postprandial glucose levels were > 5.3 and > 6.8 mmol / l, respectively, according to the french guidelines (8). antenatal visits were scheduled every 24 weeks up until 34 weeks and weekly thereafter with cardiotocogram and amniotic fluid volume assessment. detailed fetal anomaly ultrasound scan including a detailed cardiac scan was performed by a referee - accredited practitioner ; then, growth scans were performed monthly. the 37-week ultrasound scan was used for fetal weight estimation to discuss the timing and mode of delivery with the patient and obstetric staff. during labor and delivery, continuous electronic fetal heart rate monitoring was routinely used. during the 39th gestational week, labor induction (using prostaglandin e2 or oxytocin infusion) or caesarean section was decided according to obstetric history, maternal condition, and estimated fetal weight. elective caesarean section was planned if estimated fetal weight was > 4,250 g. the main end point was the occurrence of a gdm - related event. the definition of gdm - related events was based on the classical gdm - related maternal (4), fetal, and neonatal (10) complications as reported in the french guidelines. the composite criterion includes at least one of the following events : 1) preeclampsia (blood pressure 140/90 mmhg on two recordings 4 h apart and proteinuria of at least 300 mg/24 h or 2 + on dipstick testing in a random urine sample, 2) large for gestational age (lga) (birth weight > 90th percentile for a standard french population), 3) shoulder dystocia defined as the use of obstetric maneuvers (mcroberts, episiotomy after delivery of the fetal head, suprapubic pressure, posterior arm rotation to an oblique angle, rotation of the infant by 180 degrees, and delivery of the posterior arm) (12). we subsequently considered each of the previous events separately, as well as events that are classically less often associated with gdm : elective and emergency (before or during delivery) caesarean sections, lga - related caesarean section (caesarean section performed in lga babies), preterm delivery (delivery before 37 completed weeks), admission to a neonatal intensive care unit, respiratory distress syndrome (based on the clinical course, chest x - ray finding, and blood gas and acid - base values), and finally, intrauterine fetal or neonatal death (in the first 24 h of life). continuous variables were expressed as means sd and compared by one - way anova or mann - whitney u test as appropriate. logistic regression was used for the analyses of gdm effect, risk factor effect, and the analyses of the interaction between gdm and risk factor effect on gdm - related events and each classical event. statistical analyses were carried out using spss software (spss, chicago, il). a total of 20,653 women delivered in our hospital between january 2002 and december 2010. we did not include the women with known diabetes (n = 204) or those for whom the status for either one of the five risk factors was not known (n = 1674). therefore, 18,775 pregnancies were analyzed. data are routinely entered at birth for all women giving birth in our university hospital by the midwife assisting with the delivery and then checked and collected during maternity stay by a single midwife (i.p.) qualified in data management and storage. a gdm screening was performed early at 15 weeks gestation only for the subjects with a history of gdm or those who had two or more of the following criteria : family history of diabetes ; personal history of glucose intolerance ; pregravid bmi > 27 kg / m ; age > 35 years ; history of a previous pregnancy with gdm, preeclampsia, or malformation ; macrosomia (birth weight > 4 kg) and/or intrauterine fetal death ; current pregnancy with hypertension ; or estimated fetal weight > 90th percentile for gestational age on ultrasound scan. unless gdm had been found in early pregnancy, gdm screening was performed in all women at 2428 weeks gestation or later if it was not possible during this period. in both cases, gdm was diagnosed using a one - step screening and diagnostic test, with a 75-g oral glucose tolerance test (7), and was defined as a fasting plasma glucose value 5.3 mmol / l (the fasting plasma glucose target in the previous french recommendations) (8) and/or a 2-h blood glucose value 7.8 this one - step screening was decided to limit the number lost to follow - up in our population characterized by a widespread geographic origin. screening was precisely prescribed during the hospital routine follow - up visit and then performed out of the hospital. the percentage of women without screening could be considered close to 12.5% (2011 data). all women with diagnosed gdm were referred to a multidisciplinary team including a diabetologist, an obstetrician, a midwife, a dietician, and a nurse educator. these women received individualized dietary advice and instructions on how to perform self - monitoring of blood glucose levels six times a day. they received insulin therapy when fasting, and 2-h postprandial glucose levels were > 5.3 and > 6.8 mmol / l, respectively, according to the french guidelines (8). antenatal visits were scheduled every 24 weeks up until 34 weeks and weekly thereafter with cardiotocogram and amniotic fluid volume assessment. detailed fetal anomaly ultrasound scan including a detailed cardiac scan was performed by a referee - accredited practitioner ; then, growth scans were performed monthly. the 37-week ultrasound scan was used for fetal weight estimation to discuss the timing and mode of delivery with the patient and obstetric staff. during labor and delivery,, labor induction (using prostaglandin e2 or oxytocin infusion) or caesarean section was decided according to obstetric history, maternal condition, and estimated fetal weight. the definition of gdm - related events was based on the classical gdm - related maternal (4), fetal, and neonatal (10) complications as reported in the french guidelines. the composite criterion includes at least one of the following events : 1) preeclampsia (blood pressure 140/90 mmhg on two recordings 4 h apart and proteinuria of at least 300 mg/24 h or 2 + on dipstick testing in a random urine sample, 2) large for gestational age (lga) (birth weight > 90th percentile for a standard french population), 3) shoulder dystocia defined as the use of obstetric maneuvers (mcroberts, episiotomy after delivery of the fetal head, suprapubic pressure, posterior arm rotation to an oblique angle, rotation of the infant by 180 degrees, and delivery of the posterior arm) (12). we subsequently considered each of the previous events separately, as well as events that are classically less often associated with gdm : elective and emergency (before or during delivery) caesarean sections, lga - related caesarean section (caesarean section performed in lga babies), preterm delivery (delivery before 37 completed weeks), admission to a neonatal intensive care unit, respiratory distress syndrome (based on the clinical course, chest x - ray finding, and blood gas and acid - base values), and finally, intrauterine fetal or neonatal death (in the first 24 h of life). continuous variables were expressed as means sd and compared by one - way anova or mann - whitney u test as appropriate. logistic regression was used for the analyses of gdm effect, risk factor effect, and the analyses of the interaction between gdm and risk factor effect on gdm - related events and each classical event. statistical analyses were carried out using spss software (spss, chicago, il). the subjects were 29.7 5.8 years of age, their pregravid bmi was 24.1 4.9 kg / m, and 2.1% of them reported chronic hypertension, while 13.7% reported smoking before pregnancy. the cohort was multiethnic, most of the subjects being from europe (29.1%), north africa (27.8%) and sub - saharian africa (20.8%), and pakistan, india, and sri lanka (5.1%). the prevalence of each risk factor is reported in table 1. presence of risk factors in the total cohort and in women with or without gdm gdm was diagnosed in 2,710 (14.4%) women with an unchanged prevalence since 2002 (fig. a total of 10,975 (58.5%) women had at least one risk factor, with a significant progression (p < 0.001) over time. this increase in risk factor prevalence involved especially overweight, which increased from 30.8% in 2002 to 37.6% in 2010 (p < 0.0001) (fig., there was no obvious change in the population served by our hospital, particularly with regard to ethnicity, age, and parity. prevalence over the years of gdm, overweight, and the presence of at least one risk factor according to french guidelines. table 1 shows that the presence of at least one risk factor (odds ratio 1.4 [95% ci 1.31.5 ]) and the presence of either one of the risk factors were significantly associated with a higher rate of gdm. the performances of the presence of at least one risk factor were as follows : sensitivity 65.3%, specificity 42.7%, and positive and negative predictive values 16.1 and 87.9%, respectively. the higher the number of risk factors, the higher the prevalence of gdm (fig. 2). furthermore, compared with caucasian origin, north african ethnicity (1.5 [1.41.7 ]) and pakistan, india, and sri lanka origins (2.9 [2.53.4 ]) were associated with a higher gdm prevalence. prevalence of gdm and gdm - related events according to the number of risk factors. the women with gdm (gdm effect, p < 0.0001) and those with at least one risk factor (risk factor effect, p < 0.001) had a higher rate of hospitalization before delivery, with no interaction between gdm and risk factor : no gdm / no risk factors, 24.9% of hospitalization ; no gdm / risk factor, 26.8% ; gdm / no risk factors, 32.4% ; and gdm / risk factor, 34.0%. weight gain was lower (p < 0.05) in women with gdm (no risk factors 8.5 5.5 ; risk factor 8.5 5.5 kg) than in those without gdm (9.0 5.6 kg whatever the risk factor status). in women with gdm, insulin therapy was initiated in 9.7% of those without risk factors and in 12.1% of those with risk factors (p = 0.056). table 2 shows that gdm was associated with the occurrence of gdm - related events (preeclampsia, lga, and shoulder dystocia) and also with the occurrence of preeclampsia, lga, dystocia, caesarean section, and lga - related caesarean section taken separately. the presence of at least one risk factor was independently associated with the occurrence of a gdm - related event, of lga, and of lga - related caesarean section. the higher the number of risk factors, the higher the incidence of gdm - related events (fig. the subjects were 29.7 5.8 years of age, their pregravid bmi was 24.1 4.9 kg / m, and 2.1% of them reported chronic hypertension, while 13.7% reported smoking before pregnancy. the cohort was multiethnic, most of the subjects being from europe (29.1%), north africa (27.8%) and sub - saharian africa (20.8%), and pakistan, india, and sri lanka (5.1%). the prevalence of each risk factor is reported in table 1. gdm was diagnosed in 2,710 (14.4%) women with an unchanged prevalence since 2002 (fig. 1). a total of 10,975 (58.5%) women had at least one risk factor, with a significant progression (p < 0.001) over time. this increase in risk factor prevalence involved especially overweight, which increased from 30.8% in 2002 to 37.6% in 2010 (p < 0.0001) (fig., there was no obvious change in the population served by our hospital, particularly with regard to ethnicity, age, and parity. prevalence over the years of gdm, overweight, and the presence of at least one risk factor according to french guidelines. table 1 shows that the presence of at least one risk factor (odds ratio 1.4 [95% ci 1.31.5 ]) and the presence of either one of the risk factors were significantly associated with a higher rate of gdm. the performances of the presence of at least one risk factor were as follows : sensitivity 65.3%, specificity 42.7%, and positive and negative predictive values 16.1 and 87.9%, respectively. the higher the number of risk factors, the higher the prevalence of gdm (fig. 2). furthermore, compared with caucasian origin, north african ethnicity (1.5 [1.41.7 ]) and pakistan, india, and sri lanka origins (2.9 [2.53.4 ]) were associated with a higher gdm prevalence. prevalence of gdm and gdm - related events according to the number of risk factors. the women with gdm (gdm effect, p < 0.0001) and those with at least one risk factor (risk factor effect, p < 0.001) had a higher rate of hospitalization before delivery, with no interaction between gdm and risk factor : no gdm / no risk factors, 24.9% of hospitalization ; no gdm / risk factor, 26.8% ; gdm / no risk factors, 32.4% ; and gdm / risk factor, 34.0%. weight gain was lower (p < 0.05) in women with gdm (no risk factors 8.5 5.5 ; risk factor 8.5 5.5 kg) than in those without gdm (9.0 5.6 kg whatever the risk factor status). in women with gdm, insulin therapy was initiated in 9.7% of those without risk factors and in 12.1% of those with risk factors (p = 0.056). table 2 shows that gdm was associated with the occurrence of gdm - related events (preeclampsia, lga, and shoulder dystocia) and also with the occurrence of preeclampsia, lga, dystocia, caesarean section, and lga - related caesarean section taken separately. the presence of at least one risk factor was independently associated with the occurrence of a gdm - related event, of lga, and of lga - related caesarean section. the higher the number of risk factors, the higher the incidence of gdm - related events (fig. we show in the current study that a gdm screening strategy based on risk factors was able to select both the subjects with a higher risk of gdm and those who experienced more gdm - related events, with no interaction between gdm effect and risk factor effect. however, from a clinical perspective, these diagnostic and prognostic factors are not appropriate, since 34.7% of the women without risk factors actually had gdm and since those women with gdm but no risk factors, although treated, experienced more gdm - related events than women without gdm. they would probably have experienced even more complications had they been undiagnosed and thus untreated. considering that the new diagnostic criteria (13) were not yet used in 20022010, we report here a high prevalence of gdm. this is probably due not only to the diagnostic criteria we used but also to the characteristics of our population : multiethnic, with a low socioeconomic status (7). trends in gdm prevalence over the last years had never been explored in europe before the new diagnostic criteria. a recent extensive review of the literature showed an increase in the prevalence of gdm in north america, which was often less marked in caucasians (6). here we show a stability in gdm prevalence, possibly because of our diagnostic criteria. the stability in gdm prevalence was nonconcordant with the increase of risk factors in our series. this increase was essentially a result of the progression of overweight, in line with the increase of overweight and obesity in women of reproductive age in france (14). we previously reported that women 35 years old compared with those < 25 years old had a twofold increased risk of gdm (15). it is considered that nearly half the cases of gdm might be a consequence of overweight or obesity (20). a recent meta - analysis has shown that in a comparison with subjects with a normal bmi, the pooled and adjusted odds ratios of gdm were 1.8 for overweight, 3.2 for obesity grade 1, and 4.7 for obesity grades 2 and 3 (21). for each increasing kilogram per meter squared of bmi, the prevalence of gdm rose by 0.92% (21). in the current study, the odds ratios for age and bmi criteria were lower, as in a recent study (19). this might result from other factors such as the vulnerability or ethnicity, which were considered confounders in the french guidelines, whereas they might have been crucial in our current population. in particular, we show in the current study that north africa and pakistan, india, and sri lanka origins are predictive of gdm, although they are not taken into account in the french recommendations for a selective screening. (19) reported that the best cutoff values were 28 years for age and 23 kg / m for bmi. finally, the gdm diagnostic criteria in our series differed from those of the other studies, as we used low thresholds for plasma glucose values. the influence of age and bmi might be more important when glucose thresholds are higher. family history of diabetes has been reported to multiply by 1.63.0 the risk of developing gdm (6,22). in our study population, the odds ratio related to family history of diabetes was lower (1.3 [95% ci 1.11.4 ]), which could be due to some missing reports of family history of diabetes, since many women did not speak french and some others did not know the medical history of their family living abroad. personal histories of gdm and of a child with macrosomia were associated with gdm with the highest odds ratio (5.9 [5.06.9 ] (p < 0.0001). gdm is well - known to be recurrent in 3084% of cases (18,19,2224). history of macrosomia in a previous pregnancy was associated with a 1.6- to 6.2-fold increase of gdm (17,19). overall, we showed that the risk factors proposed in the french guidelines significantly predicted gdm, although the clinical relevance appears to be low, since 34.7% of the women with gdm would have been missed without universal screening, in line with previous reports (2). however, the prevalence of gdm was very low and extrapolated in their study. finally, and as shown in fig. 2, the prevalence of gdm was particularly high when the number of risk factors was greater than three. therefore, three or more risk factors would be very specific for gdm screening, although this condition concerned only 3% of the cohort. it was hypothesized that women with gdm but no risk factors would have a good prognosis and therefore that missing their diagnosis would be of little consequence. < 25 kg / m, the incidences of macrosomia and shoulder dystocia were reported to be similar regardless of whether gdm was known and treated (25). conflicting data about the prognosis of gdm detected by selective or universal gdm screening were also reported, with no (26) or beneficial (7) effects of universal screening in retrospective studies. finally, a prospective study showed a better prognosis associated with universal than with selective screening, but an earlier screening in the universal strategy was confounding (27)., gdm and risk factors were independently associated with a higher incidence of overall gdm - related events (preeclampsia, lga, and shoulder dystocia) and of lga and lga - related caesarean section considered separately. in the hyperglycemia and adverse pregnancy outcome (hapo) study, higher bmi was associated with more preeclampsia, macrosomia, caesarean section, and shoulder dystocia (28). however, there was in our series no interaction between gdm effect and risk factor effect. furthermore, from a clinical perspective, women with gdm but no risk factors, although treated, had more gdm - related events than those without gdm, which argues for universal screening. finally, the number of risk factors seems important to consider, as the incidence of gdm - related events is very high when at or above four. however, this is restricted to multiparous women, as two risk factors (macrosomia and gdm) depend on a previous pregnancy the public hospital recruitment probably included a higher proportion of women living with vulnerable conditions and was multiethnic, precluding a generalization of our results. this could also have impacted our results, as some women could not speak french and therefore may have wrongly declared their personal and family medical histories. however, this could also explain some missing reports of family history of diabetes and further argue in favor of universal screening. although the screening policy was locally universal, some women were not screened. therefore, we considered the presence of gdm in the intention - to - screen population. the proportion of unscreened women, however, was 12.5% in 2011 and was likely to be similar over the last decade. considering the prognosis issues, this study was of course not randomized, since all women with diagnosed gdm were treated. furthermore, gdm - related events could not be weighed according to the glycemic profile of the women during pregnancy. finally, the results should be confirmed with the new diagnostic criteria of gdm and in other hospitals, as this retrospective study was monocentric. to conclude, the selective screening of gdm is appealing, as it reduces the burden of unnecessary screening tests if this screening selects the women who have the highest risk of gdm - related complications. we showed, however, that in our population, such a strategy would lead to missing approximately one - third of the women with gdm. furthermore, despite appropriate treatment, women with gdm had a worse prognosis than women without gdm, even if they were free of risk factors. therefore, universal screening appears to be beneficial, at least compared with the selective screening proposed in the french guidelines. | objectivewe aimed to evaluate a selective screening strategy for gestational diabetes mellitus (gdm) based on the presence of risk factors : bmi 25 kg / m2, age 35 years, family history of diabetes, personal history of gdm, or birth of a child with macrosomia.research design and methodsof 20,630 deliveries between 2002 and 2010, we selected 18,775 deliveries in women with no known diabetes and for whom all risk factors were known. gdm was universally screened and defined as fasting plasma glucose level 5.3 mmol / l and/or 2-h postload (75 g) glucose level 7.8 mmol / l.resultsthe prevalence of at least one risk factor has increased since 2002 (p < 0.001) from 51.7 to 61.5%, with no change in the gdm prevalence (mean 14.4%, intention to screen). at least one risk factor was present in 58.5% of women who represented 65.3% of all those with gdm. the presence of risk factors was significantly associated with gdm (odds ratio 1.4 [95% ci 1.31.5 ], p < 0.001) and with gdm - related events (preeclampsia / large for gestational age / dystocia) (p < 0.001) with the following incidences : no gdm / no risk factor 8.8%, no gdm / risk factor 11.1%, gdm / no risk factor 16.7%, and gdm / risk factor 18.2%.conclusionsthe presence of risk factors increased during the last decade. this condition is predictive of gdm and gdm - related events. however, a selective screening would lead to missing one - third of the women with gdm who, even without risk factors, had more events than women without gdm. therefore, these data stand against the present selective screening currently proposed in the french guidelines. |
atrial fibrillation (af) is a common arrhythmia with an estimated clinical prevalence of approximately 1% in the general population and as high as 9% in individuals by the age of 80 years myocardial dysfunction observed in sepsis could contribute to arrhythmias and inflammation per se could induce or provoke af. we describe the management of an acute af in an elderly patient scheduled for emergency laparotomy. a 77 year - old lady (weight : 50 kg) of american society of anaesthesiologists (asa) iii was scheduled for emergency laparotomy and closure of hollow viscous perforation. she had an uneventful general anaesthesia for laparoscopic appendicectomy 2 days ago. however, she had abdominal pain and fever postoperatively in the ward for last 24 hours. on examination, she was febrile ; her pulse was 110/min, regular ; blood pressure was 106/72 mmhg and respiratory rate was 20/min. blood investigations done 6 hours prior to surgery revealed haemoglobin of 11.9 g / dl, total counts of 14,000/mm with 13% band forms, serum sodium 140 meq / l, potassium 3.6 meq / l, platelets 220,000/mm and creatinine of 1.4 mg%. electrocardiogram (ecg) showed sinus tachycardia with heart rate (hr) of 106/min. in the operating room, baseline monitors (pulsoximetry / non - invasive blood pressure / ecg) were established. ecg showed hr of 156/min in af, which was not present earlier and confirmed with 12-lead ecg., intravenous metoprolol was administered to control ventricular rate in aliquots of 1 mg to a total of 5 mg. as the patient 's blood pressure was stable, patient 's trachea was intubated with rapid sequence induction using midazolam 1 mg, fentanyl 250 mcg, propofol 60 mg and succinylcholine 60 mg. central line was inserted in right internal jugular vein (ijv) under aseptic precautions. anaesthesia was maintained with air, oxygen, sevoflurane, fentanyl, hydromorphone and rocuronium. as the hr was refractory to beta blockers, intravenous amiodarone 150 mg was administered. hr reduced to 8090/min with an improvement in blood pressure (126/86 mmhg) and rhythm reverted back to sinus within 1520 min of administration. arterial blood gas and serum electrolytes showed uncompensated metabolic acidosis (bicarbonate = 16 meq / l) and respiratory alkalosis (pco2 = 26 mmhg) with serum potassium of 3 meq / l. 20 mmol / l was administered along with 1 g of iv magnesium sulphate (serum magnesium 1.2mg / dl) and calcium chloride (serum calcium 6 mg / dl) in the icu. serum c - reactive protein (crp) and procalcitonin was found to be elevated. the patient 's trachea was extubated after 12 hours with controlled ventricular rate and sinus rhythm. on consultation with cardiologist, tab. she was advised regular follow - up and discharged home on low - dose aspirin therapy. fibrosis and loss of muscle mass of the left atrium play a major role in the development of af. genetic defects like lamin. systemic disease, e.g., inflammatory processes, sarcoidosis, autoimmune disorders, has also been linked to the development of af. it was initially suspected by the observation that inflammatory states, such as myocarditis, pericarditis, and cardiac surgeries, are frequently associated with af. there is a greater association between elevated crp and presence of af, which is supported by the findings from a large population - based cohort study of 5806 elderly individuals followed for a mean of 6.9 years. every 1 mg / dl increase in serum crp was associated with a seven fold increased risk of recurrent af and a 12-fold increased risk of permanent af compared with controls. advanced age, blunt thoracic trauma, shock (notably septic shock), use of pulmonary artery catheter, and previous treatment with calcium channel blockers are the independent risk factors of af in intensive care patients. our patient had af probably due to advanced age, underlying sepsis and electrolyte imbalances. there are multiple therapeutic strategies for the management of af, which may confuse individual practitioners. direct current cardioversion (dcc), pharmacotherapy with antiarrhythmic drugs, and anticoagulation are the mainstay treatment modalities in acute af. antiarrhythmic drugs with class i (flecainide, propafenone) and iii (amiodarone) agents are used when adopting a rhythm - controlling strategy, whereas class ii (beta blockers) and iv (diltiazem, verapamil) agents are reserved for rate - controlling measures, although class iii agents share rate - controlling properties. as the blood pressure was stable in this patient, pharmacological conversion to sinus rhythm was attempted initially with beta blocker and then with intravenous amiodarone. calcium channel blockers were not used in our patient as fatal complete heart block has been described with their use along with beta blockers. amiodarone was considered for af of acute onset and to avoid secondary complications of af like ischaemia, systolic dysfunction and embolism. airway was secured immediately in our patient to avoid hypoxia, hypercarbia, acidosis, which could further precipitate af, and to facilitate dcc if needed. in patients with ventricular rates more than 150, ongoing chest pain, or with evidence of critical perfusion : systolic bp less than 90 mmhg, heart failure, or reduced consciousness, the need to restore haemodynamic stability by the restoration of sinus rhythm is of prime importance, and guideline consensus advocates dcc as the first - line therapy to achieve this, with pharmacologic strategies being used either secondarily or in conjunction with dcc. the patient was discharged on low - dose aspirin therapy for 6 months to prevent thromboembolic complications following af. acute af in an elderly patient coming for emergency surgery with underlying sepsis and electrolyte imbalance needs immediate hr control and conversion to sinus rhythm. intraoperative vigilant monitoring, intravenous use of amiodarone, correction of electrolytes and underlying pathology played a vital role in the successful management of this patient. | atrial fibrillation (af) is a common arrhythmia with an estimated clinical prevalence of approximately 1% in the general population and as high as 9% in individuals by the age of 80 years. the aetiology is multifactorial. systemic disease, e.g., inflammatory processes, sarcoidosis, autoimmune disorders, has also been linked to the development of af. myocardial dysfunction observed in sepsis could contribute to arrhythmias and inflammation per se could induce or provoke af. we describe the successful management of an acute af in an elderly patient scheduled for emergency laparotomy and closure of hollow viscous perforation. |
when vaginal hysterectomy (vh) is not possible, many surgeons are reluctant to use laparoscopic techniques on obese patients and instead use the abdominal approach. current evidence suggests that there is no significant difference in outcomes for normal weight versus obese women undergoing laparoscopic hysterectomy (lh). however, more gynecologic surgeons will have to master new skill sets to offer this route universally. a growing body of evidence advocates the use of laparoscopic rather than open surgery in obese individuals. the general surgery literature shows a preference for performing bariatric surgery with a laparoscopic rather than open approach. a small case series in europe suggests that surgical and anesthetic complications are reduced when laparoscopy is used preferentially to abdominal approaches in gynecologic surgery. o'hanlan suggest that a study comparing the outcomes of normal weight and obese women undergoing lh and abdominal hysterectomy (ah) would contribute to the gynecologic literature. the objective of this study was to compare the surgical outcomes of obese and normal weight women having hysterectomy according to the route of the procedure. an analysis was performed of patients who had surgery by faculty offering all 3 approaches (ah, vh, lh). hysterectomy procedures from june 6, 2001 (the date of the first total laparoscopic hysterectomy offered in the institution) through february 9, 2006 were analyzed. all hysterectomies performed by these surgeons during this time period are included in this study. the indication for surgery, operative time, anesthesia time, complications, estimated blood loss, change in hematocrit, conversion of route, uterine weight, wound complications, and transfusion events were tabulated. demographics and medical history information were also collected including age, race, gravidity, parity, weight, height, and history of prior uterine scar (including cesarean delivery, myomectomy, or other incision into the uterine myometrium). the study procedures were performed by 1 of 2 faculty surgeons and a resident in obstetrics and gynecology at a tertiary care center. the faculty surgeons performing these cases trained in the same institution and operated together during the development of the total laparoscopic technique. no formal changes were made in surgical technique, such as additional training or new equipment, through the evaluation period. procedures were classified as abdominal (ah), which included total and subtotal abdominal hysterectomy ; vaginal (vh), which included total and laparoscopically assisted vaginal hysterectomy ; and laparoscopic (lh,) which included total and subtotal laparoscopic hysterectomy as well as robotically assisted laparoscopic hysterectomy. for a procedure to be designated as lh, the entire procedure including colpotomy and vaginal cuff closure had to have been performed laparoscopically. those procedures that required conversion to an abdominal route from the vaginal or laparoscopic approach were included in the group of the initial approach. obesity defined as body mass index (bmi) 30 was calculated by dividing the weight of the patient in kilograms by the patient 's height in meters squared. obesity has been defined by the world health organization as a bmi value of > 30. patients were divided into 6 groups : obese and normal weight ah, obese and normal weight vh, and obese and normal weight lh. complications were identified in the dictated operative note and from review of the medical record during the hospital stay and in clinical follow - up visits. data were compiled in microsoft excel (microsoft corporation, redmond, washington) and evaluated using the number cruncher statistical system (ncss, kaysville, utah) to perform an analysis of variance (anova). a newman - keuls multiple comparison test was used to evaluate which pairs of data differed by a statistically significant amount (f - ratio > 1). the distribution of patient race versus route of procedure was evaluated using chi - square analysis. we identified 293 cases : 68 ah, 95 vh, and 130 lh. in the ah group, 37 of 68 patients, or 54%, were obese. in the vh group, 51 of 95, or 54%, were obese. in the lh group, 69 of 130, or 53%, were obese. indications for surgery included symptomatic leiomyomata (51%), chronic pelvic pain (17%), dysfunctional uterine bleeding (17%), recurrent cervical dysplasia (8%), endometrial hyperplasia (1%), management of intraoperative hemorrhage (< 1%), management of pelvic inflammatory disease (< 1%), management of ovarian mass (< 1%), and pelvic organ prolapse (< 1%). no statistically significant difference existed in patient ages among the ah, vh, and lh groups. women in the vh group had higher average gravidity and parity than women had in the ah or lh groups, a difference that was statistically significant. the ah and lh groups were not significantly different from each other for gravidity and parity. demographic data by route of hysterectomy denotes statistical significance from other groups (race differences discussed in results). the majority of women evaluated in the study were african - american followed by caucasian and hispanic. the ah group had significantly more african - american women than did the vh or lh groups. the lh group had significantly more caucasian women than did the ah or vh groups. the rate of supracervical hysterectomy was noted to be 22% in the ah group and 15% in the lh group. the rate of salpingo - oophorectomy was 79% in the ah group, 22% in the lh group, and 20% in the vh group. conversions to an abdominal approach were experienced in 1 lh case and 4 vh cases. additional procedures performed at the same time of ah were 2 appendectomies and one suburethral sling procedure. one appendectomy, one anterior and posterior colporrhaphy, and 2 suburethral sling procedures were performed at the time of vh. one appendectomy and 2 suburethral sling procedures were performed at the time of lh. in the analysis of surgical history, the vh group had a significantly lower number of prior cesarean deliveries per patient (0.3) compared with the ah (0.6) and lh (0.8) groups. however, the lh group had significantly more total abdominal surgery per patient (2.0) than ah (1.3) or vh (1.3) had. three complications in obese women occurred : a cystotomy during vh and 2 enterotomies during ah. nine complications in normal weight women occurred : 2 cystotomies during lh, 2 cystotomies during ah, 1 cystotomy during vh, laceration of the inferior epigastric artery during lh, puncture of the sigmoid colon during lh, and 2 cases of hemorrhage requiring intraoperative transfusion during lh and ah. the ah group had a significantly higher average uterine weight (608 grams) versus the vh (159 grams) or lh (207 grams). the longest surgical times and anesthesia times were recorded in obese women in the lh group. however, these times were not significantly different from times for obese women in the ah group. obese women in the vh group had the shortest cases of the obese women in the study. similar trends were noted for normal weight women in the lh, ah, and vh groups. normal weight women having vh had the shortest anesthesia and surgery times of any group, a statistically significant difference. mean anesthesia and surgery times by route of hysterectomy and weight category hospital stay data, outlined in table 3, revealed that obese women in the lh group spent 2.5 days in the hospital on average, compared with obese women in the ah group who stayed 4.2 days, a significant difference. there was a statistically significant increase in hospital stay in the ah group versus vh or lh, for both obese and normal weight women. there was no significant difference in hospital stay in the vh and lh groups, whether obese or normal weight weight. all postoperative care was delivered by the same faculty physicians and residents in the same hospital, ensuring that the discharge criteria were consistent among all groups. hospital length of stay by route of hysterectomy and weight category estimated blood loss (ebl), outlined in table 4, was highest in normal weight women in the ah group (478ml) followed by obese women in the ah group (455ml). the blood losses seen in the obese versus normal weight lh and obese versus normal weight vh groups were not significantly different. the difference in estimated blood loss was significant between ah and the other groups for both obese and normal weight women. estimated blood loss by hysterectomy route and weight category the change in hematocrit data was not reliable for analysis. for this reason, estimated blood loss was used as the primary evaluation of surgical blood loss. surgical management of the obese patient continues to be a challenge to the gynecologic surgeon. lh appears to be a safe procedure for obese women but has a short history, leaving its role in certain clinical situations unclear. lh has been found in large randomized trials to be generally inferior to vh in terms of operative time and anesthesia complications, but superior to open procedures in recovery time and complications. a critical evaluation of these trials suggests lh is a more effective tool when surgical expertise is considered. the results from this study suggest that lh is a favorable alternative to ah in obese women who, for whatever reason, are judged unsuitable for vh. in this retrospective study, obese women having lh versus ah had significantly shorter hospital stays and less blood loss but similar surgery and anesthesia times. while recovery data were not collected for this study, the experience of the surgeons is that the lh patients return to normal activities sooner than ah patients do, whether obese or not. these data also agree with data from other studies, suggesting vh is preferable to ah or lh when possible. patients who had vh spent the fewest days in the hospital, experienced the shortest surgery and anesthesia times, and had the lowest ebl. the decision to proceed with any given surgical approach was made clinically by the surgeon. the abdominal approach would likely have been selected for a case with a high degree of anticipated difficulty due to uterine size. this evaluation period captures the introduction of laparoscopic hysterectomy at the study institution, and as such demonstrates an initial bias toward ah in patients with large uteri, until increased experience with lh made these cases feasible. the abdominal approach may also have been selected in obese patients with large uteri causing significant distortion of normal anatomic relationships. the selection bias for route of procedure in the patient with a large uterus is suggested by the much higher mean uterine weight in the ah group. the rationale for this inclusion was based on the original intention of the surgical team to remove the uterus vaginally, with a variable degree of laparoscopic assistance. in similar fashion, those vaginal or laparoscopic procedures that required conversion to laparotomy were analyzed in the group of the initial route (intent to treat). confounders such as patient disease, including diabetes and asthma, were not specifically evaluated in this study. such patient conditions may have influenced the chosen route of surgery and therefore the comparisons. the distribution of complications, favoring normal weight women in the abdominal hysterectomy group, may suggest that the selection of the abdominal approach was based on risk factors for operative difficulty (history of prior surgery or uterine scar). the vh group was more likely to have fewer such predictors for difficult surgery compared with women in the ah or lh group. this bias is consistent with studies describing larger and more symptomatic myomata in african - american women, leading to more frequent selection of the abdominal route. the vh group contained more hispanic women, but the sample size was too small to draw definitive conclusions for this finding. this distribution is consistent with the patient population served by the university in which the study was performed. the number of patients evaluated did not allow for further stratification by bmi to determine whether the severity of obesity was consistent across all groups. these data capture the experience of the faculty surgeons from the very first lh procedure performed at the institution. neither faculty surgeon received training in laparoscopic hysterectomy during residency., it did not appear that obesity biased selection of the route of surgery, as approximately half of the patients in each group were obese. intuitively, surgeons selecting patients for a new procedure might tend to choose straightforward surgical cases. however, it is promising that, even with the inclusion of the learning curve, the laparoscopic route benefited obese patients with shorter hospital stays, less blood loss, and fewer complications. these data suggest that in obese patients, vaginal hysterectomy be considered first because these patients had the shortest hospital stay, surgery, and anesthesia times. surgeons in private and academic practice environments should continue to regard vh as the route of choice in obese patients thanks to favorable outcomes and operating times. lh should be selected over ah in obese patients for whom a vaginal approach is not feasible. | objective : our objective was to compare the surgical outcomes of obese women having hysterectomy according to the route (abdominal, vaginal, or laparoscopic) of the procedure.methods:a chart review of 293 hysterectomy procedures was performed. data were collected including operative and anesthesia time, estimated blood loss, change in hematocrit, hospital stay, complications, conversion to laparotomy, transfusion, and body mass index. an analysis of variance and a newman - keuls multiple comparison test were performed.results:obese women experienced a significant decrease in hospital days (2.5 versus 4.2) and reported blood loss (204 ml versus 455 ml) in the laparoscopic hysterectomy and vaginal hysterectomy groups compared with the abdominal hysterectomy group. no significant difference was found in obese women between laparoscopic and abdominal hysterectomy for time spent in surgery and under anesthesia. for obese and normal weight women, vaginal hysterectomy offered the shortest surgery, anesthesia times, and hospital stays.conclusions:for normal and obese women, vaginal hysterectomy offered the shortest hospital stay and surgery time. in obese patients for whom vaginal hysterectomy is not possible, laparoscopic hysterectomy should be considered before abdominal hysterectomy, because the laparoscopic route reduced hospital time and blood loss. |
posterior reversible encephalopathy syndrome (pres) relates to a transient pattern of brain edema mostly involving the parietal and occipital regions. severe hypertension may lead to pres when overcoming brain autoregulation with subsequent breakthrough brain edema. we describe a case of asymmetric pres associated with the presence of a hyperplastic anterior choroidal artery (acha) in a man affected by severe hypertension. a 58-year man, with a history of mild hypertension, was referred to the emergency room for a sudden onset of visual disturbance, difficulty in speech, headache and mental confusion. clinical examination revealed increased blood pressure (220 mmhg), mild anemia (hgb, 10.5 g / dl) and hyper - cholesterolemia (250 mg / dl) on routine blood work. neurologic examination revealed left homonymous lateral hemianopia, mild difficulty in searching words, mild pronation and downward drift of the right arm. subcortical patchy hypodense foci localized in the parietal - occipital lobes, asymmetric for left predominance without enhancement after i.v. multiple hypodense confluent areas localized in the supratentorial white matter as a result of chronic ischemic cerebral disease were evident as well. mri, performed immediately after ct, showed the presence of asymmetric cortico - subcortical patchy hyperintense areas on flair images mostly localized in the left parieto - occipital lobe (fig. no signal alteration was seen in dwi images while adc maps showed high signal indicating vasogenic edema (fig. no pathological enhancement was seen after contrast administration.fig. 1the first mri (a c) showed cortical subcortical patchy hyperintense areas on t2 flair - weighted images (a) localized in the parietal occipital lobes with asymmetric representation for left predominance. no signal alteration was seen in dwi images (b) in presence of high signal on adc maps indicating vasogenic edema (c). mri exam after 3 months demonstrated complete resolution of the hyperintense areas on flair - weighted (d) images and adc maps (e). the mr - angiography of intracranic circle, performed with the 3d - tof technique (f, g), showed the presence of left hyperplastic acha (white arrows, tridimensional images f) supplying partially the distribution of the ipsilateral posterior cerebral artery (pca) (arrows, tridimensional images f). in g the same anatomical abnormalities is represented by mip reconstruction the first mri (a c) showed cortical subcortical patchy hyperintense areas on t2 flair - weighted images (a) localized in the parietal no signal alteration was seen in dwi images (b) in presence of high signal on adc maps indicating vasogenic edema (c). mri exam after 3 months demonstrated complete resolution of the hyperintense areas on flair - weighted (d) images and adc maps (e). the mr - angiography of intracranic circle, performed with the 3d - tof technique (f, g), showed the presence of left hyperplastic acha (white arrows, tridimensional images f) supplying partially the distribution of the ipsilateral posterior cerebral artery (pca) (arrows, tridimensional images f). in g the same anatomical abnormalities is represented by mip reconstruction mr - angiography, performed with the 3d - tof technique, showed the presence of left hyperplastic acha partially supplying the distribution of the ipsilateral posterior cerebral artery (pca) (fig. 1f h). because of the suspicion of acute hypertensive encephalopathy, he was treated with mannitol (100 ml 6/day), potassium canrenoate (50 mg 1cp / day), furosemid (25 mg 1cp / day). eleven days after admission, the patient was discharged with complete resolution of the symptoms, stable reduction of the blood pressure (115/70 mmhg) and a therapy composed by potassium canrenoate (50 mg 1cp / day), ramipril (10 mg 1cp / day), doxazosin mesylate (2 mg 1 cp / day), amlodipine besylate(10 mg 1 cp / day) and acetylsalicylic acid (100 mg 1cp / day). mri follow - up performed 3 months later demonstrated complete resolution of the signal alterations (fig. the acha is a branch of the internal carotid artery running between the temporal lobe and the brain stem and reaching the choroid plexus in the temporal ventricular horn. along its course, the acha supplies several territories, in particular optic tract, posterior limb of internal capsule and pulvinar. several anomalies in course and caliber have been described. in particular, enlargement of the acha has been reported with cerebral angiography in many pathological cases. in up to 1% of subjects, the acha is hyperplastic and supplies all, or a portion, of the pca territory. takahashi. described 25 hyperplastic arteries supplying part of the territory of distribution of the pca. the hyperplastic arteries were further classified into four subtypes according to the distribution area and course of the vessel. in type 3 an anomalous artery supplies the parieto - occipital and calcarine arteries, as observed in our patient. this is a result of anastomotic channels development, hemodynamically favored, between acha and pca or pcoma that determined variable degrees of hypoplasia of the pca. in our patient the hyperplastic acha supplied the territory affected by t2 signal alteration, suggesting a potential role of the vascular anomaly in its genesis. pres is an acute rapidly evolving clinical condition characterized by headache, nausea and vomiting, abnormalities of visual perception, mental status abnormalities, seizure and focal neurological signs associated with transient radiological brain anomalies. different conditions might be responsible (eclampsia, hypertensive encephalopathy, renal disease with hypertension, neurotoxicity of cyclosporine a or other immunosuppressive drugs and bone marrow transplantation) [47 ]. other rare pathologic conditions, such as intracranial hypotension, are recently discussed [8, 9 ]. the presence of hypertension and the breakthrough of cerebral autoregulation are retained as the most common causes of pres. mr diffusion - weighted imaging usually confirms the presence of vasogenic edema with high values of adc. in our patient, many findings were compatible with pres. clinical onset and symptoms (headache with nausea, visual disorders, altered mental status and seizure) the choice of using anti - hypertensive care was due to the difficulty of a fast reduction of blood pressure, avoiding other complications. the authors in this study assessed that partial or asymmetric pres was most commonly recognized in patients who have had organ transplantation and eclampsia with severe hypertension or normal blood pressure. variable expression of the pres patterns could be related to differences in arterial anatomy, as demonstrated in our case, preexisting vascular disease or regional hemispheric involvement in a clinical toxic process. acute severe hypertension with transient loss of autoregulation system (sympathetic innervations) and subsequent vasogenic edema [7, 11 ] seems the cause of pres in our patient. posterior distribution of brain alterations has been attributed to the sympathetic innervations of the cerebral vessels. this distribution has an antero - posterior gradient with a relatively reduced innervation of the posterior cerebral arterial circulation, and therefore, during acute elevation of blood pressure, posterior brain regions may be particularly susceptible to breakthrough of autoregulation. the presence of a double vascular district (acha and pca) in the same vascular territory during acute hypertensive attack might be the cause of blood hyper - influx syndrome leading to asymmetric vasogenic edema. although the presence of hyperplastic acha is well known, to our knowledge this is the first case reported in literature in which such a vascular abnormality was linked to pres syndrome. | we describe a case of asymmetric pres due to the presence of hyperplastic anterior choroidal artery (acha) in a man affected by sever hypertension. posterior reversible encephalopathy syndrome (pres) has become synonymous with a unique pattern of brain vasogenic edema and predominates in the parietal and occipital regions, accompanied by clinical neurological alterations. sever hypertension is a risk factor that exceeds the limits of brain autoregulation, leading to breakthrough brain edema. in our knowledge this is the first case reported in literature, in which a similar vascular abnormality is linked to a pres syndrome. |
electrochemical water oxidation, also known as the oxygen evolution reaction (oer), is a key energy conversion reaction in a number of clean energy technologies, including rechargeable metal air batteries, electrolysis cells, and solar fuel production. widespread commercialization of these technologies is limited in part by the scarcity and high cost of the best known catalysts for the oer, ruthenium and iridium oxides. nickel oxides (niox) present a viable alternative to precious metal oxides in alkaline environments and are currently used in commercially available alkaline electrolyzers. other nonprecious metal oxides, including manganese oxides (mnox) and cobalt oxides (coox), have also demonstrated promising oer activity under alkaline conditions. to facilitate the development of nonprecious metal oer catalysts with improved activities, it is necessary to identify the specific catalytic sites that participate in the reaction and accurately determine their turnover frequencies. although isolating site - specific turnover frequencies can be challenging in oxide electrocatalysts, the likelihood of success can be improved if the catalytic activity is studied on well - defined materials deposited on inert supports. there have been conflicting reports in the recent literature regarding the role metal supports play in the oer activity of metal oxide catalysts. a study, which used pt(111) and au(111) single crystal surfaces as supports for oer catalysts, demonstrated that the oer activity of four first row transitional metal oxides did not vary with the nature of the metal support and was linearly dependent on the coverage of the support by the metal oxide. other reports, however, have shown that the oer activities of nickel, cobalt, and manganese oxides were influenced by the nature of the underlying support, and that the oer turnover frequency of a bulk metal oxide was inferior to that of a submonolayer amount of the same metal oxide deposited on a metal support. our study, which focuses on one particular metal supported transition metal oxide system, mnox / au, conclusively demonstrates that adding a noble metal to a metal oxide oer catalyst can have a significant impact on its electrocatalytic activity. we show that this impact can not be explained simply by surface area effects and we identify interesting changes in the red - ox properties of both mnox and au when the two materials are present in one composite catalyst. using our experimental data and previous literature results, preparation of rotating disk electrode substrates : rotating disk electrode substrates were prepared from 200 mm long glassy carbon (gc) rods (diameter 5 mm, sigradur g htw hochtemperatur - werkstoffe gmbh). before deposition of mno or au nanoparticles, the rods were processed by stanford crystal shop to produce 4 mm long pieces with one side lapped and chamfered and the other side polished to a root mean square (rms) surface roughness of less than 50 nm. preparation of substrates for in situ x - ray absorption spectroscopy (xas) characterization : electrode substrates for in situ xas characterization were prepared from 200 mm long gc rods (diameter 10 mm, sigradur g htw hochtemperatur - werkstoffe gmbh). before deposition of mno or au nanoparticles, 100200 m thick wafers with one side lapped and the other side polished to a surface rms roughness of less than 50 nm. synthesis of catalyst materials : mno nanoparticles were prepared with a sputtering system (nanosys500, mantis deposition ltd.) using a glow discharge magnetron sputtering with an inert gas condensation technique, as described previously. briefly, the system consists of a nanoparticle source and the quadrupole mass filter, which filters sputtered nanoparticles by mass in situ. mn nanoparticles were size selected at approximately 10 nm and deposited at a pressure of 0.3 mtorr with a rate of 0.16 s, monitored by a quartz crystal microbalance (qcm). these size selected nanoparticles were channeled to the main chamber and deposited on substrates. after deposition, samples were transferred to the load lock chamber, which was vented with ar. gold nanoparticles were prepared using an electron beam evaporator to deposit 8 gold at a rate of 0.10.2 s monitored by a qcm. electrochemical characterization : the oer activity of the catalyst samples was characterized using cyclic voltammetry in a three electrode electrochemical cell in a rotating disk electrode (rde) configuration. characterization was performed in 0.1 m koh electrolyte using a scan rate of 20 mvs, at room temperature. a carbon rod (ted pella) was used as the counter electrode, while ag|agcl (fisher scientific) was used as the reference electrode. the potential scale was calibrated to a reversible hydrogen electrode (rhe), and all the potentials were ir - compensated to 85% and reported vs rhe. the average measured resistance between the working and reference electrodes was 40 for all samples. the oer activity was determined by scanning the potential from 0.05 v to 1.71.8 v in a n2 saturated environment. to prepare surfaces for ex situ xas characterization, the potential was scanned from 0.05 v to a vertex potential of 1.65 v at 20 mvs and held at 1.65 v for 30 min. physical and chemical characterization of nanoparticles : the size and morphology of the catalytic materials were investigated using scanning electron microscopy (sem, fei magellan 400xhr). the images were obtained using a secondary electron detector, a beam current of 25 pa, and beam voltage of 5 kv. the oxidation state of the mnox nanoparticles was characterized using ex situ and in situ x - ray absorption spectroscopy (xas) at stanford synchrotron radiation lightsource (ssrl). ex situ measurements were performed on the 31-pole wiggler beamline 101 at the ssrl using a ring current of 350 ma and a 1000 lmm spherical grating monochromator with 40 m entrance and exit slits, providing 10 phs at 0.3 ev resolution in a 1 mm beam spot. all data were acquired in a single load at room temperature and under ultrahigh vacuum (10 torr) in total or auger electron yield (tey, aey) modes. the measurements were performed on mnox nanoparticles and five powder standards (mnf2, mno, mn3o4, mn2o3, and -mno2) attached to an aluminum sample holder using conductive carbon. -mno2 powder was prepared by dissolving 0.5 g of kmno4 in 30 ml of millipore water, followed by dropwise addition of ethanol under stirring, drying the resulting powder at 60 c overnight, and calcining the powder at 400 c for 3 h. mnf2, mno, mn3o4, and mn2o3 powders were purchased from sigma - aldrich and used as received. the energy was carefully calibrated in two steps, by first correcting the energy scale for the drift in the beam energy and then aligning the energy of the first peak of the mn3o4 powder control with a literature value of 639.6 ev. hard x - ray xas measurements were carried out at the ssrl at beamline 6 - 2 es2. the beamline was operated with a double - crystal si(311) monochromator with a rh - coated mirror to reject the high order harmonics. the x - ray beam was focused with a parabolic mirror to a spot size of 230 m fwhm (v) by 400 m fwhm (h) at the sample position. the x - ray energy was calibrated to the pre - edge absorption peak (6543.3 ev) of potassium permanganate. the mn k1 signals were resolved by 6 spherically bent analyzer crystals ge 333 installed on the high energy resolution x - ray emission spectrometer and detected by a silicon drift detector in photon counting mode. all spectra were normalized to have an edge jump of unity after linear backgrounds were subtracted from the raw data. the error bars reported in the spectra and the residuals represent one standard deviation based on poisson statistics and standard error propagation. in situ completely separate electrochemical cells (custom built cells, adams & chittenden), reference electrodes (ag|agcl, basi), and counter electrodes (100 mm length/5 mm diameter glassy carbon rod, si gradur g htw hochtemperatur - werkstoffe gmbh) were used with the mno - gc and mno / au - gc samples to avoid the possibility of contaminating the mno - gc sample with trace amounts of au. the working electrodes (gc wafers with deposited nanoparticulate catalysts) were glued to the window of the electrochemical cell using epoxy. the schematic of the in situ electrochemical cell is shown in supporting information, figure s1. prior to performing xas measurements, cyclic voltammograms were collected at 20 mvs in nitrogen - saturated 0.1 m koh electrolyte by first scanning the potential from 0.05 to 1.1 v and then from 0.05 to 1.65 v. to record in situ mn xanes spectra at an oer relevant potential, the potential was scanned from 0.05 v to a vertex potential of 1.65 v at 20 mvs and held at 1.65 v for 15 min. all measurements were ir - compensated to 85% and are reported vs rhe by assuming a potential shift of 0.960 v. the average measured resistance between the working and the reference electrodes was 70. effect of au : to further study the effect of au on the oer activity of mno - gc nanoparticles, additional electrochemical experiments were carried out. for these experiments, the potential was scanned on a mno sample and bare gc support from 0.05 v to 1.8 v in a n2 saturated environment. then 0.1 mm hau(iii)cl4 hydrate (sigma aldrich, 99.999% metals basis) was added to each electrolyte and the samples were repeatedly scanned starting in the oer region, 1.2 to 1.8 v, with the cathodic scan progressively reaching more reductive potentials with each cycle to deposit increasing amounts of au on the surface of the catalyst. schematic (a) and sem images (b) of the three catalytic samples, mno - gc, mno / au - gc, and au / mno - gc, illustrating morphology and coverage of glassy carbon support by mno and au nanoparticles. because the influence of the underlying substrate on the oer activity of mnox has not yet been clearly established in literature, we prepared nanoparticulate catalysts on inert gc supports to directly evaluate the impact of au. the first sample consisted of only mno deposited on gc (mno - gc). the second and third samples consisted of both mno and au nanoparticles : one sample with mno sputtered on top of au nanoparticles evaporated onto gc (mno / au - gc), while in the other au was evaporated on top of the mno - gc catalyst (au / mno - gc). schematic representations and sem images of the mno - gc, mno / au - gc, and au / mno - gc catalysts are shown in figure 1a, b. from the images, we can estimate that the mno loading is less than 1 g (calculations are presented in the supporting information). total electron yield (tey) mn l - edge xas characterization of the mn surface oxidation state in the three catalysts is presented in figure 2. in the figure, the xas spectra of the composite samples consisting of both mno and au are compared to the spectra of mno - gc and two powder controls : mnf2 and mno. in these experiments, mnf2 was used as a mn powder standard in addition to commercially purchased mno powder because of the known surface oxidation of the mno phase in air. comparison of the spectra of the powder standards to the spectra of the as - deposited mno / au - gc and au / mno - gc catalysts using this surface - sensitive technique illustrates that after the addition of au, mno nanoparticles exhibit the oxidized form rather than the reduced form of the mn phase. it is not clear whether this slight difference in the starting surface oxidation state results in a change in the crystal structure of the material to form a different bulk phase. our attempts to characterize the crystallinity of the catalysts with and without the addition of au using conventional and synchrotron grazing incidence x - ray diffraction (xrd) yielded only reflections corresponding to the gc support (supporting information, figures s2 and s3). this indicated that either both types of samples were amorphous or that the crystallinity of the nanoparticles could not be detected using grazing incidence xrd. total electron yield of mn l - edge xas of the three catalytic samples, mno - gc, mno / au - gc, and au / mno - gc, and two powder standards, mnf2 and mno. the surface of mno, which is known to oxidize in air, was not sputtered prior to characterization and represents oxidized mno. electrochemical characterization of the catalysts in 0.1 m koh electrolyte shown in figure 3 demonstrates that the composite catalysts, consisting of both mno and au nanoparticles, have significant oer activity, while the oer current density of the mno - gc catalyst is negligible by comparison. the supporting information (figure s4) also presents characterization of au evaporated onto gc in the absence of mno (au - gc) to rule out simple additive effects of mno - gc and au - gc. from the inset in figure s4 it is clear that the oer current of the composite samples is significantly greater than the sum of the individual activities of mno - gc and au - gc. table 1 compares the oer activity metrics of mno - gc, mno / au - gc, and au / mno - gc, including geometric current density, mass activity, and turnover frequency (tof), all calculated at either 300 or 400 mv overpotential (), to the activity metrics of some of the best mnox electrocatalysts reported in literature. although table 1 focuses purely on electrocatalysts, supporting information, table s1 presents a similar comparison to exceptional manganese oxide photochemical water oxidation catalysts. the tabulated activity metrics for manganese oxide catalysts reveal that the catalytic behavior of mno - gc in the absence of au is consistent with expectations. although its current density normalized to the geometric surface area is low in magnitude, the mass activity and tof values of the catalyst match the performance of the best mnox oer catalysts. furthermore, normalization of mno - gc oer current by its capacitance produces similar surface - area normalized activity to that of the previously reported mno thin film catalyst (supporting information, figure s6) deposited by atomic layer deposition (ald). from tables 1 and s1, it is also apparent the two composite catalysts consisting of both mno and au nanoparticles exhibit unusually high oer activities. each mass activity and tof is about an order of magnitude higher than the corresponding metrics for the previously reported highly active thin film mnox catalysts. the only other mnox catalyst with similar activity was published by subbaraman and co - workers, who electro - deposited mnox islands on a pt(111) support. although the authors did not include a cv of mnox demonstrating its oer activity, they reported an oer current (on a geometric basis) of 5 macm at ca. = 560 (1.78 v). this geometric current density is similar to the geometric current densities of our composite catalysts after the background oer current of pt(111) substrate is subtracted. from the results of subbaraman and co - workers, it is not clear whether or not the use of a noble metal support has an impact on the measured oer activity of mnox catalyst. direct comparison between the activity metrics of mno - gc and au / mno - gc catalysts in the present report, however, convincingly demonstrates that the addition of a noble metal to mno nanoparticles sputtered onto a gc substrate will lead to a significant enhancement in their oer activity. the influence of adding au to the oer activity of mnox could occur either through a modification of the mno phase of the starting catalyst, thus creating a bulk mnox phase with intrinsically higher oer activity than the original catalyst, or through the formation of active sites at the interface of mnox and au. to probe this further, we characterized the catalytic surface after exposure to oer conditions. in figure 4, we present ex situ tey mn l - edge xas spectra of mno - gc, mno / au - gc, and au / mno - gc catalysts previously exposed to an oer relevant potential of 1.65 v. the corresponding chronoamperometry curves used to generate the oer catalytic surfaces are shown in supporting information, figure s7. as expected, exposure to oxidative potentials leads to oxidation of mno nanoparticles in all three cases. the presence of au, however, favors formation of less oxidized mnox nanoparticles. furthermore, in the au / mnox - gc catalyst where au is deposited on top of the mno nanoparticles, mn assumes the lowest oxidation state among the three oer samples (supporting information, figure s8). one potential explanation for this result is that after exposure to oer conditions, the mn oxidation state may vary as a function of a distance from au, with mnox located at the interface with au assuming a more reduced state than mnox located away from au. to further study the possible interface effect in the composite samples, we compared mn l - edge xas spectra of mno / au - gc and mno - gc samples in tey and auger electron yield (aey) detection modes. although both aey and tey detection modes are surface sensitive, the probing depth of aey is shallower than that of tey. if an interface effect is present, one would expect the measured oxidation state of the oxidized mnox nanoparticles in the mno / au - gc sample to vary as a function of probe depth as illustrated in the schematic in supporting information, figure s9, resulting in different tey and aey spectra. figure s9 also presents the aey and the tey spectra of mno - gc and mno / au - gc for the as - deposited and for the oer samples. comparison of aey and tey spectra of mnox in the absence of au (the mno - gc sample) in figure s9 demonstrates a lack of variation in mn oxidation state as a function of distance from the gc support, both before and after applying an oer relevant potential. making the same comparisons with the mno / au - gc sample, however, shows that while the as - deposited sample has a negligible difference in the mn spectra between aey and tey mode, a more significant spectral difference is observed for the sample exposed to oer relevant potentials. in that sample, the top of the nanoparticle, which is located away from the au - mnox interface, is likely more oxidized, resembling the spectra of the mno - gc sample exposed to oer conditions. these results support the hypothesis that there is an interface effect between au and mnox after mno is exposed to high oxidative potentials. in conventional heterogeneous catalysis, such an interface effect has been previously observed with mno2 and other reducible metal oxides, such as tio2 and ce2o3, in contact with au. the ex situ nature of the mn l - edge xas characterization, however, does not allow us to differentiate between an interface effect that emerges when the catalyst is under oer conditions or an interface effect that emerges after the catalyst is removed from the electrochemical cell. to answer this question, in situ methods cyclic voltammetry characterization of the three catalytic samples, mno - gc, mno / au - gc, and au / mno - gc. characterization was performed in 0.1 m koh electrolyte, at 20 mv / s, and 1600 rpm. in situ characterization was performed with a bulk - sensitive technique using mn k - edge xas. our measurements focused on the x - ray absorption near edge structure (xanes) region, which probes the bulk electronic structure of the catalyst. in the experiments, mno and au nanoparticles were deposited on 200 m thick gc wafers to allow x - ray illumination of the backside of the electrodes. we characterized two samples, mno - gc and mno / au - gc, with a slightly reduced loading of mno compared to the ex situ samples (supporting information, figure s10). these samples were held at 1.65 v during the mn k - edge xas measurement. despite the mno / au - gc sample having a higher oer current throughout the entire experiment (figure s10), our in situ xas characterization shown in figure 5 does not reveal a measurable difference between the xanes spectra of mn in the mno - gc and the mno / au - gc samples. the observation of the same average mn oxidation during the electrochemical evolution of oxygen for both samples suggests that a small subset of sites rather than the bulk phase is critical to the enhanced oer catalysis in the samples containing au nanoparticles ; for example, this could occur by means of some of the mnox sites locally interacting with au in such a way as to form oer active sites with modified properties during electrochemical characterization. total electron yield mn l - edge xas of the three catalytic samples, mno - gc, mno / au - gc, and au / mno - gc, and two powder standards, mno2 and mn2o3. to further explore this hypothesis we performed electrochemical characterization of the mno - gc catalyst before and after adding 0.1 mm of hau(iii)cl4 to the electrolyte. figure 6 shows that as we electrodeposit more au onto the surface by sweeping to more reductive potentials, the oer activity increases, eventually reaching a current density greater than 10 macm at 1.8 v. when the same experiment was performed with a bare gc substrate, we also observed an increase in the oer activity with increased electrodeposition of au, but the activity remained low, below 1 macm at 1.8 v. these experiments demonstrate that the addition of au salt to the characterization electrolyte will lead to a similar enhancement in oer activity of mno - gc catalyst as evaporation of au onto the catalytic surface prior to electrochemical characterization. some dissolution of au into electrolyte is expected to occur at the highly oxidative potentials necessary for the oer. we took advantage of this fact and also performed experiments with the mno - gc sample in the setup previously used to characterize samples containing evaporated au nanoparticles, but without cleaning the cell. thus, trace au was expected to be present to form a sample which we refer to as trace - au / mno - gc. supporting information, figure s11 shows that although the morphology of the mno nanoparticles in trace - au / mno - gc had not changed while evaluating oer activity, the oer current increased with time during a potential hold at 1.65 v for 30 min (after first cycling the catalyst from 0.05 to 1.65 v at 20 mvs), ultimately rising to the same level of oer activity as that for the au / mno - gc sample. this result further illustrates that the electrolyte conditions can have a significant effect on the activity of mnox, with the addition of only a trace amount of au leading to a significant increase in the oer activity. supporting information, figure s12 compares cyclic voltammograms of the au - gc, au / mno - gc, and trace - au / mno - gc samples. analysis of electrochemical features of the samples confirms the presence of au in trace - au / mno - gc sample and reveals that the presence of mnox has an influence on the red - ox properties of au. specifically, the addition of mnox leads to formation of multiple oxidation / reduction peaks as opposed to the one clear oxidation peak and the one clear reduction peak observed in the au - gc sample. the presence of multiple oxidation / reduction peaks in both au / mno - gc and trace - au / mno - gc could indicate that the interactions between au and mnox have led to a formation of au sites with different strengths of au since reducible metal oxides such as mno2 are known to interact with noble metals to form a more oxidized noble metal at the interface of the two materials, this result is consistent the presence of interfacial effects between mnox and au. mn k - edge xas of two catalytic samples, mno - gc and mno / au - gc, and the difference between the two spectra, demonstrating that mn has a similar oxidation state under oer conditions in the presence and absence of au. after taking into account our ex situ and in situ spectroscopic characterization as well as electrochemical studies, we propose that the observed enhancement in the oer activity of mnox in the presence of au is caused by the local participation of au in the oer catalysis, for example through dissolution of au and its re - deposition onto a subset of mnox sites, rather than a bulk change in the starting properties of mnox. this interpretation is consistent with all of the results in our study and is also supported by the published literature in the area of oer catalysis for bulk mnox materials. as described previously, table 1 convincingly demonstrates that the samples consisting of both mnox and au had anomalously high oer activity when compared to other active mnox oer catalysts. the only mnox catalyst that had been reported to have similar oer activity was a manganese oxyhydroxide prepared using a completely different method, an electrodeposition, but also in the presence of a noble metal, pt(111). similar to au, pt is known to interact with transition metal oxides to form a reduced oxide and an oxidized metal at the interface of the two materials and dissolve at oxidizing potentials relevant to the oer. therefore, the presence of pt during the oer could lead to an enhancement in the oer activity of mnox that is similar to the enhancement observed in the presence of au. additionally, a recent work from najafpour and sedigh reports that several of mnox phases, including mn3o4, -mn2o3, -mno2, and k - birnessite can convert to the same layered mnox structure under water oxidizing conditions. as discussed by the authors, this observation deemphasizes the nature of the starting mnox phase in the oer catalysis and highlights the importance of the electrolyte conditions during water oxidation. therefore, the presence of a noble metal in the catalyst sample or the electrolyte could have a greater influence on the formation of the oer active sites than the as - deposited phase of mnox. (a) cyclic voltammetry of mno - gc and bare gc support in the presence and absence of trace amount of au (0.1 mm hau(iii)cl4) demonstrating the increasing oer activity with increasing deposition of au ; (b) 10-fold magnification of panel a focusing on the oer region. the effect of electrolyte conditions on the oer activity of electrocatalysts is not limited to mnox. trotochaud and co - workers have recently reported transformation of niox thin film catalyst into a layered hydroxide / oxyhydroxide oer catalyst during electrochemical characterization. during the transformation, the catalyst scavenged fe impurities from the solution incorporating them into the final active catalytic structure. it is possible that like mnox, niox could also interact with noble metal additives to form oer active sites with enhanced tofs. for example, in a different study, yeo and co - workers have already demonstrated a higher tof with a catalyst consisting of only 0.14 monolayers of niox deposited on au relative to the tof of a bulk niox catalyst, indicating that an interaction between niox and au exists. although trotochaud and co - workers have also attempted to assess the role of au by characterizing niox catalysts on au / ti and ito supports, their study involved conformal niox thin films with limited electrochemical accessibility of the au support. in contrast, in the study by yeo and co - workers, the au oxidation / reduction features were still present in the submonolayer sample, which meant that au had an opportunity to locally impart oer activity by either interacting with the adjacent niox or by dissolving into the electrolyte at the high oxidizing potentials necessary for the oer and subsequently integrating into the niox catalyst. thus, the electrochemical accessibility of the au likely plays a role and should be an important consideration when designing catalyst morphologies for future studies. in an investigation of catalysts for the oxygen evolution reaction (oer), we have shown that a composite catalyst of mno nanoparticles in the presence of au exhibited an enhancement in electrocatalytic activity by 1 order of magnitude over the best reported pure mnox catalysts. a difference in the mn l - edge x - ray absorption spectra of as - deposited catalysts originally indicated that a change in the starting properties of mnox could be responsible for the observed increase in the oer current. however, subsequent electrochemical measurements as well as ex situ and in situ mn xas characterization of the oer relevant samples identified more likely possibilities : namely local, interfacial effects between the au and mnox. even trace amounts of au were sufficient to activate the catalyst for the oer, further indicating that a local interaction between au and mnox, impacting only a subset of mnox sites, is the likely cause of the observed oer enhancement. as the intrinsic oer activity of other nonprecious metal oxides such as coox and niox (supporting information, table s2) are even higher than that of mnox, investigating the role of au in the oer activities of a number of nonprecious metal - based transition metal oxide catalysts could lead to even higher - performance materials | to develop active nonprecious metal - based electrocatalysts for the oxygen evolution reaction (oer), a limiting reaction in several emerging renewable energy technologies, a deeper understanding of the activity of the first row transition metal oxides is needed. previous studies of these catalysts have reported conflicting results on the influence of noble metal supports on the oer activity of the transition metal oxides. our study aims to clarify the interactions between a transition metal oxide catalyst and its metal support in turning over this reaction. to achieve this goal, we examine a catalytic system comprising nanoparticulate au, a common electrocatalytic support, and nanoparticulate mnox, a promising oer catalyst. we conclusively demonstrate that adding au to mnox significantly enhances oer activity relative to mnox in the absence of au, producing an order of magnitude higher turnover frequency (tof) than the tof of the best pure mnox catalysts reported to date. we also provide evidence that it is a local rather than bulk interaction between au and mnox that leads to the observed enhancement in the oer activity. engineering improvements in nonprecious metal - based catalysts by the addition of au or other noble metals could still represent a scalable catalyst as even trace amounts of au are shown to lead a significant enhancement in the oer activity of mnox. |
there is concern that older people with reduced respiratory muscle strength may be misclassified as having copd on the basis of spirometric results and receive unnecessary and potentially harmful treatment. with increasing age, the smaller pulmonary airways more readily collapse and forced vital capacity (fvc), forced expiratory volume in 1 second (fev1), and forced expiratory flow decrease ; there is an increase in functional residual capacity and expiratory reserve volume, residual volume increases, and there is little change in total lung capacity.13 in addition with aging, there is a decreased respiratory response to hypoxemia and hypercapnia and a reduced perception of raised resistance of the airway.2 there is a loss of synovial joints between the sternum and the costal cartilages reducing chest wall compliance and tension forces, leading to reduced maximal respiratory pressure and decreased muscle mass and strength of accessory respiratory muscles.4 it has also been observed that there is a significant decrease by 25% in the diaphragm strength in the older compared with young adults.5 one study has observed that inspiratory (mainly diaphragmatic) strength has been considered to be the most relevant in relation to function, fatigue, and training.6 with aging, there is a triad of loss of muscle mass, strength, and function defined as sarcopenia. this is common and has serious consequences including increased risk of current and future falls, dependency, hospitalization, and mortality. grip strength is recommended as a simple standardized clinical measure used in the diagnosis of sarcopenia.7 age - associated skeletal muscle changes may impact respiratory muscle function, and a positive correlation between grip strength and inspiratory and expiratory muscle strength has been demonstrated.810 reduced lung function (in particular fev1 and fvc) after adjusting for age, smoking, and physical activity has been demonstrated with reduced skeletal muscle mass in korea.11 copd is defined by the global initiative for chronic obstructive lung disease (gold) by symptoms, exposure to risk factors (namely smoking), family history, and confirmation with spirometry with a postbronchodilator fev1/fvc 70 years, a history of never smoking or trivial smoking 70 years, a history of never smoking or trivial smoking 1 year, or acutely ill were excluded. the significance of this study s findings has to be taken within this context and are preliminary findings. recruiting participants based on the researcher s availability may have led to potential eligible patients not being included but would not have resulted in observer bias. this study examined individuals far older than in previous studies and this older group had difficulty maintaining the consistency to conform to the american thoracic society (ats) guidelines for spirometry. we reported the maximum spirometry values rather than the mean values recognizing that with repeated testing some patients may fatigue. this could be standardized by setting limits to exclude lung function tests, for example, with fev1/fvc > 90% or < 60% and abnormal spirogram patterns. assessment of svc in all participants would have been beneficial ; however, this was not possible to obtain due to participants being discharged home. further evaluation of svc to measure airway obstruction particularly with those limited by coughing may be useful as a research tool and in practice. we performed it with a single researcher with trained competency in collecting data confirmed with intraobserver variability assessment. the use of a standardized method for measuring grip strength was advantageous and is not usually the case in other studies in older persons. similarly, we performed spirometry in a standardized way that would allow easy comparison to other studies. we considered the appropriateness of performing spirometry in hospitalized patients given the potential instability of this population. previous studies have highlighted variability in spirometry results particularly in older participants with cognitive impairment (due to an inability to coordinate or perform the test in a standardized manner), those acutely ill, and smokers (secondary to pathophysiologic changes).20 therefore, we decided to use rigorous exclusion criteria to standardize recruitment and enable comparison with other studies, although we acknowledge its limitation on sample size. this study aimed to evaluate the relationship between grip strength and lung function in older persons and whether current guidelines may inadvertently lead to clinicians misdiagnosing and potentially treating older nonsmokers with unnecessary and potentially harmful treatment. a larger community - based study using the same rigorous methodology may provide a better understanding of this relationship. it could be that a larger number of older persons in the community would benefit from this research due to the increasing screening programs in primary care. it may also be easier to perform grip strength and spirometry within an outpatient setting in a more consistent fashion. it has also been demonstrated in older adults that fixed ratio fev1/fvc may better identify subjects at risk of death or hospitalization than using the lower limit of normal spirometry values.32 hence, this area of debate may be more complicated than simply disease classification. future work may consider review of grip strength to lung function expressed in terms of the diagnostic cutoff values for copd using both the lower limit of normal and the fixed ratio criteria. there may be other avenues of assessing the relationship between grip strength and spirometry in older nonsmokers using additional biochemical markers for aging and measuring fev at the earlier part of forced expiration to increase accuracy of results before fatigability sets in. similarly resistance training has been recognized as important in maintaining muscle mass and strength in older people. there may be the potential for pulmonary rehabilitation programs for older people in the future to maintain respiratory muscle mass and strength.32 men had significantly stronger grip strength and larger fev1, fvc, pefr, and svc compared with women probably because of larger body size. there was a significant association between grip strength and pefr and grip strength and svc after adjustment for age, height, and weight in women, which may reflect stronger patients generating higher intra - thoracic pressure at the start of spirometry and pushing harder against thoracic cage recoil at end - expiration. in men, a significant relationship was found between grip strength and fev1, which was attenuated after adjustment for age, height, and weight, and this requires further evaluation. | objectiveolder people with reduced respiratory muscle strength may be misclassified as having copd on the basis of spirometric results. we aimed to evaluate the relationship between lung function and grip strength in older hospitalized patients without known airways disease.methodspatients in acute medical wards were recruited who were aged 70 years ; no history, symptoms, or signs of respiratory disease ; mini mental state examination 24 ; willing and able to consent to participate ; and able to perform hand grip and forced spirometry. data including lung function (forced expiratory volume in 1 second [fev1 ], forced vital capacity [fvc ], fev1/fvc, peak expiratory flow rate [pefr ], and slow vital capacity [svc ]), grip strength, age, weight, and height were recorded. data were analyzed using descriptive statistics and linear regression unadjusted and adjusted (for age, height, and weight).resultsa total of 50 patients (20 men) were recruited. stronger grip strength in men was significantly associated with greater fev1, but this was attenuated by adjustment for age, height, and weight. significant positive associations were found in women between grip strength and both pefr and svc, both of which remained robust to adjustment.conclusionthe association between grip strength and pefr and svc may reflect stronger patients generating higher intrathoracic pressure at the start of spirometry and pushing harder against thoracic cage recoil at end - expiration. conversely, patients with weaker grip strength had lower pefr and svc. these patients may be misclassified as having copd on the basis of spirometric results. |
focal choroidal excavation (fce) was first described by jampol as an anomalous excavation of the choroid ; it is typically observed by optical coherence tomography (oct).1,2 this condition has been associated with several chorioretinal conditions and asymptomatic status, including central serous chorioretinopathy, choroidal neovascularization, and best vitelliform macular dystrophy.112 however, the mechanisms underlying the formation of fce remain uncertain. wakabayashi described two patterns of fce : 1) conforming pattern excavations that involved the outer retinal layers up to and including the external limiting membrane ; and 2) nonconforming pattern excavations that involved only the retinal pigment epithelium (rpe). here we describe a clinical course of fce associated with vkh before and after treatment and compare the morphological findings of fce in four subjects with different chorioretinal conditions. a 55-year - old man (subject 1) presented with bilateral metamorphopsia, with decimal visual acuity being 0.8 in the right eye and 1.2 in the left. the clinical diagnosis of vkh was made based on bilateral panuveitis and multifocal exudative retinal detachments at the posterior poles (figure 1). the serous detachments entirely resolved within 9 weeks and visual acuity in the right eye improved to 1.2. serial spectral - domain oct (sd - oct) images obtained at the preuveitic, uveitic, and posttreatment phase are shown in figure 2. sd - oct obtained 1 year before the onset of vkh due to symptoms of a floater and visual disturbance demonstrated a dome - shaped posterior protrusion of the rpe and outer retinal layers into the choroidal cavity (ie, conforming fce) at the fovea in the right eye. sd - oct at the uveitic phase identified multiple bilateral sensory retinal detachments, with the fce now involving only the rpe (ie, nonconforming fce). the fce pattern and its alteration during treatment of the case with vkh were compared to those of four subjects with fce associated with other chorioretinal conditions (figure 3). this comparison group consisted of two patients with age - related macular degeneration (amd ; subjects 2 and 3), one subject with a macular hole (mh ; subject 4), and one asymptomatic individual (subject 5). treatment with intravitreal ranibizumab injections and vitrectomy had been performed in the patients with amd and mh, respectively. a conforming fce was observed in three patients (subjects 2, 3, and 5) and pattern alteration from nonconforming to conforming fce was found after treatment in one subject (subject 4). in the two subjects with amd, a hyperreflective material around the fce was absorbed, resulting in a well - demarcated conforming fce after treatment. a detailed clinical course of fce at the fovea was documented in a case with vkh disease. sd - oct images obtained at the preuveitic, uveitic, and posttreatment phase suggested the preexistence of fce before the onset of vkh, and the fce pattern change (from conforming to nonconforming pattern during the period of choroidal inflammation) was observed during the follow - up. the fce identified at the preuveitic phase in our case supported the congenital / acquired posterior pole malformation, as previously suggested.1,4,9,11,12 on the other hand, hashida speculated that a direct pressure effect on the choroidal layer by subretinal fibrin disrupted the choroidal integrity and focal choroidal atrophy / thinning following inflammation and resulted in a formation of fce in a case with vkh.4,7,13 although such a remarkable subretinal fibrin was not detected, the association between the fce formation and choroidal inflammation could not be excluded entirely in our case, considering other possible subclinical inflammatory events prior to this disease history. an alteration of the fce pattern observed in vkh was also found in the comparison group with other chorioretinal conditions. in subject 4 with an mh, the nonconforming fce at presentation associated with vitreomacular traction changed to conforming fce after treatment. in two subjects with amd, a hyperreflective material around the fce was observed to be absorbed, resulting in a well - demarcated conforming fce via treatment (subjects 2 and 3). these findings suggest that the cavity within fce can be filled with subretinal fluid associated with inflammation and exudative material generated by choroidal neovascularization. given the proximity between the fce and the active lesions, the involved retina around the active lesion may well show disorganization of retinal layers in the pathological process ; then the conforming pattern is hard to maintain, unsurprisingly. in addition, it is possible that the cavity within fce may potentially accelerate the pathophysiological changes of chorioretinal disorders. | we describe focal choroidal excavation (fce) in a case of vogt koyanagi harada (vkh) disease and compare the findings with different chorioretinal conditions. a 55-year - old man was diagnosed with vkh based on panuveitis and exudative retinal detachments. spectral - domain optical coherence tomography demonstrated a dome - shaped protrusion with a nonconforming pattern at the fovea, which had been detected as a conforming pattern 1 year before the onset. the fce pattern returned into a conforming pattern following corticosteroid therapy. these findings suggest that the natively existent fce could be affected by pathophysiological changes of vkh as well as other chorioretinal conditions. |
levocetirizine (lcz), an active enantiomer of cetirizine, is a second - generation antihistamine that was approved by the us food and drug administration in 2008 for the relief of symptoms associated with allergic diseases in adults and children [1 - 3 ]. although it has some side effects such as sleepiness, headache, dry mouth, vision problems, palpitation, and fatigue it is considered to be effective and safe treating allergic disease. we present the first report of lcz - induced liver injury with histologic confirmation in a 48-year - old man without a history of hepatobiliary disease. a 48-year - old man without significant medical history was referred from the dermatology department due to jaundice and generalized pruritus. the patient did not report any fatigue, anorexia, nausea, abdominal discomfort, fever, rash, myalgia, muscle weakness, swelling, and recent illness. medication in recent use included lcz 5 mg daily for 2 months, and the patient had been taking bepotastine besilate 10 mg daily until 2 months ago. he did not report taking any other medications including statins, over - the - counter medications, herbal medications including red ginseng or any extract or nutritional supplements. specific history of exposure to hepatotoxic chemicals or familial history of liver disease was not identified. initial blood pressure was 110/70 mmhg, pulse rate 88/min, respiratory rate 18/min, and body temperature 36.7c. laboratory findings revealed prothrombin time 11.8 s, international normalized ratio 0.88, aspartate aminotransferase (ast) 1,208 iu / l, alanine aminotransferase (alt) 2,043 iu / l, total bilirubin 8.68 mg / dl, direct bilirubin 7.23 mg / dl, alkaline phosphatase 184 u / l, lactate dehydrogenase 463 iu / l, protein 5.9 g / dl, albumin 3.9 g / dl, and creatine phosphokinase 21 iu / l. serologic viral marker evaluations revealed hepatitis b surface antigen negative, antibody to hepatitis b surface antigen positive, antibody to hepatitis b core antigen negative, anti - hepatitis c virus negative and anti - human immunodeficiency virus negative. serum anti - mitochondrial antibody, anti - smooth muscle antibody, anti - nuclear antibody, anti - dsdna antibody, anti - neutrophil cytoplasm antibody, and anti - liver kidney microsome antibody were negative. an abdominal sonography revealed no evidence of extrahepatic obstruction, biliary ductal disease, cholelithiasis, or hepatic parenchymal abnormalities. for histologic evaluation, a percutaneous ultrasonography - guided liver biopsy was performed without complications. there was an increased amount of connective tissue extending from the portal area and inflammation around apoptotic hepatocytes (fig. the patient stopped taking lcz immediately after evaluation and all kinds of liver enzymes were normalized within 3 weeks after discontinuation (fig. the patient visited the outpatient department several times after discharge and has maintained normal liver function. drug - induced liver injury (dili) is a rare but potentially life - threatening adverse drug reaction. a large number of drugs have deleterious effects on the liver, and dili is a major cause of acute liver injury and liver transplantation. dili is responsible for ~10% of all adverse drug reactions in the united states. in korea, the annual extrapolated incidence of dili in hospitalized patients at a university hospital was 12/100,000 persons / year. the mechanism of dili is unknown, but it is not associated with medication dose, particularly idiosyncratic dili. because of its low incidence, idiosyncratic dili can not be detected in preclinical testing or clinical trials. the r - enantiomer of cetirizine dihydrochloride, which has favorable pharmacodynamic and pharmacokinetic characteristics has been demonstrated to be safe and effective for the treatment of allergic diseases, as evidenced by the low number of adverse effects in clinical trials. a similar number of patients reported treatment - emergent adverse events with lcz and placebo. the most common treatment - emergent adverse events are headache (4.6%), fatigue (1.8%), and dry throat (1.4%). one patient in the lcz group showed cellulitis (0.4%) as a serious adverse event. a previous report of lcz - induced hepatitis showed less severe hepatic inflammation compared with our case (ast 113 iu / ml and alt 115 iu / ml vs. ast 1,208 iu / ml and alt 2,043 iu / ml, respectively). this difference of severity was originated in which the previous case was detected by routine liver function test after using lcz only for 2 weeks, but our patient exhibited jaundice after taking lcz for 2 months. because hepatotoxicity is not included in the lcz prescribing information and only one case of lcz - induced liver injury has been reported, hepatotoxicity was not monitored in our case. therefore, any drug could cause dili. patients with dili can present with symptoms similar to those of patients with hepatobiliary disease of other causes, and therefore it is important to maintain a high index of clinical suspicion in determining the diagnosis. the clinical presentations of dili are acute hepatitis and/or cholestasis, although idiosyncratic reactions frequently occur in a background of higher incidence rates of mild asymptomatic liver injury. our patient did not have a specific past medical history, nor show serologic or radiologic findings. based on the timing of lcz administration and the clinical course, we could suspect lcz - induced liver injury. because the patient conducted liver biopsy and recovered after withdrawal of lcz numerous drugs have been reported to cause liver injury, but only one case of lcz - induced liver injury, which lacked histologic confirmation, has been described. furthermore, unlike the previous case of lcz - induced liver injury, our patient showed > 20-fold elevation of serum ast and alt levels. to our knowledge, this is the first reported case of lcz - induced liver injury confirmed by liver biopsy. lcz is generally well tolerated and widely used, and it has consistently demonstrated high response rates and a favorable side - effect profile. although its hepatotoxicity is not established, lcz could be associated with acute liver injury. in conclusion, lcz is an effective and well - tolerated drug for the treatment of allergic diseases ; however, clinicians should be aware of its potential hepatotoxicity. | levocetirizine is a second - generation nonsedative antihistaminic agent that has been demonstrated to be safe and effective for treating allergic disease. there was only one case report of levocetirizine - induced liver toxicity, but a liver biopsy was not performed. in this article, we present the first case of levocetirizine - induced liver injury with histologic findings. a 48-year - old man was hospitalized with jaundice and generalized pruritus that had developed after 2 months of therapy with levocetirizine for prurigo nodularis. laboratory findings revealed acute hepatitis with cholestasis. a liver biopsy demonstrated portal inflammation and hepatitis with apoptotic hepatocytes. the patient fully recovered 3 weeks after withdrawing levocetirizine. although levocetirizine is safe and effective, physicians should be aware of its potential hepatotoxicity. |
bacterial vaginosis (bv) is a common condition causing various symptoms such as vaginal discharge, odor and irritation, and has been associated with increased acquisition of many sexually transmitted diseases [1, 2 ]. pregnant women with bv have an increased risk of preterm labor and preterm delivery with the potential for neonatal morbidity and mortality. early research concerning bv characterized the vaginal microbiological flora using gram stain and culture [4, 5 ]. more recent studies have focused on host defense factors and the local immune response that may mediate the relationship between vaginal flora and adverse reproductive and pregnancy outcomes [6, 7 ]. (il-1), interleukin 6 (il-6), interleukin 8 (il-8), and the host defense molecule secretory leukocyte protease inhibitor (slpi) have been of particular interest [810 ]. subjects are routinely asked to avoid vaginal intercourse and the use of intravaginal products that might affect test results prior to having specimens collected. however, compliance with these requests is difficult to assess. when vaginal fluid is collected to measure cytokine concentrations, it is important to determine what effect, if any, there may be on the results if semen is present. the objective of this analysis was to determine whether semen present in vaginal fluid alters proinflammatory cytokine or slpi concentrations. we hypothesized that the presence of semen would increase the concentrations of vaginal proinflammatory cytokines and slpi. this secondary analysis included data from 138 pregnant women, between 7 and 20 weeks gestation, who participated in a prospective observational cohort study of the effects of bv on pregnancy outcome. subjects were recruited from the prenatal clinics associated with the university of washington medical center in seattle, wash, usa. participation in the study was limited to those subjects who met the following criteria : singleton pregnancy less than 20 weeks gestation, no prior preterm birth or major medical problems such as chronic hypertension or pre - existing diabetes, and no recent antibiotic use. the study was approved by the university of washington and the centers for disease control and prevention institutional review boards and all subjects provided written, informed consent. we compared subject history, gram stain for sperm and detection of acid phosphatase as predictors for the presence of semen in vaginal fluid. acid phosphatase was considered to be the reference as it is an enzyme present in high concentrations in semen, but not found in other secretions such as vaginal fluid, saliva, or mucus. we then compared the concentrations of proinflammatory cytokines and slpi in samples from women with and without semen detected in vaginal fluid. subjects were asked to abstain from vaginal intercourse and the use of intravaginal products for 48 hours prior to their study visit. subjects completed a structured interview with questions regarding demographics, reproductive history, behavioral habits, and time of last intercourse. a physical exam was conducted including notation of amsel criteria as well as a vaginal wet mount and gram stain. two dacron swabs were used to collect vaginal fluid from the posterior vaginal fornix and placed in cryotubes containing 0.9 ml phosphate buffered saline. swabs were frozen at 80 degrees and stored for later cytokine and slpi testing. an additional dacron swab was used to collect vaginal fluid to prepare an air - dried microscope slide which was then gram stained and read at 100x magnification for the presence of semen and determination of bv score by nugent criteria. vaginal fluid from the frozen samples was aliquoted and used to measure proinflammatory cytokine and slpi concentrations by enzyme immunoassay. for acid phosphatase detection, vaginal fluid was spotted to whatman no.1 filter paper and then placed in a chemical fume hood and sprayed until wet with the prepared reagent. development of a purple color within 1 minute was considered a positive test for the presence of acid phosphatase [15, 16 ]. the reagent was prepared by mixing 10 ml of stock solution a (1 gram fast blue b, 20 grams sodium acetate trihydrate, 10 ml glacial acetic acid, 100 ml dh20) and 1.0 ml of stock solution b (0.4 grams sodium alpha naphthyl acid phosphate, 5 ml dh20) in a spray bottle. the prepared reagent has a shelf life of 7 days, while stock solutions a and b are stable for up to six months at 4 degrees. we used the chi square test or fisher s exact test for categorical variables. analyses were stratified by presence or absence of bv on gram stain, given that proinflammatory cytokines and slpi may vary with bv status [17, 18 ]. of 138 subjects, 36 (26%) reported vaginal intercourse within the past two days, despite instructions to abstain. 28 (20%) had acid phosphatase detected in their vaginal fluid, and 6 (4%) had sperm seen on gram stain. women with detectable acid phosphatase were more likely than those without acid phosphatase to report recent vaginal intercourse (68% versus 15%, p <.0001), yet subject history and acid phosphatase detection did not have high concordance. subjects with and without detectable semen by acid phosphatase were similar with respect to age, race / ethnicity, gestational age, and presence of genital infections (see table 1). the remaining analyses compared vaginal cytokine and slpi concentrations among women with and without acid phosphatase detected in vaginal fluid. there was no significant difference in il-1, il-6, or il-8 concentrations among women with or without acid phosphatase, when stratified for bv (see figures 1(a), 1(b), 1(c)). however, slpi concentrations were significantly higher in the acid phosphatase positive group regardless of bv status (see figure 1(d)). there was a significant increase in il-1 and decrease in slpi among women with bv (see figures 1(a), 1(d)) in this cohort of pregnant women, detection of semen by acid phosphatase was associated with higher slpi concentrations in vaginal fluid. these results are not surprising, in that slpi is a known component of seminal fluid, however, the presence of semen did not appear to influence the vaginal proinflammatory cytokine concentrations. proinflammatory cytokines can also be detected in semen but are usually not present at high concentrations except in the context of a sexually transmitted infection in the male partner. increased cytokine levels in semen have been established in hiv infection (il-1), and genital infections such as chlamydia trachomatis (il-8) and neisseria gonorrhoeae (il-6, il-8). we did find a high (26%) prevalence of recent vaginal intercourse by history, despite instructions to abstain prior to the study visit. we also found that patient history is not a highly sensitive or specific marker for the detection of acid phosphatase, as only 68% of those with acid phosphatase present reported recent intercourse. gram stain detection of sperm is limited by the concentration of sperm as well as duration of persistence in the vaginal fluid. in addition, we found that gram stain is not a useful method to screen for recent vaginal intercourse, with a sensitivity of only 11%. while other tests such as prostate specific antigen (psa) may be more sensitive, the cost and implementation of equipment and methodology to perform this elisa test may be prohibitive to many laboratories. it is necessary to note that while samples were immediately frozen for storage after collection and care taken to minimize the number of freeze / thaw cycles when aliquoting for testing, we can not determine if there was any degradation of cytokine concentrations over time or due to the presence of semen. results from this study suggest that the use of acid phosphatase to detect semen in vaginal fluid samples can provide useful information and this testing can be performed using a simple and inexpensive method. vaginal fluid samples with acid phosphatase should not have slpi measured, as there is likely a substantial contribution from seminal fluid. however, measurement of proinflammatory cytokines is probably not influenced by the presence of semen. the acid phosphatase detection test may be a useful adjunct for analysis of the effect of seminal fluid on other host defense and immune factors in future studies. | the presence of semen in vaginal fluid, as identified by an acid phosphatase spot test, does not influence vaginal proinflammatory cytokine concentrations. objective : determine whether semen, as detected by acid phosphatase, influences vaginal cytokines or secretory leukocyte protease inhibitor concentrations. methods : 138 pregnant women had vaginal fluid collected for gram stain, acid phosphatase detection by colorimetric assay, and interleukin 1-beta, interleukin-6, interleukin-8, and secretory leukocyte protease inhibitor measurement by enzyme immunoassay. results for women with and without acid phosphatase were compared by mann - whitney test. results : of 138 subjects, 28 (20%) had acid phosphatase detected ; of these, only 19 (68%) reported recent intercourse and 3 (11%) had sperm seen on gram stain. there were no significant differences in proinflammatory cytokine concentrations ; however, secretory leukocyte protease inhibitor concentrations were significantly higher among women with acid phosphatase. conclusions : proinflammatory cytokine measurement does not appear to be affected by the presence of semen, but secretory leukocyte protease inhibitor is significantly higher when semen is present. detection of semen by acid phosphatase was associated with higher vaginal slpi concentrations, however, the presence of semen did not appear to influence vaginal proinflammatory cytokine concentrations. |
longipalpis were obtained from santarm and camar colonies, maintained at the department of entomology, oswaldo cruz foundation, rio de janeiro (rj), brazil. salivary glands of three - five days - old non blood - fed adult female lu. longipalpis were dissected, transferred to microtubes containing phosphate buffer saline (pbs) and lysed by sonication. the homogenates were centrifuged at 10,000 rpm for 1 min to remove debris and the supernatants were transferred to new tubes and stored to -80c until use. parasites - leishmania amazonensis (mhom / br/75/josefa) was maintained on schneider insect 's medium supplemented with 10% foetal calf serum and 2% human urine at 26c. briefly, stationary promastigotes of five - six day cultures were collected, washed and resuspended at 1 x 10 /ml density in rpmi-1640. parasites (4 ml) were then overlaid on 4 ml of ficoll (10%) in pbs and centrifuged at room temperature (rt) for 10 min at 1,300 g with the brake off. metacyclic forms were collected in the 10% ficoll layer, washed twice in pbs and resuspended in pbs for assays. reagents - factor xa was purchased from calbiochem (san diego, ca, usa). human factor va and factor x were purchased from haematologic technologies (essex junction, vt, usa). human factor ixa was purchased from american diagnostica (greenwich, ct, usa). human fviii (advate) was purchased from baxter healthcare corporation (westlake village, ca, usa) and was activated with human thrombin as previously described (astermark. chromogenic substrates for factor xa (s-2765, n--benzyloxycarbonyl - d - arg - gly - arg - p - nitroanilide) and thrombin (s-2238, h - d - phenylalanyl - l - pipecolyl - l - arginine - p - nitroanilinedihydrochloride) were purchased from diapharma (westchester, oh, usa). activated partial thromboplastin time (aptt) (cephalin plus kaolin) and prothrombin time (pt) (thromboplastin with calcium) reagents were from biomrieux (rj, brazil). l--phosphatidylcholine (pc) and ps were purchased from sigma chemical co (st louis, mo, usa). phospholipid vesicles (pc / ps) composed of 75% pc, 25% ps (w / w) were prepared by sonication. briefly, phospholipids in chloroform were dried with a stream of n2, resuspended in 50 mm tris - hcl, 150 mm nacl, ph 7.5 and sonicated for 2 min. evaluation of plasma coagulation by recalcification time - plasma coagulation assays by recalcification time were performed on an amelung kc4a coagulometer (labcon, heppenheim, germany) using plastic tubes. human blood samples were collected from healthy donors in 3.8% trisodium citrate (9:1, v / v) and platelet poor plasma was obtained by further centrifugation at 2,000 g for 10 min. the procoagulant activity of l. amazonensis was evaluated by incubation of plasma (50 l) with 50 l of tris buffered saline containing different concentrations of metacyclic promastigotes. after 1 min incubation at 37c, plasma clotting was initiated by addition of 100 l of 12.5 mm cacl 2 and coagulation time was recorded. longipalpis sgl was evaluated by incubating 50 l of pbs containing 1 sgl with plasma (50 l). after 1 min at 37c, plasma clotting was initiated by addition of 100 l of 12.5 mm cacl 2 and coagulation time was recorded. for control, assays were also performed upon incubation of sgl (various dilutions) with different concentrations of metacyclic forms of l. amazonensis. activated partial aptt and pt tests - clotting times of human plasma were recorded on a coagulometer after activation of either the intrinsic or the extrinsic coagulation pathways. platelet - poor plasma was incubated with 50 l of pbs containing or not 1 sgl and 50 l of aptt reagent for 2 min at 37c. plasma clotting was started by addition of 100 l of 12.5 mm cacl 2 and coagulation time was recorded. for pt assays, 50 l of platelet - poor plasma were incubated with 50 l of pbs containing or not 1 sgl for 1 min at 37c. reaction was started by addition of 100 l of pt reagent and the plasma clotting was recorded. flow cytometric analysis - ps exposure by metacyclic forms of l. amazonensis was evaluated by flow cytometric analysis. parasites were resuspended in annexin v binding buffer (10 mm hepes, 150 mm nacl, 2.5 mm cacl 2, ph 7.2) and further incubated for 15 min at rt with annexin v - alexa-488 (molecular probes, eugene, or, usa) according to the instructions of the manufacturer. propidium iodide (pi) was added to the samples at the moment of acquisition (0.4 g / ml, final concentration). the samples (10,000 events) were acquired on facscalibur and analysed on cell quest software. prothrombin activation assay - activation of prothrombin by the prothrombinase complex (factor xa / factor va / calcium) was performed in 50 mm hepes, 100 mm nacl, 5 mm cacl 2, 1 mg / ml bovine serum albumin (bsa), ph 7.5 (hepes - bsa buffer), using a previously described discontinuous assay (fernandes. 2006). factor xa (10 pm, final concentration) was incubated for 2 min at 37c with factor va (1 nm, final concentration) and pc / ps (20 m, final concentration) in the presence of serially diluted sgl. reaction was initiated by addition of prothrombin (0.5 m, final concentration) and aliquots of 10 l were transferred into microplate wells containing 40 l of tris - ethylenediamine tetraacetic acid (edta) buffer (50 mm tris - hcl, 150 mm nacl, 20 mm edta, 1 mg / ml polyethyleneglycol 6,000, ph 7.5). after addition of 50 l of 100 m s-2238 prepared in tris - edta buffer, absorbance at 405 nm was recorded at 37c for 20 min at 6 s intervals using a versamax microplate reader (molecular devices, menlo park, ca, usa) equipped with a microplate mixer and heating system. the ability of metacyclic promastigotes in assembling the prothrombinase complex was evaluated by the incubation of factor xa (10 pm, final concentration) and factor va (1 nm, final concentration) for 2 min at 37c with l. amazonensis in hepes - bsa buffer. reaction was initiated by addition of prothrombin (0.5 m, final concentration) and thrombin formation was evaluated as described. the inhibitory effect of sgl on prothrombinase complex assembled in either pc / ps or l. amazonensis was also evaluated. in this case, sgl (at various concentrations) was incubated with factor xa, factor va and either pc / ps or l. amazo - nensis, before addition of prothrombin. factor x activation assay - activation of factor x to factor xa by the intrinsic tenase complex (factor ixa / factor viiia / calcium) was performed in hepes - bsa buffer, using a previously described discontinuous assay (fernandes. factor ixa (0.2 nm, final concentration) was incubated for 2 min at 37c with factor viiia (10 iu / ml, final concentration) in the presence of serially diluted sgl. reaction was initiated by addition of factor x (50 nm, final concentration) and after 5 min at rt, aliquots of 25 l were transferred into microplate wells containing 25 l of tris - edta. after addition of 50 l of 200 m s-2765 prepared in tris - edta buffer, absorbance at 405 nm was recorded, at 37c, for 20 min at 6 s intervals using a versamax microplate reader. chromogenic assay for measuring factor xa activity - the ability of sgl in interfering with factor xa catalytic activity was evaluated using a chromogenic assay. fxa (1 nm, final concentration) was incubated for 10 min at rt in the presence of sgl serially diluted in hepes - bsa buffer. after addition of 50 l of 200 m s-2765, absorbance at 405 nm was recorded at 37c for 20 min at 6 s intervals in a versamax microplate reader. the analysis was performed using prism 3.0 (graphpad software). in all cases, l. amazonesis metacyclic promastigotes activate blood coagulation in a ps - dependent manner - a number of studies have reported presence of ps on leishmania parasites (wassef. however this phenomenon has been refuted by other groups that were unable to detect this phospholipid on viable parasites (zufferey. we demonstrate that metacyclic promastigote forms of l. amazonensis expose ps in viable parasites. 1a shows a parasite population that stains for annexin v, but is negative for labelling with pi, a known marker of cell membrane rupture. ps exposure by leishmania amastigote forms accelerate blood coagulation in vitro, as previously demonstrated in parasites isolated from balb / c mice lesions (balanco. 1b shows that incubation of human plasma with 10 and 10 parasites reduced the human plasma coagulation time by 32% and 28%, respectively, as compared to control, thus evidencing a parasite - number dependent procoagulant effect. parasites were analysed by flow cytometry in a dot plot of alexa 488 conjugated - annexin v vs. propidium iodide (pi) double staining. data are representative of two independent experiments ; b : recalcification time of human plasma was measured in the absence (ct) or in the presence of different numbers of metacyclic promastigotes. results from 10 independent experiments are expressed as mean standard error of the means (sem). asterisks mean p < 0.01 relative to ct ; c : l. amazonensis metacyclic promastigotes support the prothrombinase complex assembly. activation of prothrombin into thrombin by factor xa / factor va was performed in the absence or in the presence of logarithmic or metacyclic promastigotes. the amount of thrombin formed was quantified as described in the materials and methods section. data from three independent experiments are expressed as mean sem ; d : annexin v blocks the assembly of the prothrombinase complex on l. amazonensis. metacyclic promastigotes were pre - incubated with different concentrations of annexin v and further assayed for thrombin generation in the presence of factor xa / factor va as described in the materials and methods section. it is generated by the cleavage of prothrombin by prothrombinase complex, which consists in a calcium - dependent, membrane - assembled complex between the enzyme factor xa and its cofactor factor va. assembly of the prothrombinase complex on ps - containing membranes is essential to achieve a high catalytic efficiency (mann 1999). in order to confirm ps exposure in l. amazonensis, we further evaluated the ability of parasites in promoting thrombin formation by the prothrombinase complex. 1c shows that metacyclic promastigotes, but not logarithmic promastigotes, support the coagulation complex assembly resulting in prothrombin activation into thrombin. this process was highly dependent on ps exposure, since pre - incubation of parasites with annexin v completely blocked thrombin formation (fig. longipalpis saliva counteracts l. amazonensis procoagulant activity - a number of anticoagulant proteins have been identified in lu. the presence of one sgl caused a 2.5-fold increase in plasma coagulation time, as compared to control in the absence of sgl (fig. next, we investigated the putative coagulation pathways inhibited by sgl by means of the activated partial aptt and pt tests. our data showed that the coagulation time was not altered in the presence or in the absence of one sgl as evaluated by the pt test (fig. 2a), which contains tissue factor and is used to screen the extrinsic pathway. on the other hand, the aptt test, which is used to evaluate the intrinsic pathway, showed an increase of 25% in the coagulation time when one sgl was added to the reaction medium (fig. 2:anticoagulant effect of lutzomyia longipalpis saliva counteracts procoagulant activity of leishmania amazonensis metacyclic promastigotes. a : anticoagulant activity of sandfly saliva. recalcification time of human plasma was measured in the absence (ct) or in the presence of salivary gland lysate (sgl). prothrombin time and thromboplastin time assays were also performed in the absence (ct) or in the presence of sgl. in all cases, data from three independent experiments are expressed as mean standard error of the means (sem). asterisks mean p < 0.001 or p < 0.01 relative to ct ; b : recalcification time of human plasma was measured in the absence (plasma alone) or in the presence of different numbers of promastigotes, co - incubated or not with 0.5 sgl ; c : recalcification time of human plasma was measured in the absence (plasma alone) or in the presence of 10 6 promastigotes, co - incubated or not with different numbers of sgl. in b and c, data were analysed by student t test. : p < 0.05 ; : p < 0.01 (relative to control). considering that the procoagulant activity of l. amazonensis metacyclic forms might be deleterious to vector blood feeding (kimblin. 2b shows that 0.5 sgl efficiently counteracted the procoagulant effect of either 10 or 10 parasites. alternatively, assays performed at a fixed concentration of 10 parasites and varying sgl amounts showed a dose - dependent inhibitory effect of sandfly saliva (fig. 2c). sandfly saliva inhibits prothrombinase complex activity - plasma coagulation results from the enzymatic cleavage of soluble fibrinogen into an insoluble fibrin polymer. this reaction is specifically catalysed by thrombin in a reaction that is subsequent to massive prothrombin activation by membrane - assembled prothrombinase complex (mann 1999). in this context, we further evaluated the ability of lu. this was assessed by incubating saliva with factor xa, factor va and prothrombin in the presence of calcium ions and artificial pc / ps membranes. as shown in fig. 3a, the presence of increasing sgl concentrations progressively inhibited thrombin formation by pc / ps - assembled prothrombinase complex. we next examined the ability of sandfly saliva to inhibit l. amazonensis -assembled prothrombinase complex. 3b shows that the presence of one sgl efficiently counteracted thrombin formation in all parasite concentrations tested. a : activation of prothrombin into thrombin was carried out in the presence of factor xa, factor va and l - -phosphatidylcholine (pc)/phosphatidylserine (ps) vesicles as described in the materials and methods section. assays were performed in the presence of serially diluted salivary gland lysate (sgl) or hepes - bovine serum albumin buffer alone (pc / ps). data from five independent experiments are expressed as mean standard error of the means (sem) [: p < 0.001 ; : p < 0.05 (relative to control) (pc / ps) ] ; b : inhibition of prothrombinase complex by sgl (0.5 equivalent per assay) in the presence of different numbers of leishmania amazonensis metacyclic promastigotes. a negative control of thrombin generation in the presence of factor xa / factor va alone and in the absence of parasites and sgl sandfly saliva inhibits tenase complex and fxa activity - activation of zymogen factor x into factor xa is efficiently catalysed by the intrinsic tenase complex, which consists in a calcium - dependent, membrane - assembled complex between the enzyme factor ixa and its cofactor factor viiia (mann 1999). in order to evaluate whether the intrinsic tenase complex is inhibited by sandfly saliva, we incubated factor ixa, factor viiia and pc / ps vesicles with serially diluted sandfly saliva. 4:inhibition of intrinsic tenase complex and factor xa catalytic activity by lutzomyia longipalpis saliva. a : activation of factor x was carried out in the presence of factor viiia, factor ixa and l - -phosphatidylcholine / phosphatidylserine vesicles as described in the materials and methods section. assays were performed in the presence of serially diluted salivary gland lysate (sgl) or hepes - bovine serum albumin (bsa) buffer alone (ct). data from three independent experiments are expressed as mean standard error of the means (sem) (asterisk means p < 0.05 relative to ct) ; b : factor xa catalytic activity was assayed by measuring the cleavage of s-2765 chromogenic substrate in the absence (ct) or in the presence of different numbers of sgl serially diluted in hepes - bsa buffer. data were analysed by student t test. : p < 0.01 ; : p < 0.05 (relative to ct). in general, many arthropods anticoagulants target thrombin or factor x and/or factor xa, this last a nexus of the intrinsic and extrinsic pathways of blood coagulation (champagne 2005). in order to evaluate whether the sandfly saliva anticoagulants have a direct effect on factor xa and can thus contribute to extend the observed coagulation time, chromogenic assays were performed with factor xa and sandfly saliva. 4b, the presence of 0.5 sgl caused a 50% inhibition in factor xa catalytic activity, thus indicating that the anticoagulant effect of sandfly saliva is, at least in part, related to direct inhibition of factor xa activity. the loss of membrane asymmetry during programmed cell death, i.e. apoptosis, is typically associated with ps exposure. the presence of ps in leishmania parasites has been reported by a number of groups (wassef. 1985, henriques. 2003, wanderley. 2006, 2009, yoneyama. 2006, frana - costa. 2013) and the phenomenon of ps exposure by viable parasites has been named " apoptotic mimicry ". this process is often referred to as a strategy of the parasites to evade host inflammatory and immune responses (wanderley. 2006, mercer & heleniu 2008, laliberte & moss 2009, santos. this process, which resembles ps exposure during apoptosis, consists in the externalisation of ps on living parasite 's surface. in the case of leishmania it is believed that most of the amastigote forms expose ps without evidences of apoptotic death (balanco. 2001, wanderley. 2006). interestingly, it has been reported a differential ps exposure between amastigotes isolated from either susceptible or resistant mice (wanderley. the recognition of ps - exposing metacyclic promastigotes by phagocytic cells modulates host immune response by eliciting the production of transforming growth factor - beta and interleukin-10, which facilitate the establishment and maintenance of the infection by the non - ps exposing metacyclic forms (van zandbergen. we demonstrate that metacyclic promastigote forms of l. amazonensis expose ps, while not presenting evidences of cell membrane rupture. ps exposure by activated platelets is an essential phenomenon during physiological haemostasis (heemskerk. in fact, ps - containing membranes allow the assembly of multimolecular enzymatic complexes that ultimately lead to thrombin generation and fibrin deposition (mann 1999). as a result of ps exposure, we herein demonstrate that l. amazonensis accelerates plasma clotting and supports the assembly of the prothrombin activating complex, prothrombinase. this process was fully dependent of ps exposure since annexin v completely reverted thrombin formation in the presence of l. amazonensis. the ability of l. amazo - nensis in recruiting and activating coagulation proteins might be correlated with non - haemostatic properties of these proteins. in this context it has been demonstrated that activation of protease - activated receptor-1, known as the thrombin receptor, contribute to l. amazonensis infection in macrophages (rana. 2011). saliva of haematophagous arthropods contains a diverse mixture of compounds that counteracts host haemostasis (ribeiro & francischetti 2003). these compounds are essential for achieving a successful blood feeding. in this context, an anti - clotting molecule containing a carbohydrate - recognition domain - like structure, with a c - type (ca -dependent) lectin domain, similar to the snake venom active proteins has been identified (kini 2006). six other members of a family of putative anticoagulants were identified on secreted proteins from lu. however, the effect of saliva towards extrinsic pathway can be eventually underestimated, due to the large amount of thromboplastin reagent that is used in the pt test. this observation agrees with the estimated concentration of the factor xa inhibitor, lufaxin, which has been recently characterised in the salivary gland of lu. our results demonstrate that the anticoagulant effect of sandfly saliva is partially due to the direct inhibition of factor xa activity, resulting in decreased thrombin formation by prothrombinase complex and decreased factor xa activity towards a chromogenic substrate. in this context, lufaxin specifically inhibits factor xa in a tight, reversible, ca -independent and non - competitive manner (collin. 2012). remarkably, sandfly saliva counteracts l. amazonensis procoagulant activity as well as thrombin formation by l. amazonensis -assembled prothrombinase complex. it is important to emphasise that other anticoagulant proteins might exist in the saliva of lu. longipalpis. while anophelines display thrombin - directed anticoagulants, culicine mosquitoes display factor xa - directed anticoagulants (stark & james 1996, prez de len albimanus, anophelin, a 6.5 kda saliva - derived peptide, functions as a specific and tight - binding thrombin inhibitor (francischetti. in addition, a kazal - type thrombin inhibitor has been also characterised in aedes aegypti (watanabe. aedes albopictus, the protein alboserpin, a factor xa inhibitor, was characterised as an atypical serpin, which binds tightly to factor xa and also contains high affinity for heparin and interacts with pc and phosphatidyl ethanolamine (calvo. altogether we conclude that the previously described immunosuppressive properties evoked by ps exposure on l. amazonensis parasites are concomitant with procoagulant responses. on the other hand, the potent anticoagulant effect of leishmania parasites ' vector 's saliva most of the anticoagulant properties of haematophagous organisms are driven against host procoagulant enzymes or multimolecular coagulation complexes. therefore it is conceivable that vector 's saliva impairs procoagulant, but not immunosuppressive properties of ps exposing parasites in the first steps of infection. on the other hand, the subsequent spread of the parasite on the vertebrate host possibly unmasks the procoagulant properties of ps exposing parasites which may evoke important biological responses during leishmania infection. | leishmania parasites expose phosphatidylserine (ps) on their surface, a process that has been associated with regulation of host 's immune responses. in this study we demonstrate that ps exposure by metacyclic promastigotes of leishmania amazonensis favours blood coagulation. l. amazonensis accelerates in vitro coagulation of human plasma. in addition, l. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. this process was reversed by annexin v which blocks ps binding sites. during blood meal, lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. since saliva and parasites are co - injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of l. amazonensis. lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. it also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (pc)/ps vesicles or in l. amazonensis. sandfly saliva inhibits factor x activation by the intrinsic tenase complex assembled on pc / ps vesicles and blocks factor xa catalytic activity. altogether our results show that metacyclic promastigotes of l. amazonensis are procoagulant due to ps exposure. notably, this effect is efficiently counteracted by sandfly saliva. |
eligible patients were drawn from 2 truven marketscan databases : commercial claims & encounters and medicare supplemental & coordination of benefits. large selfinsured employers, health plans, hospitals, and medicare contribute claims data to these databases. the full population includes 26 million people annually in the commercial data and 4 million people annually in the medicare claims. the medicare population includes people 65 and older with employersponsored comprehensive hmo coverage through medicare advantage or supplemental coverage through medigap plans. marketscan data are widely used in health services research to characterize patterns of care.18, 19, 20, 21 compared to the whole us population, the marketscan population is generally younger, more likely to be employed, and thus wealthier. previous comparisons have shown that marketscan represents the under65, noncapitated population well.22 this study was deemed not human subjects research by the health care policy compliance office, in accordance with the office of human research administration, harvard longwood medical area policy, and federal regulations [45 cfr 46.102(f) ]. we matched inpatient and outpatient claims using dates of service to form episodes of care that included any international classification of disease, 9th revision clinical modification (icd9cm) procedure codes or current procedural terminology codes related to the 3 devices of interest. though we focused primarily on crtp and crtd implants, we identified icd implants to provide context according to overall device utilization. we limited the sample to adults (18 years old) receiving new implants between 2008 and 2013 by excluding episodes with codes that indicated removals, revisions, and generator changes, and then classified each new implant as icd, crtd, or crtp (figure 1). tables s1 and s2 list the specific codes used to select new implants and classify devices. we required 6 months of continuous enrollment prior to implant in order to measure comorbidities and medications. cohort selection method. cpt indicates current procedural terminology ; crtd, crt with icd backup ; crtp, cardiac resynchronization therapy pacemaker ; icd, implantable cardioverter defibrillator ; icd9, international classification of disease, 9th revision. we measured characteristics of patients heart failure using claims in the 6 months prior to implant. two of these (arrhythmias and congestive heart failure) use codes from the 2013 centers for medicare and medicaid services hierarchical condition categories.23 the other 3 (atrial fibrillation / flutter, cardiac failure, and conduction disorders) use small subsets of diagnosis codes, listed in table s3. we also measured charlson index comorbidities in the 6 months prior to implant : cerebrovascular disease, coronary disease, dementia, diabetes mellitus, hiv / aids, kidney disease, liver disease, paralysis, peripheral vascular disease, pulmonary disease, rheumatoid arthritis, and stomach ulcers. we used the deyo adaptation with several codes that reflect the romano adaptation ; specific icd9cm diagnosis codes are found in table s3.24, 25, 26 we defined medication use as at least 90 days of medication supply in the 6 months prior to implant. we measured drugs in the following categories : blockers, angiotensinconverting enzyme inhibitors, and angiotensin receptor blockers. this 90day window prior to implant is consistent with the lower bound of a 3 to 6month course recommended in the 2010 comprehensive heart failure practice guideline27 and used in previous studies.28 specific drug names are found in table s4. we defined optimal medical therapy as use of any blocker combined with any angiotensinconverting enzyme inhibitor or angiotensin receptor blocker. some individuals in the database lack claims for prescription drugs (either because they do not have prescription drug coverage or their employer / insurer does not contribute these claims to the data). thus, we restrict our measures of medication use to people with any drug claim in the period (75% of all patients), a conservative definition of the subpopulation with both prescription drug coverage and available claims. we summarized patient characteristics by device type using counts and percentages for binary variables, and median, 25th, and 75th percentiles for continuous variables. we computed standardized mean differences in binary patient characteristics for all pairwise combinations of the 3 devices. we computed unadjusted implant rates using the entire marketscan population. in our main analyses, we combine the commercial and medicare advantage populations. to highlight the differences in clinical characteristics and medications among patients receiving each device, we plot standardized mean differences (with 95% cis) in figure 2. these are considered substantial when they exceed 0.10 in absolute value.29 clinical differences between implant groups. points (intervals) are standardized mean differences (95% ci) for each characteristic. positive numbers indicate higher prevalence in the device listed first in each panel, measured using the 6 months of claims prior to implant. ace indicates angiotensinconverting enzyme ; arb, angiotensin receptor blockers ; av, atrioventricular ; crtd, crt with icd backup ; crtp, cardiac resynchronization therapy pacemaker ; icd, implantable cardioverter defibrillator. to evaluate changes over time and geography in the relative proportions of each device type among new crt implants, we modeled device receipt using year, age quintiles, sex, and all 2 and 3way interactions among them (ie, a saturated model). for the 3way comparison of each device type among all implants, we fit a multinomial model ; for crtp receipt versus crtd, we used a logistic model. these models produce fitted probabilities of receiving each device type adjusted for age, sex, and year. we fit a version of the logistic model that also included state fixed effects ; for that model, we excluded 4 states (north dakota, puerto rico, south dakota, and vermont) with no crtp implants. we present results as fitted device percentages (out of all new implants) in selected patient subgroups, rather than regression coefficients, for ease of interpretation. eligible patients were drawn from 2 truven marketscan databases : commercial claims & encounters and medicare supplemental & coordination of benefits. large selfinsured employers, health plans, hospitals, and medicare contribute claims data to these databases. the full population includes 26 million people annually in the commercial data and 4 million people annually in the medicare claims. the medicare population includes people 65 and older with employersponsored comprehensive hmo coverage through medicare advantage or supplemental coverage through medigap plans. marketscan data are widely used in health services research to characterize patterns of care.18, 19, 20, 21 compared to the whole us population, the marketscan population is generally younger, more likely to be employed, and thus wealthier. previous comparisons have shown that marketscan represents the under65, noncapitated population well.22 this study was deemed not human subjects research by the health care policy compliance office, in accordance with the office of human research administration, harvard longwood medical area policy, and federal regulations [45 cfr 46.102(f) ]. we matched inpatient and outpatient claims using dates of service to form episodes of care that included any international classification of disease, 9th revision clinical modification (icd9cm) procedure codes or current procedural terminology codes related to the 3 devices of interest. though we focused primarily on crtp and crtd implants, we identified icd implants to provide context according to overall device utilization. we limited the sample to adults (18 years old) receiving new implants between 2008 and 2013 by excluding episodes with codes that indicated removals, revisions, and generator changes, and then classified each new implant as icd, crtd, or crtp (figure 1). tables s1 and s2 list the specific codes used to select new implants and classify devices. we required 6 months of continuous enrollment prior to implant in order to measure comorbidities and medications. cpt indicates current procedural terminology ; crtd, crt with icd backup ; crtp, cardiac resynchronization therapy pacemaker ; icd, implantable cardioverter defibrillator ; icd9, international classification of disease, 9th revision. we measured characteristics of patients heart failure using claims in the 6 months prior to implant. two of these (arrhythmias and congestive heart failure) use codes from the 2013 centers for medicare and medicaid services hierarchical condition categories.23 the other 3 (atrial fibrillation / flutter, cardiac failure, and conduction disorders) use small subsets of diagnosis codes, listed in table s3. we also measured charlson index comorbidities in the 6 months prior to implant : cerebrovascular disease, coronary disease, dementia, diabetes mellitus, hiv / aids, kidney disease, liver disease, paralysis, peripheral vascular disease, pulmonary disease, rheumatoid arthritis, and stomach ulcers. we used the deyo adaptation with several codes that reflect the romano adaptation ; specific icd9cm diagnosis codes are found in table s3.24, 25, 26 we defined medication use as at least 90 days of medication supply in the 6 months prior to implant. we measured drugs in the following categories : blockers, angiotensinconverting enzyme inhibitors, and angiotensin receptor blockers. this 90day window prior to implant is consistent with the lower bound of a 3 to 6month course recommended in the 2010 comprehensive heart failure practice guideline27 and used in previous studies.28 specific drug names are found in table s4. we defined optimal medical therapy as use of any blocker combined with any angiotensinconverting enzyme inhibitor or angiotensin receptor blocker. some individuals in the database lack claims for prescription drugs (either because they do not have prescription drug coverage or their employer / insurer does not contribute these claims to the data). thus, we restrict our measures of medication use to people with any drug claim in the period (75% of all patients), a conservative definition of the subpopulation with both prescription drug coverage and available claims. we summarized patient characteristics by device type using counts and percentages for binary variables, and median, 25th, and 75th percentiles for continuous variables. we computed standardized mean differences in binary patient characteristics for all pairwise combinations of the 3 devices. we computed unadjusted implant rates using the entire marketscan population. in our main analyses, we combine the commercial and medicare advantage populations. to highlight the differences in clinical characteristics and medications among patients receiving each device, we plot standardized mean differences (with 95% cis) in figure 2. these are considered substantial when they exceed 0.10 in absolute value.29 clinical differences between implant groups. points (intervals) are standardized mean differences (95% ci) for each characteristic. positive numbers indicate higher prevalence in the device listed first in each panel, measured using the 6 months of claims prior to implant. ace indicates angiotensinconverting enzyme ; arb, angiotensin receptor blockers ; av, atrioventricular ; crtd, crt with icd backup ; crtp, cardiac resynchronization therapy pacemaker ; icd, implantable cardioverter defibrillator. to evaluate changes over time and geography in the relative proportions of each device type among new crt implants, we modeled device receipt using year, age quintiles, sex, and all 2 and 3way interactions among them (ie, a saturated model). for the 3way comparison of each device type among all implants, we fit a multinomial model ; for crtp receipt versus crtd, we used a logistic model. these models produce fitted probabilities of receiving each device type adjusted for age, sex, and year. we fit a version of the logistic model that also included state fixed effects ; for that model, we excluded 4 states (north dakota, puerto rico, south dakota, and vermont) with no crtp implants. we present results as fitted device percentages (out of all new implants) in selected patient subgroups, rather than regression coefficients, for ease of interpretation. we identified a cohort of 55 044 patients implanted with new crt or icd devices over 6 years (figure 1). the majority (68%) were implanted with an icd, 27% with a crtd, and 5% with a crtp. among crt devices, crtd were the vast majority (85%). table shows the differences in demographics, heart failure characteristics, comorbidities, and medication use across the 3 device types. implants of crtp devices were most likely to be implanted in the outpatient setting (55%), followed by crtd (53%), and icd (48%). the crtp group had the highest proportion of women (40%), followed by crtd (30%), and icd (26%). patients receiving crtp devices were older at implant (median 76 years) than those receiving crtds (67 years) and icds (62 years). baseline characteristics of patients with new implants ace indicates angiotensinconverting enzyme ; arb, angiotensin receptor blocker ; crtd, cardiac resynchronization therapy with icd backup ; crtp, cardiac resynchronization therapy pacemaker ; icd, implantable cardioverter defibrillator ; optimal medical therapy, blocker and ace inhibitor or arb. the most common cardiovascular diagnosis was congestive heart failure, found in the majority of patients (crtp 55%, crtd 84%, icd 66%). we also found high proportions with atrial fibrillation / flutter (crtp 59%, crtd 29%, icd 23%). cardiac conduction disorders were common only among patients with crtp (21%) and crtd devices (27%), but not those with icd devices (8%). the proportion of patients receiving both blockers and angiotensinconverting enzyme inhibitors or angiotensin receptor blockers prior to implant appeared low : only 31% of crtp, 46% of crtd, and 40% of icd patients. these proportions did not change appreciably when limited to the population with a diagnosis of congestive heart failure. the biggest distinctions between patients who received crtp (compared to crtd) were higher prevalence of atrial fibrillation / flutter and arrhythmias, and much lower prevalence of congestive heart failure and cardiac failure (figure 2). figure 3 displays the percentage of people who received new implants out of the entire marketscan population in each year. medicare advantage enrollees, who are older than commercial enrollees, had higher proportions of all implant types. in 2013, for every 100 000 medicare enrollees, there were 11 crtp recipients, 33.8 crtd recipients, and 58 icd recipients. in the commercial population, these numbers were 0.4, 2.9, and 10.6, respectively. for the remaining results, we combined the commercial and medicare populations. each line represents the number of patients receiving each new implant type divided by the total number of enrollees in each year. crtd indicates crt with icd backup ; crtp, cardiac resynchronization therapy pacemaker ; icd, implantable cardioverter defibrillator. among new implants, icds dominated but decreased slightly over the study period, from a high of 72% of all implants in 2008 to 66% in 2013. crts represented 32% of new implants overall, of which the fraction of crtp devices grew from 12% to 20% over the study period. figure s1 contains the results from a multinomial model for receipt of each device out of all new implants. the results from the binomial model for crtp receipt (out of new crt implants) from a model with year, age (in quintiles), sex, and all interactions are shown in figure 4. all 3 predictors are influential. for example, among people in the middleage quintile (ages 6168) in 2010, women were 2.2 times as likely as men to receive crtp (18% versus 8% of new crt implants). in that same year, men in the oldestage quintile (77 and older) were 2.9 times more likely to receive crtp than the youngest (age 1854) : 23% versus 8% of new implants. the trend in increasing crtp implants over the study period was stronger at older ages and among women. for example, crtp proportion among men ages 18 to 54 remained essentially constant at 8% between 2008 and 2013. among men 77 years and older, the crtp percentage increased from 18% to 27%. among women, the crtp share in women 18 to 45 years also remained nearly constant at 10%, while the share in those 77 and older increased from 25% to 43% of new implants. each line shows the modeled probability of receiving crtp (conditional on receiving a crt device) by year (x axis), age (quintile, each line is a different quintile), sex (men in the top row, women in the bottom row) from a saturated logistic regression model. crtd indicates crt with icd backup ; crtp, cardiac resynchronization therapy pacemaker ; icd, implantable cardioverter defibrillator. adding state to this model allowed us to examine patterns across states, adjusted for age, sex, and year. the state fixed effects from a binomial model of crtp implants (out of all new crt implants) illustrate the geographic variation (figure 5). among states with at least an average of 100 new implants per year, we identified particularly high crtp percentages in south carolina and wisconsin, and particularly low shares in new york and indiana. these state differences are sufficient to change the fitted proportions substantially. for example, the fitted percentage of crtp (out of all new crt implants) among women age 62 to 68 in 2013 is 26% in south carolina versus 12% in new york. each point shows the state fixed effect from a binomial model for cardiac resynchronization therapy pacemaker (crtp) devices out of all new crt implants with segments extending 2 se on either side of the estimate. numbers in parentheses give the average total annual implants in each state ; we display only results for states with at least 20 crtp implants. in addition, we examined how residuals from the binomial model with state effects changed over time. figure 6 shows the difference between the modeled and observed patterns of crtp proportions (out of all new crt implants) for women age 62 to 68 in 2013, limited to results for states with an average of at least 100 annual total implants. kansas appears to be increasing relative to national trends, while minnesota and oregon are decreasing. lines connect the difference between each state 's annual cardiac resynchronization therapy pacemaker (crtp) percentage (out of all new crt implants) and the fit from a multinomial model that includes age, sex, year, and state. the states are arranged in order of the overall trend of the residuals from most negative (top left) to most positive (bottom right). we identified a cohort of 55 044 patients implanted with new crt or icd devices over 6 years (figure 1). the majority (68%) were implanted with an icd, 27% with a crtd, and 5% with a crtp. among crt devices, crtd were the vast majority (85%). table shows the differences in demographics, heart failure characteristics, comorbidities, and medication use across the 3 device types. implants of crtp devices were most likely to be implanted in the outpatient setting (55%), followed by crtd (53%), and icd (48%). the crtp group had the highest proportion of women (40%), followed by crtd (30%), and icd (26%). patients receiving crtp devices were older at implant (median 76 years) than those receiving crtds (67 years) and icds (62 years). baseline characteristics of patients with new implants ace indicates angiotensinconverting enzyme ; arb, angiotensin receptor blocker ; crtd, cardiac resynchronization therapy with icd backup ; crtp, cardiac resynchronization therapy pacemaker ; icd, implantable cardioverter defibrillator ; optimal medical therapy, blocker and ace inhibitor or arb. the most common cardiovascular diagnosis was congestive heart failure, found in the majority of patients (crtp 55%, crtd 84%, icd 66%). we also found high proportions with atrial fibrillation / flutter (crtp 59%, crtd 29%, icd 23%). cardiac conduction disorders were common only among patients with crtp (21%) and crtd devices (27%), but not those with icd devices (8%). the proportion of patients receiving both blockers and angiotensinconverting enzyme inhibitors or angiotensin receptor blockers prior to implant appeared low : only 31% of crtp, 46% of crtd, and 40% of icd patients. these proportions did not change appreciably when limited to the population with a diagnosis of congestive heart failure. the biggest distinctions between patients who received crtp (compared to crtd) were higher prevalence of atrial fibrillation / flutter and arrhythmias, and much lower prevalence of congestive heart failure and cardiac failure (figure 2). figure 3 displays the percentage of people who received new implants out of the entire marketscan population in each year. medicare advantage enrollees, who are older than commercial enrollees, had higher proportions of all implant types. in 2013, for every 100 000 medicare enrollees, there were 11 crtp recipients, 33.8 crtd recipients, and 58 icd recipients. in the commercial population, these numbers were 0.4, 2.9, and 10.6, respectively. for the remaining results, we combined the commercial and medicare populations. each line represents the number of patients receiving each new implant type divided by the total number of enrollees in each year. crtd indicates crt with icd backup ; crtp, cardiac resynchronization therapy pacemaker ; icd, implantable cardioverter defibrillator. among new implants, icds dominated but decreased slightly over the study period, from a high of 72% of all implants in 2008 to 66% in 2013. crts represented 32% of new implants overall, of which the fraction of crtp devices grew from 12% to 20% over the study period. figure s1 contains the results from a multinomial model for receipt of each device out of all new implants. the results from the binomial model for crtp receipt (out of new crt implants) from a model with year, age (in quintiles), sex, and all interactions are shown in figure 4. all 3 predictors are influential. for example, among people in the middleage quintile (ages 6168) in 2010, women were 2.2 times as likely as men to receive crtp (18% versus 8% of new crt implants). in that same year, men in the oldestage quintile (77 and older) were 2.9 times more likely to receive crtp than the youngest (age 1854) : 23% versus 8% of new implants. the trend in increasing crtp implants over the study period was stronger at older ages and among women. for example, crtp proportion among men ages 18 to 54 remained essentially constant at 8% between 2008 and 2013. among men 77 years and older, the crtp percentage increased from 18% to 27%. among women, the crtp share in women 18 to 45 years also remained nearly constant at 10%, while the share in those 77 and older increased from 25% to 43% of new implants. each line shows the modeled probability of receiving crtp (conditional on receiving a crt device) by year (x axis), age (quintile, each line is a different quintile), sex (men in the top row, women in the bottom row) from a saturated logistic regression model. crtd indicates crt with icd backup ; crtp, cardiac resynchronization therapy pacemaker ; icd, implantable cardioverter defibrillator. adding state to this model allowed us to examine patterns across states, adjusted for age, sex, and year. the state fixed effects from a binomial model of crtp implants (out of all new crt implants) illustrate the geographic variation (figure 5). among states with at least an average of 100 new implants per year, we identified particularly high crtp percentages in south carolina and wisconsin, and particularly low shares in new york and indiana. these state differences are sufficient to change the fitted proportions substantially. for example, the fitted percentage of crtp (out of all new crt implants) among women age 62 to 68 in 2013 is 26% in south carolina versus 12% in new york. each point shows the state fixed effect from a binomial model for cardiac resynchronization therapy pacemaker (crtp) devices out of all new crt implants with segments extending 2 se on either side of the estimate. numbers in parentheses give the average total annual implants in each state ; we display only results for states with at least 20 crtp implants. in addition, we examined how residuals from the binomial model with state effects changed over time. figure 6 shows the difference between the modeled and observed patterns of crtp proportions (out of all new crt implants) for women age 62 to 68 in 2013, limited to results for states with an average of at least 100 annual total implants. kansas appears to be increasing relative to national trends, while minnesota and oregon are decreasing. lines connect the difference between each state 's annual cardiac resynchronization therapy pacemaker (crtp) percentage (out of all new crt implants) and the fit from a multinomial model that includes age, sex, year, and state. the states are arranged in order of the overall trend of the residuals from most negative (top left) to most positive (bottom right). this study characterizes cardiac implantable electric device use in a large, nationwide sample of cardiac patients. we found that crtp devices comprise a growing minority of new crt implants (1220% from 2008 to 2013), with significant variation across states. while the fraction of each implant type in the entire marketscan population does not reflect trends in the eligible populations, our study is among the largest samples of realworld inpatient and outpatient crt implants in the united states. in addition, our finding of relatively low rates of appropriate neurohormonal blockade among device recipients raises questions about effective application of optimal medical therapy in these patients prior to device implantation. the overwhelming preference for crtd devices among all crt recipients in our study contrasts sharply with the application of crt in other countries, particularly in europe. a large multinational comparison of crtd and crtp implant rates reveal that among the 10 countries with the highest per capita rates of crt implantation, crtp devices range from 17% (italy) to 49% (hungary) of crt devices.30 in the swedish heart failure registry,31 63% of patients receiving crt were implanted with crtp.32 our data accord with a smaller, singlecenter study from the cleveland clinic, which demonstrated that among class iii / iv heart failure patients receiving crt, 95% received crtd versus crtp.33 even among patients aged > 80, 86% received crtd.34 what drives these striking national differences in device selection is not clear. american and european guidelines for crt are generally similar, including guidance indicating insufficient evidence that crtd is superior to crtp.35 some believe that older or frailer patients are better candidates for crtp rather than crtd or icd.36 our study finds that while crtp recipients were older, they were otherwise not more likely to have identified comorbidities compared to crtd recipients. in practice, patients receiving crtp devices have worse survival than those receiving icd or crtd.37 although older patients experience more inhospital mortality and complications of device implants,38 and may realize diminished survival benefit from icds,39 the existing evidence provides little guidance on how to select crtp versus crtd devices for individual patients.40 even the existing clinical trial data characterizes outcomes for patients younger than those found in registries and in our sample, with trial populations having a mean age of 69.4 years and only 6.4% of studied patients aged 85 or older.41 there are few data characterizing the differences between those who receive crt (of either type) and those who are apparently eligible but either decline or are never offered crt. identifying such patients may help elucidate the rationale for device selection, if in fact patient characteristics guide these decisions. studies currently under way are evaluating how patients assess their options for devicebased therapy and whether decision support tools can streamline that process.42 several possible mechanisms may explain the increasing proportion of crtp devices among crt implants over the study period. for example, changes in patient and provider preferences, population demographics, and evidence in favor of ablate and pace strategies may contribute. in our sample, 32% of crtp recipients had av node ablation on the day of implant or in the 3 months after. because providers and patients appear to favor crtp at older ages, demographic changes are relevant. early signs of crt benefit in patients with atrial fibrillation began to appear in the literature43, 44 before they were established by the blockhf trial in 2013.45 in addition, there is increasing recognition of pacingrelated cardiomyopathy, which generally responds well to upgrade to crtp.46 although our sample was restricted to de novo implants, increasing recognition of risk factors for likely future pacingrelated deterioration may create a pool of patients who become eligible for crtp implant but likely not crtd. finally, federal audits of icd implants were launched in 2012 to investigate icd implants in patients excluded in the 40 days after acute myocardial infarction and 3 months following percutaneous coronary intervention. the period of inquiry extended back to 2003 and although icds were the subject of this initial inquiry, dualchamber pacemaker indications are also being actively scrutinized, and pci is likely to receive similar attention.47 increased scrutiny on these implants may have encouraged providers to adopt more conservative devicebased therapies. we hypothesized that there would be geographic variation in crtp versus crtd utilization, as significant variability in practice patterns have been identified for other cardiac interventions. previous studies have found wide variation in icd implant rates by patient, hospital, and region.48, 49, 50, 51 otherwise eligible patients who are older, african american, and admitted to smaller hospitals are less likely to receive an icd, while larger, academic hospitals with the capability to perform other cardiac interventions are more likely to implant icd devices. in keeping with these results, our study found substantial variation in implant rates by geography, computed as the fraction of each device type out of the total share of devices. the patterns of differences across states adjusted for age, sex, and national time trends were not changing appreciably over time in all but a handful of states. thus, how crt decisions are made locally and what drives this regional variability requires further investigation. optimal medical therapy reported in registry studies is much higher than what we find here.52, 53 one study found 70% of patients without contraindications were prescribed a blocker and angiotensinconverting enzyme inhibitors / angiotensin receptor blockers following implantation with a crtd device. back calculating from the study, we can see that in their cohort of 45 392 patients receiving a crtd implant, 96% to 99% of patients had no contraindication. these studies both used reported discharge on optimal medical therapy from the national cardiovascular data registry icd registry. a more recent study examined guidelinerecommended medical therapy prior to primary prevention icd implant using part d claims linked to the national cardiovascular data registry icd registry.28 like that study, our claimsbased analysis reflects prescription fills rather than prescriptions, and therefore may reflect actual medication use. the roth study distinguished between any claim for guidelinedirected therapy (61% of patients) and an adequate supply (28% of patients), which they defined as 80% coverage of the 90 days leading up to implant. our definition of drug use requires 90 days supply in the 6 months prior to implant, which is somewhere between these 2 definitions. further work to reconcile these apparent differences will be important for characterizing whether, in fact, these patients are receiving guidelinebased care prior to and concurrent with their device therapy. our study has several potential limitations, arising in part from our methodological strengths, including the broadly representative population of commercially insured and medicare enrollees. the icd registry does not include crtp devices, so we are uniquely positioned to study crtp, crtd, and icd devices. however, in this data source, diagnosis and procedure codes may not accurately reflect a patient 's clinical characteristics. in particular, our inability to measure left ventricular ejection fraction and qrs morphology and duration are significant limitations of our claimsbased analysis. registrybased studies contain much more comprehensive information about each patient and device implant, but lack followup information about outcomes and subsequent healthcare use. we are also limited in our ability to adjust for additional patient characteristics, such as comorbidities, in models for geographic and temporal variation. even in such a large database as this, the total number of patients implanted with crtp devices was quite small. for example, in a mediumsized state like maryland (153 average annual total implants), there are only 1 or 2 crtp implants in each sexage group each year, sometimes none. following publication of the blockhf trial in 2013,45 it is reasonable to expect increasing use of crtp devices for patients with av block who do not have indications for icd therapy. our study period precedes publication of this trial, but we have no left ventricular ejection fraction measurements or data beyond 2013, so we can not directly address the question of changing practice in response to blockhf. although large randomized trials comparing crtp and crtd devices are unlikely, research to identify variation in patient responses to crt therapy can leverage geographic variation in practice to perform quasiexperimental analyses and identify subgroups of patients most likely to benefit from crtp devices. furthermore, recent developments in behavioral economics provide examples of effective means to improve adherence with guidelinerecommended practice, such as a sufficient course of optimal medical therapy prior to device implant. for example, a recent study found that inappropriate antibiotic prescribing could be reduced by regular emails to physicians describing their performance as top performing or not.54 electronic health records could facilitate these interventions by enabling automatic report generation for physicians and patients. in sum, our large, national study of cardiac implantable electric device use in a commercially insured population demonstrates that, among crt recipients, crtd is overwhelmingly selected, though a small increase in crtp use has been seen in recent years, especially among older patients and women. in future work, we will describe outcomes of device therapy that are measurable in claims, particularly subsequent hospitalbased healthcare use and the frequency with which each device type is upgraded, downgraded, or removed (for example, icd downgrades to pacemakers).55 in ongoing work, we are studying trends in crtp use among the feeforservice medicare population. the longitudinal dynamic treatment regimens for patients with implanted devices have not been previously studied. we also plan to fit and validate a model of eligibility in a rich data source such as a registry and apply it to the claims data using observables and statistical methods for incorporating external data.56 this will clarify the treatment selection patterns within the larger population of potentially eligible recipients. hatfield and normand received funding from us food and drug administration grant (u01 fd00449301). kramer is supported by a career development award from the paul beeson scholars program (k23ag045963). each line shows the modeled probability of receiving a particular device (conditional on receiving any new implant) by year (x axis), age (quintile, each line is a different quintile), sex (men in the top row, women in the bottom row) from a saturated multinomial regression model. crtd indicates cardiac resynchronization therapy with icd backup ; crtp, cardiac resynchronization therapy pacemaker, icd, implantable cardioverter defibrillation. | backgroundcardiac implantable electric devices are commonly used to treat heart failure. little is known about temporal and geographic variation in use of cardiac resynchronization therapy (crt) devices in usual care settings.methods and resultswe identified new crt with pacemaker (crtp) or defibrillator generators (crtd) implanted between 2008 and 2013 in the united states from a commercial claims database. for each implant, we characterized prior medication use, comorbidities, and geography. among 17 780 patients with crt devices (median age 69, 31% women), crtps were a small and increasing share of crt devices, growing from 12% to 20% in this study period. compared to crtd recipients, crtp recipients were older (median age 76 versus 67), and more likely to be female (40% versus 30%). preimplant use of blockers and angiotensinconverting enzyme inhibitors or angiotensin ii receptor blockers was low in both crtd (46%) and crtp (31%) patients. the fraction of crtp devices among all new implants varied widely across states. compared to the increasing national trend, the share of crtp implants was relatively increasing in kansas and relatively decreasing in minnesota and oregon.conclusionsin this large, contemporary heart failure population, crtd use dwarfed crtp, though the latter nearly doubled over 6 years. practice patterns vary substantially across states and over time. medical therapy appears suboptimal in realworld practice. |
lateral elbow pain is common with a population prevalence of 1%3%.1 the peak incidence occurs at around 4050 years of age group and in women aged 4246 years, incidence increases to 10%.23 in the uk, the netherlands, and scandinavia, the annual incidence of lateral elbow pain in general practice is 4 - 7/1000 population.345 the term was coined in 1883 as lawn - tennis elbow.6 inflammatory cells are not found in the tendon tissues ; therefore, nirschl. coined the term angiofibroblastic tendinosis to describe this condition.78 its common name, tennis elbow, is somewhat of a misnomer because the condition is often work related and occurs in athletes and nonathletes alike.9 the condition starts as a micro - tear in extensor carpi radialis brevis.10 acute onset of symptoms occurs more often in young athletes ; chronic, recalcitrant symptoms typically occur in older patients.9 conservative measures are undertaken initially because symptoms in most patients improve with time and rest. those who fail to respond to conservative therapy are considered for surgical treatment.11 the goals of nonoperative treatment are to revitalize the unhealthy pain producing tendinosis tissue.10 nonoperative treatment includes rehabilitative resistance exercise with progression of the exercise program,10 corticosteroid injection,12 autologous blood injection,13 extracorporeal shock wave therapy,14 botulinum toxin injection,15 and hyaluronic acid with chondroitin sulfate injection.16 platelets, an important reservoir of growth factors in the body, play an important role in many processes such as coagulation, immune response, angiogenesis, and the healing of damaged tissues. numerous proteins are contained in the alpha - granules of platelets : platelet - derived growth factor, transforming growth factor, platelet factor interleukin, platelet - derived angiogenesis factor, vascular endothelial growth factor, epidermal growth factor, insulin - like growth factor, and fibronectin.17 single or multiple injections of platelet - rich plasma (prp) have been shown to be of significance in the management of tennis elbow. randomized controlled trial comparing efficacy of prp with other modalities will validate the usefulness of prp in lateral epicondylitis (le).18 the study was conducted from august 2013 to may 2015 at our institution after getting ethical committee clearance. patients in the age group of 2050 years of either sex who were clinically having symptoms suggestive of tennis elbow were included in the study. patients who had received any previous treatment in the form of local injections of steroid were excluded from the study. patients who were suffering from symptoms of pain around the elbow due to other reasons such as inflammatory arthropathies, posterior interosseous nerve syndrome, osteochondritis dissecans of elbow, elbow pain referred from cervical spine, or ipsilateral shoulder were excluded from the study. all patients were subjected to routine blood investigation including the markers for inflammatory arthropathy and radiographic examinations of cervical spine, ipsilateral shoulder, and the elbow under study. patients clinically diagnosed to have tennis elbow and after excluding all other causes of elbow pain were subjected to ultrasonographic examination of the elbow under study to confirm the diagnosis of tennis elbow. the findings involve hypoechoic signal from the extensor tendons suggestive of edema of the extensor tendon in all cases. first group of patients was given autologous prp, the second group was given steroid triamcinolone, and the third group of patients was given normal saline injection as the placebo. the results were recorded by visual analog scale (vas) score and facial pain scale (fps). the scores were recorded in the prepared pro forma on the day of injection before giving the injection, then after 12 weeks and after 24 weeks. patients were advised for rest during initial 2 weeks in the form of refraining from strenuous activities by the extremity under study after the injection. bilateral cases were injected simultaneously, and the postinjection protocol was same. to prepare prp around 15 ml of patient 's blood was obtained by drawing blood through a scalp vein catheter to avoid turbulence while drawing the blood. the lower halves of the supernatant from all the three tubes are transferred into another plain tube for the second spin. one milliliter of lower half was taken into a 1 ml syringe having 0.1 ml of calcium chloride. at the end of preparation of prp the samples were sent for platelet count and the count compared with patient 's platelet count. the second group was given 1 ml of triamcinolone, and the third group was given 1 ml of normal saline. the site of injection was 5 mm distal to the lateral epicondyle in the extensor tendons, particularly extensor carpi radialis brevis tendon [figure 1 ]. one milliliter of 2% lignocaine with adrenaline was injected at the injection site after giving test dose. if any resistance was felt during the injection, the needle is withdrawn a bit and again injected. eighty patients including 49 females and 31 males in the age group of 2040 years with ninety elbows were included in the study. out of the 49 females with 53 elbows, 18 received prp, 18 received triamcinolone, and 17 received normal saline. out of the 31 males with 37 elbows under study, 12 received prp, 12 received triamcinolone, and 13 received normal saline. for statistical analysis, 1, no. 2, and no. 3 to represent prp, triamcinolone, and normal saline. kruskal wallis test was used to compare the result among all the three groups as shown in tables 1 and 2. if we analyze the mean ranks, we can see that the mean ranks in all the three groups are similar on the day of injection for both vas and fps scores. at 12-week followup, the vas score mean ranks in prp group and triamcinolone group improved by 33% and 28.5%, respectively [figures 2 and 3 ]. similarly, at 12 weeks, the improvements in fps scores were 34% and 23%, respectively, in prp and triamcinolone group. at 24-week followup, the vas score mean ranks in prp group and triamcinolone group improved by 49% and 5%, respectively. similarly, at 24 weeks, the improvements in fps scores were 50% and 3%, respectively, in prp and triamcinolone group. the normal saline group showed worsening of results in vas score and in fps score at 12 weeks and 24 weeks. hence, the pain scores improved in both prp and triamcinolone group but worsened in the normal saline group at both 12- and 24-week followup. at 24 weeks, the improvement in pain was more with prp than with triamcinolone where most patients had a relapse of pain. kruskal - wallis test to compare the result among all the three groups kruskal - wallis test to compare the result among all the three groups a line diagram showing mean visual analog scale for different time points between groups a line diagram showing mean facial pain scale for different time points between groups we calculated the p values in table 2 which showed that on the day of injection, it is not significant for both vas score (p = 0.517) and fps score (p = 0.960). hence, on the day of injection, there was no significant difference in pain relief. however, at both 12 weeks and 24 weeks as both prp and triamcinolone showed improvement in mean ranks, we conclude that prp and triamcinolone showed statistically significant improvement in pain scores than placebo at 12 weeks and 24 weeks. whitney u test was used to compare vas and fps scores between prp and triamcinolone. the mean ranks of both vas and fps scores in prp group showed 1.138% and 4% improvement at 12 weeks, and at 24 weeks, 25% and 28% improvement, respectively. the differences in result were not significant at 12 weeks (p = 0.955 and 0.496), but at 24 weeks, significant (p < 0.001) for fps scores and for vas scores (p = 0.001). mann - whitney u - test ranks of visual analog scale and facial pain scale between platelet - rich plasma as one and triamcinolone as two mann - whitney u - test statistics to compare visual analogue scale and facial pain scale between platelet - rich plasma and triamcinolone (grouping variable : injection given) similarly, when we compared the vas and fps scores between prp and placebo with mann whitney u test [tables 5 and 6 ], we found that the mean ranks of both vas and fps scores in prp group showed 47.4% and 47.19% improvement at 12 weeks, and at 24 weeks, 48.17% and 46.86% improvement, respectively. however, the mean ranks of both vas and fps scores in normal saline group showed worsening at 12 weeks and at 24 weeks. the differences in result were significant (p < 0.001) at both 12 weeks and 24 weeks for both vas and fps scores. mann - whitney u - test ranks to compare visual analogue scale and facial pain scale between platelet - rich plasma as one and placebo as three mann - whitney u - test statistics to compare visual analogue scale and facial pain scale between platelet - rich plasma and placebo (grouping variable : injection given) table 7 shows p values among different groups on comparison. summary of p values among different pairs of groups post hoc analysis was done for the study as shown in figures 2 and 3, which showed a significant difference between vas and fps scores of prp group than triamcinolone and normal saline group at 24 weeks, but at 12 weeks, the scores were not significant between prp and triamcinolone groups. consort diagram (flow chart) of the study no complications were found in the group receiving prp and placebo. however, out of thirty elbows given triamcinolone hypopigmentation at the injection site was found in 13 patients with associated subdermal atrophy in 3 patients [figure 5 ]. le is the most common cause of lateral elbow pain in adults that is encountered in day - to - day practice by most orthopedic surgeons. although it is typically a self - limiting process, there are many nonsurgical and surgical treatment options available if le becomes chronic and continues to cause pain.9 with evolution of various nonsurgical options available for treatment of tennis elbow, prp injection has been shown to be a promising option in various multicenter studies. however, there are conflicting reports that state that prp might not be as effective as predicted. krogh. in their study concluded that at 3-month followup, there was no significant reduction in pain in any of the three groups. the injection of prp was the most painful.19 however, brkljac. in their study concluded that an injection of prp improves pain and function in patients suffering from le where conservative management has failed.20 similarly, raeissadat. in their study found that prp and autologous whole blood injections are both effective methods to treat chronic le and their efficacy persisted during long term followup. prp was not superior to awb in long term followup.21 peerbooms. in their study after 1-year followup found that treatment of patients with chronic le with prp reduces pain and significantly increases function, exceeding the effect of corticosteroid injection.22 gosens. in their study concluded that treatment of patients with chronic le with prp reduces pain and increases function significantly, exceeding the effect of corticosteroid injection even after a followup of 2 years. in their study concluded that prp injection can improve pain and lower the risk of complications, whereas autologous blood injection can improve pain, disabilities scores, and pressure pain threshold but has a higher risk of complications.24 in our study, we found that at 12-week followup, the pain relief was better in both prp and corticosteroid injection groups as compared with the normal saline group, but at 24-week followup, the pain relief was maintained better with prp than corticosteroid. patients who had received steroid were asymptomatic at 3-month followup, but at 6-month followup, 33.33% patients complained of a recurrence of pain symptoms that was more than 50% of the initial vas and fps score. in prp group, only 13.33% of patients were symptomatic with vas score and fps score more than 50% of the initial value. krogh. in their study concluded that the injection of prp was the most painful.19 mishra and pavelko in their study concluded that treatment of patients with chronic elbow tendinosis with buffered prp reduced pain significantly. they initially injected bupivacaine with epinephrine into the skin and subcutaneous tissue as a local field block and then 0.5 ml directly into the area of maximum tenderness. then, 23 ml prp was injected using a 22-gauge needle into the common extensor tendon using a peppering technique. this technique involved a single skin portal and then five penetrations of the tendon.25 in our study, we used 2% xylocaine local infiltration before injection in all three groups and injection was given at the common extensor tendon using peppering technique. concluded that prp appeared to enable biological healing of the lesion, whereas corticosteroid appeared to provide short term, symptomatic relief but resulted in tendon degeneration.26 park. in their study concluded that 1.3%4% people develop hypopigmentation which develops over the initial 14 months after the injection and resolves spontaneously over 630 months. subcutaneous fat atrophy is known to last for 612 months after corticosteroid injection, and it is known to be reversible and resolved within 1 year.27 our study found that 13 patients out of the thirty patients who received corticosteroid suffered from hypopigmentation at injection site, and three patients suffered from subdermal atrophy. the limitation of the study is that sample size needed for the study was not calculated. the efficacy of single injection of prp to relieve the pain of tennis elbow is better than triamcinolone or placebo over a short term followup period. however, still more studies are required at different centers by different research groups to establish the efficacy of prp over long term followup period, and multicenter randomized controlled trial would further strengthen evidence - based practice in treatment of le or tennis elbow. | background : lateral elbow pain is common with a population prevalence of 1%3%. the study was a comparative trial to validate the efficacy of single injection of platelet - rich plasma (prp) for tennis elbow as compared with single injections of triamcinolone and placebo (normal saline) over a short term period.materials and methods : comparative trial with 3- and 6-month followup evaluated with visual analog scale (vas) and facial pain scale (fps). our study included a total of eighty patients with unilateral or bilateral tennis elbows. the study population included patients between 20 and 40 years age group belonging to either sex with seventy unilateral and ten bilateral affections for more than 3-month duration. patients suffering from elbow pain due to other problems or those who have received any form of injection were excluded from the study. one milliliter of 2% xylocaine injection was given before injecting the proposed formulation under trial. vas and fps were used for scoring pain. kruskal wallis test and mann whitney u - tests were used for statistical analyses at 12 and 24 weeks.results:overall, 49 females and 31 males were included with thirty elbows in each group. both the prp and triamcinolone groups had better pain relief at 3 and 6 months as compared to normal saline group (p < 0.05), but at 6 months followup, the prp group had statistically significant better pain relief than triamcinolone group. in the triamcinolone group, 13 patients had injection site hypopigmentation and 3 patients had subdermal atrophy.conclusion:over a short term period, prp gives better pain relief than triamcinolone or normal saline in tennis elbow which needs to be validated over long term period by further studies. |
arsenic is recognized as one of the most serious inorganic contaminants in soil and groundwater worldwide, with significant public health implications. arsenic often makes its way into soil and water courses by the natural processes of weathering and dissolution of minerals such as arsenian pyrite, fe(as, s)2, and arsenopyrite, feass. anthropogenic activities, particularly mineral extraction and processing can also introduce arsenic - rich effluents into the environment if not carefully monitored and controlled. the effects of arsenic on human health are highly detrimental, with arsenic poisoning being linked to neurological disorders, dermatological and gastrointestinal problems, and it is also a known carcinogen. arsenic can exist in a range of oxidation states from 3 to + 5, although in aqueous solutions it is most commonly found as as(iii) or as(v) oxyacids. as(iii) is both more toxic (2065 times) and more mobile (being able to travel five to six times faster) than as(v) and is one of the main toxic species in natural waters. analyses of hydrothermal fluids show that as is transported mainly as as(iii), and the uptake of as(iii) from aqueous solutions is reported to occur via neutral molecules, which suggest that arsenous acid (as(oh)3) or related species could be the common form of arsenic in contaminated waters. an understanding of the geochemistry of as(oh)3 in low temperature anoxic sedimentary environments is therefore crucial to the development of safe drinking water and food supplies in many countries. of the processes influencing arsenite mobility, reactive mineral water interfaces exert control on the bioavailability of contaminant arsenic species in natural aqueous systems. the adsorption of arsenic species onto mineral surfaces strongly affects their concentrations in aqueous environments. in recent years, iron sulfide mackinawite (fes), has attracted significant interests for environmental remediation due to its natural abundance and high treatment efficiency in anoxic environments. fes is a layered iron sulfide mineral that crystallizes in the tetragonal structure shown in figure 1, and it is known to be the first crystalline ferrous sulfide phase to form under sulfate reducing conditions. fes is a nontoxic mineral and a precursor to other stable iron sulfide minerals, such as greigite and pyrite. like other 2d layered materials, for example, mos2, fes possesses a high specific surface area and reactive surfaces that are ideal for the uptake of aqueous contaminants. furthermore, fes nanoparticles can be synthesized easily, which makes it a promising candidate for the treatment of groundwater and soil contaminated with arsenic, selenium, and heavy metals, including mercury, and chromium. layered structure of mackinawite, with the tetragonal unit cell highlighted by dash lines. owing to its unique structure and surface chemical properties, mackinawite has been reported to be very effective in immobilizing divalent metals such as mg, ca, mn, ni, cd, and hg from aqueous solutions. fes has also been shown to have a high removal capacity for inorganic oxyanions, including as under anoxic conditions. it has been reported that mackinawite suspensions and synthetic nanoparticulate mackinawite can effectively remove as(iii) at a ph range of 510. a comparative study of the removal capacity of as(iii) and as(v) in aqueous solutions by goethite, lepidocrocite, mackinawite, and pyrite, by farquhar. has shown that mackinawite was more efficient than iron - oxide phases or pyrite. their results suggested that the arsenic uptake by freshly prepared mackinawite was due to outer - sphere complexation, but fundamental aspects of this process, including the registries of the adsorption complexes, adsorption energies, and structural parameters remain unclear. such information can not be obtained directly from experimental work and the underlying physical driving forces that control the reactivity of arsenic species with the fes surfaces remain poorly understood. the diverse interactions and reactions occurring at the mineral however, molecular simulations provide an alternative way to gain fundamental insight into these processes. calculations based on the density functional theory (dft) have become indispensable in unravelling the interactions of organic and inorganic molecules with solid surfaces as they are capable of accurately predicting lowest - energy adsorption geometries and identifying charge transfer and other electronic effects. for example, dft - based studies have been instrumental in elucidating the complex adsorption processes of arsenic and arsenate on iron oxide mineral surfaces. goffinet and mason employed spin - polarized dft calculations to study the inner - sphere as(iii) complexes on hydrated -fe2o3(0001) surface models. blanchard and co - workers have modeled arsenate adsorption on the hydrated (1012) hematite surface, investigating charged inner- and outer - sphere complexes using dft calculations. to date, no systematic theoretical study has been conducted to investigate the detailed adsorption mechanism of arsenous acid at the water, the structures and properties of the adsorption complexes of as(oh)3 on hydrated mackinawite (fes) surfaces were studied using dispersion - corrected density functional theory calculations (dft - d2). the energetically preferred as(oh)3 surface complexes on the hydrated (001), (011), and (111) surfaces of mackinawite have been identified. detailed structural analysis of the adsorption complexes and insight into the nature of adsorption on the different surfaces was determined via analysis of projected density of states and differential charge density iso - surfaces. vibrational frequency assignment of the different identified adsorption complexes of as(oh)3 was carried out, which will be useful for comparison with any future experimental studies. the calculations were carried out using the vasp code, which employs a basis set of plane - waves to solve the kohn sham (ks) equations of the density functional theory (dft) in a periodic system. long - range dispersion forces were accounted for in our calculations using the grimme dft - d2 method, which is essential for the accurate description of the fes interlayer interactions, as well as the interactions between the as(oh)3 molecule and the water the d2 method was used in this study to remain consistent with previous work and to ensure that direct comparison could be made with our earlier studies. however, we have carried out a number of test calculations using the dft - d3 method, as mentioned in the text where relevant, but no significant differences between the two methods were observed. the generalized gradient approximation (gga), with the pw91 functional was used to calculate the total free energies. the interactions between the valence electrons and the cores were described with the projected augmented wave (paw) method in the implementation of kresse and joubert. the on - site potential, gga+u, was not employed for these calculations as previous studies on fes using vasp have shown that the extra localization of the d - electrons through the inclusion of a + u correction term provides inadequate structural optimizations. an energy cutoff of 400 ev for the plane - wave basis set was tested to be sufficient to converge the total energy of mackinawite to within 0.0001 ev and the brillouin zone was sampled using 11 11 7 and 5 5 1 monkhorst pack k - points mesh for bulk and surface calculations, respectively, which ensures electronic and ionic convergence. geometry optimizations were performed using the conjugate gradient minimization algorithm until the magnitude of the residual hellman the bulk fes was modeled in the tetragonal structure (figure 1). from a full geometry optimization, the equilibrium lattice parameters were predicted to be a = 3.587, c = 4.908, and c / a = 1.368, which agree well with those measured experimentally (a = 3.674, c = 5.033, and c / a = 1.370). similar results were obtained within the dft - d3 scheme, which predicted the lattice parameters to be a = 3.590, c = 4.992, and c / a = 1.390. from the fully relaxed bulk structure, we created the (001), (011), and (111) surfaces of fes, which are the commonly observed facets in mackinawite nanoparticles. the surfaces were created using the metadise code, which generates nonpolar supercells, avoiding dipole moments perpendicular to the surface plane, as is required for reliable and realistic surface calculations. for each surface, a minimum slab thicknesses of 10 was used in each simulation cell, and a vacuum region of 15 was tested to be sufficient to avoid interactions between periodic slabs. the converged slab thickness used to model the (001), (011), and (111) surfaces because the processes take place in an aqueous environment, the fes surfaces were hydrated through associative adsorption of a monolayer of water, to provide a realistic picture of the as(oh)3 complexation in natural aqueous systems at the mackinawite water interface. in an earlier study, we showed that dissociative water adsorption did not occur spontaneously at fes surfaces. we considered that a monolayer of water was obtained when all surface cations / anions had been terminated by water. the hydrated (001), (011), and (111) surfaces are modeled by large slabs constructed as (3 3)9water, (4 2)8water, and (3 2)6water supercells, respectively. these simulation supercells are large enough to minimize lateral interaction between the as(oh)3 molecules in neighboring image cells. different binding modes of the as(oh)3 molecule were considered, for example, monodentate or bidentate adsorption configurations, in order to obtain the lowest - energy adsorption complexes. the adsorption energy (eads) of the as(oh)3 on the hydrated fes surfaces was calculated as follows:1where ewater surf represents the total energy of the relevant hydrated fes substrate, and eas(oh)3 is the energy of the free as(oh)3 molecule. differences in the adsorption energies reflect trends in surface reactivity, thus eads is useful for characterizing activity trends and relative energetics. a bader population analysis was carried out for all the as(oh)3water fes complexes, using the code developed by henkelman and co - workers in order to quantify any charge transfer between the substrate surfaces and the adsorbate molecule. vibrational frequency assignment of the as o and o h bond stretching modes was performed within the framework of the self - consistent density functional perturbation theory. prior to studying the adsorption and surface reactions of as(oh)3, we have characterized the interaction of water with the (001), (011), and (111) surfaces of fes and how hydration affects their relative stabilities. shown in figure 2 are the optimized structures of the hydrated (001), (011), and (111) surfaces. the relaxed surface energies (r) of the pure symmetric stoichiometric slabs were calculated using the equation:2where eslabrelaxed and eslabunrelaxed are the energies of the relaxed and unrelaxed slabs, respectively, nebulk is the energy of an equal number (n) of bulk fes units, and a is the area of one side of the slab. considering that the adsorption of water on the fes surfaces may affect their stability, we have also calculated the surface energies of the surfaces after water adsorption using eqs 3.3where eslab + waterrelaxed is the energy of the surface with adsorbed water molecules and newater is the energy of an equivalent number of free water molecules. side view of the geometry - optimized structures of hydrated fes (a) (001), (b) (011), and (111) surfaces. (color scheme : fe = gray, s = yellow, o = red, and h = white). the calculated surface energies of different surfaces (pristine and hydrated) as listed in table 1, show that the order of increasing surface energies, and therefore decreasing stability, before and after hydration is (001) < (011) < (111). all the fes surfaces were stabilized through hydration, as is perhaps to be expected because the adsorbed water molecules stabilize the low - coordinated surface atoms. at the fes(001) surface, we found that the water molecules were only physisorbed with the hydrogen atoms pointing toward the terminating surface sulfur ions (figure 2a), similar to results obtained from previous dft, and molecular dynamics (md) simulations of the structure and dynamics of water at the fes(001) surface. the shortest h s distance is calculated at 2.319, which is larger than the typical hydrogen - bond length in water of 1.97, and therefore suggests that dispersion forces may play an important role in stabilizing the water molecule on the fes(001) surface. in a previous study, we showed that the dispersion interactions contribute approximately 87% of the total adsorption energy of water on the fes(001). the average hydrogen to oxygen (h --- o) interatomic distance between the water molecules on the (001) surface is calculated at 1.824. compared to the (001) surface, the water molecules on the (011) surface are oriented in such a way that now the o atoms are closest to the surface fe sites (average fe o = 2.253) as shown in figure 2b. the hydrogen atoms are oriented toward the sulfur ions in the next fes layer at an average distance of 2.703, which is larger than the average fe o bond length of 2.253 and therefore suggests that the major interactions between the adsorbing water molecules and the (011) surface is through the interaction of their oxygen ions with surface fe ions. in the case of the water fes(111) complex (figure 2c), the water molecules are located above the bridge sites between adjacent fe ions (average fe o = 2.205). the hydrogen atoms are oriented toward the sulfur ions in the next fes layer at an average distance of 2.043, compared to 2.703 at the fes(011) surface, which indicates stronger hydrogen - bonding at the fes(111) surface than at the fes(011). generally, the fes surfaces were found to undergo modest relaxations relative to the bulk interlayer spacings upon hydration, where the topmost three percentage relaxations of the interlayer spacings are calculated to be + 6.5%, + 3.3%, and 3.4% for the (001), 24.1%, + 10.9%, and 2.3% for the (011), and + 29.3%, + 12.1%, and 6.6% for the (111). the multilayer relaxations for the hydrated surfaces were calculated as the percentage difference in the surface interlayer spacing, dij - hydrated, from the layer spacing of the same orientation in the geometry of the unrelaxed surface structure, dij - unrelaxed, created from the equilibrium bulk material. in these simulations, since the models are constructed from the optimized bulk structure, the required surface layer spacing is given by the spacing of the unrelaxed bulk - terminated slab structure.4 within this definition, negative values correspond to inward relaxation (contraction) and positive values denote outward relaxation (dilation) of the interlayer spacings. arsenous acid (as(oh)3) exists in two conformations in the gas phase with either c1 or c3 symmetry. the optimized geometries of the c1 and c3 conformations are shown in figure 3 and the calculated interatomic bond distances and bond angles along with earlier theoretical results are listed in table 2. from our geometry optimization calculations, we found that the c1 symmetry is 0.03 ev more stable than the c3 symmetry, in agreement with earlier theoretical results of ramrez - sols. and tossell. we show from climbing - image nudged elastic band (cneb) calculations that the c1 conformation has to overcome an activation barrier of 0.34 ev to transform to the higher - energy c3 conformation. the three as o bond distances of the c1 and the c3 conformers do not differ significantly, calculated to be 1.798, 1.801, and 1.811 for the c1 symmetry and 1.810, 1.811, and 1.813 for the c3 symmetry. our calculated bond distances (as o and o h) and angles (o as o and as o h) show good agreement with earlier theoretical results and with those obtained from x - ray absorption and exafs analysis. in our study, we have explored several possible adsorption structures including monodentate or bidentate binding geometries on the different hydrated fes surfaces. (color scheme : as = green, o = red and h = white). several possible modes of adsorption sites and configurations were studied for as(oh)3 adsorption at the water - fes(001) interfaces but only the lowest - energy structure (denoted as up outer) is shown in figure 4a (the remaining conformations and calculated binding energies are given in the supporting information (si) figure s1 and table s1, respectively). in the lowest - energy as up outer complex, the as(oh)3 is adsorbed outside the water layer with the as atom pointing upward, while the hydroxyl groups form hydrogen - bonded interactions with the surface - bound water molecules. the adsorption energy of this structure is 1.14 ev, which is 0.2 ev more favorable than the as up inner - sphere complex (si figure s1b), in which the as(oh)3 molecule is adsorbed within the water layer by displacing some of the water molecules during the adsorption process. in the case of as down configurations, the inner - sphere complex (si figure s1c) is found to be energetically more favorable than the outer - sphere complex (si figure s1d) by 0.23 ev. in all adsorption geometries, we observe only small elongations in the as o and o h bonds (table 3 and si table s1) compared to the structural data of the free as(oh)3 molecule (table 2), which may be attributed to the hydrogen - bonded interactions with the surface water molecules. in the lowest - energy outer - sphere as up complex, the three hydrogen atoms of the as(oh)3 molecule interact with three different surface water molecules at hmol owat distances of 1.702, 1.747, and 1.960. we also observe hydrogen - bonded interactions between hydrogen atoms of two water molecules and o atoms of as(oh)3 at hwat omol distances of 1.639 and 1.783. the hmol owat and hwat omol bond lengths calculated in the present study compare closely with the typical hydrogen - bond length in water of 1.97, which therefore suggests that hydrogen - bonded interactions contribute significantly to the stabilization of as(oh)3 at the water lowest - energy adsorption complexes of as(oh)3 at the (a) (001), (b) (011), and (c) (111) water fes interfaces, in side (top) and top (bottom) views. (color scheme : fe = grey, s = yellow, as = green, owater = red, oas(iii) = pink, and h = white). as with the water fes(001) surface, we have considered different possible adsorption structures for as(oh)3 on the water some of the water molecules were displaced from the surface by the as(oh)3, enabling direct stronger interactions with the surface cations sites. shown in figure 4b is the lowest - energy adsorption configuration identified (the remaining conformations are given in the si figure s2, whereas the calculated adsorption energies and optimized structural parameters are reported in table 3 and si table s2. fes(011) interface is calculated to be a bidentate fe aso fe complex (figure 4b), wherein the as(oh)3 molecule interacts with the surface fe atoms via the as and one o atom. the adsorption energy of this structure is calculated at 1.82 ev, compared to the adsorption energy of 1.43 ev for the monodentate fe o complex (si figure s2b), wherein the as(oh)3 molecule interacts with the surface fe atoms via only one o atom, 1.06 ev for the monodentate fe as complex (si figure s2c), wherein the as(oh)3 molecule interacts with the surface fe atoms via the as atom, and 0.89 ev for the as bridge complex (si figure s2d), wherein the as(oh)3 is adsorbed in a bridging position between the fes layers and stabilized through hydrogen - bonded interactions with the surface water molecules. s interatomic distances are calculated in the range of 2.9604.147, whereas the as fe are calculated in the range of 2.2693.787 (table 3 and si table s2). similar interatomic distances were reported from spectroscopic and extended x - ray absorption fine structure (exafs) data fitting of as(iii) sorbed on mackinawite (as s = 3.1 and as fe = 3.43.5) in aqueous solution. again, we have explored several possible sites and modes of adsorption of as(oh)3 on the water fes(011) surface, some of the water molecules were displaced by as(oh)3 during the adsorption process, which allows for the formation of direct interactions with the surface cation sites. displayed in figure 4c is the lowest - energy adsorption complex identified (the remaining conformations are given in the si figure s3).the lowest - energy as(oh)3 adsorption configuration at the water fes(111) interface was calculated to be the fe o fe complex (figure 4c), wherein the as(oh)3 molecule adsorbs at the bridge site between adjacent surface fe atoms via one o atom. the adsorption energy of this structure (fe o fe complex) is calculated at 1.76 ev, whereas the energies of the other stable adsorption configurations are calculated at 1.57 ev for the fe aso fe complex (si figure s3b), 1.17 ev for the fe as complex (si figure s3c), and 0.86 ev for the hwat oh ssurf complex (si figure s3d). in the lowest - energy fe o fe complex, the bridging o fe distances were calculated at 2.159 and 2.138, and the average values are reported in table 3. the as s and as fe interatomic distances are converged at 3.675 and 3.365, respectively. similar interatomic distances were calculated for the as atom interacting with the surface s and fe ions in the other adsorption configurations (si table s3). at all three water fes interfaces, we have observed elongations in the as o bonds in all adsorption complexes (1.7651.946), especially in the complexes in which the o atom interacts directly with the surface fe ions. o h bond elongations were also observed (0.9761.046), which can be attributed to the presence of hydrogen - bonded interactions between the hydrogen atom of as(oh)3 and the o atom of the surface water molecules as reported in table 3 and si tables s1s3. to gain insight into the nature of the interactions between the as(oh)3 molecule and the different hydrated fes surfaces, we have carried out an atom - by - atom projected density of states (pdos) analysis of the free molecule and compared it to those of the adsorbed states. the pdos for the free as(oh)3 molecule is shown in figure 5a1, whereas those for the lowest - energy adsorption configurations at the water fes (001), (011), and (111) interfaces are shown in figures 5b1, c1, and d1, respectively. in the free as(oh)3 pdos, we note that the states around the fermi level are dominated by p - states of as and o, which are associated with the lone pair electron density of the as and o atoms as shown in the highest occupied molecular orbital (homo) in figure 5a2. these orbitals are therefore expected to interact strongly with the orbitals of the surface species during sorption processes at the mineral surfaces. fes (011) and (111) interfaces where the as(oh)3 interacts directly with the surface fe ions, we observe disappearance or reduction of the as - p and o - p states of as(oh)3 around the fermi level, due to their strong hybridization with the interacting surface fe - d states (figures 5c1 and d1). at the water fes(001) interface, however, we only observe a shift toward lower energy levels (figure 5b1), which signifies stabilization of the as(oh)3 via physisorption. the pdos for the interacting surface fe - d - states before and after the adsorption of as(oh)3 at the water fes(011) interface, and for the interacting surface s - p - states at the water we found that the electronic properties of the surfaces were essentially preserved after the adsorption of as(oh)3, with only small shifts in the peak positions and heights, which indicates adsorption induced changes due to the interactions between the as(oh)3 species and the water substrate systems were determined through analyses of the iso - surface plots of the differential charge density, which is obtained by subtracting from the charge density of the total adsorbate substrate complex, the sum of the charge densities of the as(oh)3 molecule and the hydrated fes surface. the atomic positions of the water fes surface and of the as(oh)3 molecule are kept the same as those of the total adsorbate substrate system. in this way, the presentation highlights local electron density rearrangement and bond formation in the as(oh)3water fes complexes. shown in figures 5b2, c2, and d2 are the isosurfaces of the electron density differences due to as(oh)3 adsorption at the water fes (001), (011), and (111) interfaces, respectively. an inspection of the iso - surfaces reveals electron density accumulation within the bonding regions between as(oh)3 and the water fes (011) and (111) interacting surface fe ions, which is consistent with the formation of new bonds. in the case of the as(oh)3water fes (001) complex, we see electron density accumulation between the hydrogen and o atoms indicative of hydrogen - bonded interactions. despite the strong electron density redistribution within the as(oh)3water fes complexes, only little charge transfer occurs from the interacting surface species to the adsorbed as(oh)3 molecule, as revealed from our bader charge population analyses (tables 3 and si tables s1s3). the charge gained by the as(oh)3 in the different adsorption complexes is calculated to be in the range of 0.010.04 e at the water (right) pdos for as(oh)3 in the (a) free state and adsorbed in the lowest - energy geometry at the water (left) the corresponding isosurfaces of the differential charge density, where the purple and orange contours indicate electron density increase and decrease by 0.02 e /, respectively. pdos for the interacting surface fe d - states before and after the adsorption of as(oh)3 at the (a) water fes(011) interface, and (c) for the interacting surface s - p - states at the water fes(001) interface. in order to propose an assignment for the as o and o h stretching vibrational modes of the adsorbed as(oh)3, which can serve as a guide for future experimental identification of the different adsorption complexes of as(oh)3 at the water fes interfaces, we have computed the wavenumbers of the normal modes of all the stable adsorption complexes at the different water fes interfaces (table 4 and si table s4). h stretching vibrational modes for the free as(oh)3 molecule compare closely with experimental data, as shown in table 4, which ensures the reliability and accuracy of our approximate assignments. the three as o stretching vibrational modes for the free as(oh)3 molecule were calculated at 700.8, 639.1, and 638.3, which compares with the experimental values of 710.0, 655.0, and 655.0 cm. h stretching vibrational modes are calculated at 3743.5, 3715.3, and 3674.6 cm which are similar to the o compared to the free as(oh)3 molecule, we observe a reduction in the stretching vibrational modes of the as o bonds upon as(oh)3 adsorption, indicative of weakening of these bonds, in agreement with the elongated as o bonds calculated for the as(oh)3 adsorption complexes at the different water for example, the three stretching as o bands of as(oh)3 adsorbed in the lowest - energy configuration at the water fes(111) surfaces can be assigned at 580.2, 501.5, 488.9 cm and 673.5, 616.5, 456.1 cm, respectively, which are lower than the gas phase stretching as h bands of the adsorbed as(oh)3 compared to the free unbound state (table 4), which can be attributed to the formations of hydrogen - bonded interactions with the oxygen ions of the surface water molecules. the unique information provided by our atomic - level investigations provide fundamental insights into the structure property relationships of fes water fes(001) interface through a network of hydrogen - bond interactions with water molecules at the surface. fes(011) and water fes (111) interfaces, which is characterized by hybridization between the s - p and o - p states of as(oh)3 and the surface fe - d states. our calculated as fe and as s interatomic distances in the lowest - energy adsorption complexes at the various water - mackinawite interfaces (as fe = 2.2693.369 and as s = 3.3823.675) show good agreement with those obtained from k - edge exafs and xanes spectroscopic data (as fe = 3.43.5 and as s = 3.1). the long distances obtained from experiment clearly suggest as interactions via outer sphere complexes at the fes surface. however, from our simulation results, the short as fe distances (2.2172.530) calculated for the fe aso fe and fe as adsorption complexes at the water - fes (011) and (111) interfaces indicate that, depending on the surface structure and composition, inner - sphere complexation with respect to the as atom is also possible at the water - mackinawite interface. future investigations will expand the work presented here to include classical md simulations which will provide a complete description of the dynamic processes occurring at the as(oh)3water fes interfaces. the calculated interatomic distances and adsorption energies from this work will be useful in the derivation of force fields to be employed in the classical md simulations to simulate more complex systems, including single and multiple as(oh)3 species adsorption from an explicit 3-dimensional aqueous environment. | reactive mineral water interfaces exert control on the bioavailability of contaminant arsenic species in natural aqueous systems. however, the ability to accurately predict as surface complexation is limited by the lack of molecular - level understanding of as water mineral interactions. in the present study, we report the structures and properties of the adsorption complexes of arsenous acid (as(oh)3) on hydrated mackinawite (fes) surfaces, obtained from density functional theory (dft) calculations. the fundamental aspects of the adsorption, including the registries of the adsorption complexes, adsorption energies, and structural parameters are presented. the fes surfaces are shown to be stabilized by hydration, as is perhaps to be expected because the adsorbed water molecules stabilize the low - coordinated surface atoms. as(oh)3 adsorbs weakly at the water fes(001) interface through a network of hydrogen - bonded interactions with water molecules on the surface, with the lowest - energy structure calculated to be an as up outer - sphere complex. compared to the water fes(001) interface, stronger adsorption was calculated for as(oh)3 on the water fes(011) and water fes(111) interfaces, characterized by strong hybridization between the s - p and o - p states of as(oh)3 and the surface fe - d states. the as(oh)3 molecule displayed a variety of chemisorption geometries on the water fes(011) and water fes(111) interfaces, where the most stable configuration at the water fes(011) interface is a bidentate fe aso fe complex, but on the water fes(111) interface, a monodentate fe o fe complex was found. detailed information regarding the adsorption mechanisms has been obtained via projected density of states (pdos) and electron density difference iso - surface analyses and vibrational frequency assignments of the adsorbed as(oh)3 molecule. |
the incidence of parenchymal brain metastasis (pbm) from primary systemic cancer has been increasing along with prolonged survival of the patients with cancer. recent report showed that up to 30% of cancer patients can be expected to develop pbm according to the types of primary cancer6,12). a population - based estimate of the incidence proportion of pbm was highest for lung cancer followed by melanoma, renal-, and breast cancer3). however, leptomeningeal carcinomatosis (lmc), which is one of terminal manifestations of central nervous system (cns) metastasis from systemic malignancy, remains incurable without a discernible advance in treatment over the past decades. lmc also has been diagnosed more frequently with the advancement of neuroimaging and prolonged survival of cancer patients8,13,29). among the pathogenesis of lmc, spread of cancer cells followed by surgical resection (sr) has been suggested from several clinical observations of solid tumors including malignant brain tumors2,11,16,22,23,34). although the overall incidence of lmc is difficult to estimate according to pbm status, it is apparent that the primary cancer showing a high incidence of pbm, such as lung cancer, breast cancer or melanoma, develops into a high proportion of lmc among those cancer patients5,35). several reports suggested that the risk for development of lmc may increase in patients with pbm who underwent sr compared with non - surgical treatment, and at the same time, the risk may also be affected by factors such as location of the tumor, treatment modality and the histology of primary cancer21,23,24,30 - 33). however, to our knowledge, neither a controlled - prospective study nor any study which attempted to adjust these variables to evaluate the significant risk factors for lmc has been reported. breast cancer is known to be the second most common cancer associated with pbm and is also, one of the most common primary cancers that causes lmc5,6,38). thus, we retrospectively collected data on patients with pbm from one primary histology of breast cancer. the primary objective of the study was to estimate the incidence of lmc after sr or whole brain radiation therapy (wbrt) as the initial treatment in a cohort of breast cancer patients with pbm. further, we examined if sr confers a higher risk for the development of lmc than wbrt in these patients, and also, analyzed the influence of the above mentioned clinical factors on the development of lmc. from our institutional medical record database, we identified 274 cases of sr or wbrt as the initial treatment for pbm from breast cancer between august, 2001 and december, 2010. eleven breast cancer patients, who received radiosurgery as the initial treatment for their pbm, were not included in this cohort. patients were excluded if they were diagnosed with lmc before the index procedure (12 patients), did not have a mri follow - up at our institution after the index procedure (76 patients), or had incomplete data (3 patients) on entities under study. twenty - seven patients (15%) underwent sr of their tumors, and the remaining 156 patients (85%) received wbrt. among 27 sr group, 11 patients underwent wbrt within one month after sr as an adjuvant treatment, 10 patients received wbrt after local or distant recurrence of their brain metastasis, and the remaining 6 patients were observed without wbrt. patients were followed up with mri every 3 to 6 months, until the patients died or no longer returned to our hospital. we reviewed the following factors from medical records and conventionally categorized them for the analysis : type of index procedure (sr or wbrt), age, time from diagnosis to brain metastasis, volume of tumor, number of brain metastatic lesions (single or multiple), location (supratentorial or infratentorial) of the brain metastatic lesion, adjuvant chemotherapy, karnofsky performance status (kps) scale and systemic disease status (no evidence of disease, stable, or progressing). the primary outcome was the incidence of lmc, as diagnosed by cytological csf analysis or neuroimaging findings such as " clear leptomeningeal enhancement extending into the cerebral sulci or into the folia of the cerebellum, spinal cord, cauda equina or subependyma"13). all patients were evaluated by brain mri at least once for monitoring local or cerebral recurrence. spinal mri and csf cytology was examined only if the patient had symptoms of lmc. time to event (lmc) was defined as the time from the index procedure to the diagnosis of lmc or the last follow - up image. wbrt group patients were censored when they received sr for the management of recurrent tumor to avoid overlapped exposure to higher risk. the differences in the distributions of categorical variables for sr and wbrt groups as well as the incidence of lmc were analyzed using the chi - square test or fisher exact test, as appropriate. a p - value of 0.05 or less was considered statistically significant. for the analysis of the cumulative incidences of lmc, patients were censored at the end of observation or lost to follow - up. in the univariable analysis, the cox proportional hazard model was used to estimate the hazard and corresponding 95% confidence interval (ci) of developing lmc for each of the risk factors. factors that showed at least marginal significance from the univariable analysis (p - value 0.05). the median age of the patients at the time of the index procedure was 52 years (range, 28 - 80 years), and the median time from diagnosis of the primary cancer to brain metastasis was 41 months (range, 1 - 271 months). the median tumor volume was 4.0 cm (range, 0.01 - 105.0 cm), and it was significantly different between two modes of treatment : the median tumor volume of the sr group was 10.9 cm while that of the wbrt group was 3.1 cm (p 4.0 ml), number of lesions (single versus multiple), location of tumor (supratentorial versus infratentorial), adjuvant chemotherapy, kps (70 versus 0.05). the median age of the patients at the time of the index procedure was 52 years (range, 28 - 80 years), and the median time from diagnosis of the primary cancer to brain metastasis was 41 months (range, 1 - 271 months). the median tumor volume was 4.0 cm (range, 0.01 - 105.0 cm), and it was significantly different between two modes of treatment : the median tumor volume of the sr group was 10.9 cm while that of the wbrt group was 3.1 cm (p 4.0 ml), number of lesions (single versus multiple), location of tumor (supratentorial versus infratentorial), adjuvant chemotherapy, kps (70 versus < 70) and systemic disease status (ned or stable versus progressing) were not significantly associated with the incidence of lmc in univariable analysis. among the factors analyzed univariately for the cumulative incidence of lmc, variables with a p - value less than 0.2 were included in the multivariable cox proportional hazards model (table 3). among the preoperative characteristics, the index procedure was still significant and the younger age group gained statistical significance in this multivariable analysis. the administration of adjuvant chemotherapy and ned / stable systemic disease status significantly reduced the cumulative incidence of lmc in multivariable analysis even though these variables were not significant at the p - value 0.05 in the univariable analysis. compared to the wbrt group, the hr of the sr group was 4.03 (95% ci, 1.51 - 10.73 ; p=0.005). the hr of the younger age (< 40) group was 2.89 (95% ci, 1.16 - 7.19 ; p=0.022), adjuvant chemotherapy group decreased the risk for development of lmc at a hr of 0.43 (95% ci, 0.19 - 0.99 ; p=0.048), whereas patients with progressing systemic disease showed increased risk of lmc at a hr of 3.53 compared with ned or stable systemic disease (95% ci, 1.35 - 9.23 ; p=0.010). to evaluate the differential effect of treatment modality on the development of lmc according to the age group, we performed the subgroup analysis presented in table 4. for the different modes of treatment, wbrt group showed lower incidence of lmc in the old age (40) group (fisher 's exact test, p=0.005), whereas the difference according to the index procedure became not significant in the young age (< 40) group due to high incidence of lmc regardless of the index procedures. however, occasionally it occurs in the absence of systemic disease, and it can even be the first manifestation of cancer5). thus, lmc can develop independently from systemic malignancy without pbm, but some studies reported that patients who underwent sr for their pbm had a higher incidence of lmc compared with that of patients received non surgical treatments31 - 33). it is unclear how pbm spreads and causes lmc, but we could observe the surgical spillage or the drop - metastasis of malignant brain tumors2,25,35). the incidence of lmc in patients with pbm compared with that in patients without pbm are yet to be studied, and even reports investigating the incidence of lmc in patients with pbm are rare17,18,30). kim.17) reported that in 400 patients with cns metastases of breast cancer, 318 patients (79.5%) had pbm only, 52 patients (13%) had both pbm and lmc, and 30 patients (7.5%) had lmc only. the number of breast cancer patients diagnosed with histologic confirmation during the same period was 10172 in that study, and based on this, we inferred the incidence of lmc in patients with pbm as 52/370 (14%), which was much higher than that in patients without pbm (30/9, 802, 0.3%). lee.18) reported that the incidence of lmc among cns metastases of breast cancer was 25% (68 of 272 patients), which was apparently higher than the known incidence proportion of lmc in breast cancer patients which is about to 1 - 5%5). however, this simple comparison should be understood with caution because many clinical factors including systemic disease status may affect the incidence. the hematogenous pathway can be a route for both pbm and lmc as tumor cells overcome blood - brain and blood - csf barrier28). although it is not clear whether systemic chemotherapy can prevent either pbm or lmc, several studies indicated that systemic chemotherapy could prolong patients ' survival with pbm from breast cancer and intra - csf chemotherapy is currently considered the best treatment option for lmc14,17,26,28,36). it is noteworthy that systemic disease status and chemotherapy were significant factors affecting the incidence of lmc in our study. in view of the poor prognosis of patients with lmc, early diagnosis of lmc with prompt initiation of treatment is important to prolong survival, especially in patients with good performance status and neurological function8). physicians should be alerted to a differential risk of the condition depending on certain characteristics, and the method of treatment ; early appropriate actions to prevent or treat lmc in higher - risk patients should be taken. recent trends in diagnosing lmc with mri are highly sensitive although it is not definitive13). a strong adherence of malignant cells to the leptomeninges or the presence of a focal rather than widespread leptomeningeal tumor, can contribute to false - negative cytological results15,37). although the neuroimaging - based diagnosis of lmc without csf cytology is not generally accepted as a definitive diagnosis, a consensus is reached to allow for the treatment of lmc diagnosed by radiographic evidence only in known cancer patients. in our study, 10 patients were diagnosed with lmc based only on an mri. four of them revealed a negative csf finding and in the remaining 6 patients, csf testing was not performed. it is known that certain biological features of breast cancer affect clinical finding including lmc. several reports have suggested a higher risk for brain metastasis in her-2 positive and triple negative (tn) breast subtypes than in hormone receptor (hr) positive subtypes6,19,20). lee.18) reported a different distribution of subtypes of breast cancer between pbm with lmc and pbm without lmc, and a worse prognosis of patients with tn. however, the direct comparison of incidence of lmc according to the subtypes of breast cancer was not made. several studies suggested that mechanical spread of tumor cells to the csf space could contribute to the development of lmc. some studies reported an increased incidence of lmc after posterior fossa tumor surgery compared with supratentorial lesions25,35). other studies observed a higher incidence of lmc after sr compared with non - surgical treatment31 - 33). norris.25) suggested that the more chance of csf exposure in patients receiving posterior fossa tumor removal was responsible for the increased development of lmc. suki.33) reported increased incidence of lmc in patients with pbm, whose tumor was removed in a piecemeal manner than en - bloc excision. thus, authors investigated the incidence of lmc after sr of pbm according to the proximity to csf pathway and suggested the pbm not entirely surrounded by brain parenchyma should be removed in caution not to spill the tumor by piecemeal removal or using cavitron ulatrsonographic aspirator1). in our study, the sr group showed a significantly higher incidence of lmc than the wbrt group, which is in accordance with previous finding. however, the pbm location whether or not the lesions involved posterior fossa, did not affect the risk of development of lmc. this result may reflect not tumor location itself but a chance for csf exposure to be responsible for development of lmc. known prognostic factors for the survival of breast cancer patients include axillary nodal status, tumor size / type / grade, lymphatic / vascular invasion, proliferation markers, ethnicity and patient age at diagnosis. many studies, which evaluated the influence of age on outcome in breast cancer have been small and had conflicting results. two relatively large trials have, however, demonstrated a worse prognosis for patients younger than 35 years of age, even after adjustment for other prognostic factors4,39). a different distribution of molecular phenotypes in the population of young women with breast cancer compared to the general population of women with breast cancer was reported7). young age appears to be an independent risk factor and the median age of patients with brain metastases is about 5 years younger than those without brain metastases6). in our study, an age of younger than 40 years was an independent risk factor for development of lmc in patients with pbm from breast cancer. this propensity is in accordance with the observation of lee.18), where the age of pbm from breast cancer patients with lmc was significantly younger than those without lmc although the variables were not adjusted. the role of systemic chemotherapy to prevent cns metastasis has not yet been proven. however, a recent study reported a favorable outcome showing significantly prolonged survival in patients with pbm from breast cancer who received chemotherapy, compared with the patients without chemotherapy16). these finding leave a possibility that the chemotherapy directly reduce pbm from breast cancer or lmc, in addition to prolonging patient 's survival by decreasing the systemic cancer burden. in our study, the patients in a direction of reduced systemic cancer burden (i.e. patients with stable or ned of systemic cancer and patients with systemic chemotherapy) showed a decreased risk for the development of lmc after treatment for their pbm from breast cancer. the role of wbrt for preventing cns recurrence after sr of pbm was proven in a randomized controlled clinical trial27). however, it is unclear whether or not this " cns recurrence " included lmc, and we could not find any study that compared not the local recurrence rate, but the risk of lmc after sr in patients with or without wbrt9,10). an 11% incidence of lmc in the wbrt only group in our study is the first presented figure indicating the natural risk for the development of lmc without sr. but in this cohort, we did not have a group of patients, who had pbm from breast cancer without wbrt. thus, to evaluate the role of wbrt for preventing the development of lmc, a further well controlled cohort study is needed. however, occasionally it occurs in the absence of systemic disease, and it can even be the first manifestation of cancer5). thus, lmc can develop independently from systemic malignancy without pbm, but some studies reported that patients who underwent sr for their pbm had a higher incidence of lmc compared with that of patients received non surgical treatments31 - 33). it is unclear how pbm spreads and causes lmc, but we could observe the surgical spillage or the drop - metastasis of malignant brain tumors2,25,35). the incidence of lmc in patients with pbm compared with that in patients without pbm are yet to be studied, and even reports investigating the incidence of lmc in patients with pbm are rare17,18,30). kim.17) reported that in 400 patients with cns metastases of breast cancer, 318 patients (79.5%) had pbm only, 52 patients (13%) had both pbm and lmc, and 30 patients (7.5%) had lmc only. the number of breast cancer patients diagnosed with histologic confirmation during the same period was 10172 in that study, and based on this, we inferred the incidence of lmc in patients with pbm as 52/370 (14%), which was much higher than that in patients without pbm (30/9, 802, 0.3%). lee.18) reported that the incidence of lmc among cns metastases of breast cancer was 25% (68 of 272 patients), which was apparently higher than the known incidence proportion of lmc in breast cancer patients which is about to 1 - 5%5). however, this simple comparison should be understood with caution because many clinical factors including systemic disease status may affect the incidence. the hematogenous pathway can be a route for both pbm and lmc as tumor cells overcome blood - brain and blood - csf barrier28). although it is not clear whether systemic chemotherapy can prevent either pbm or lmc, several studies indicated that systemic chemotherapy could prolong patients ' survival with pbm from breast cancer and intra - csf chemotherapy is currently considered the best treatment option for lmc14,17,26,28,36). it is noteworthy that systemic disease status and chemotherapy were significant factors affecting the incidence of lmc in our study. in view of the poor prognosis of patients with lmc, early diagnosis of lmc with prompt initiation of treatment is important to prolong survival, especially in patients with good performance status and neurological function8). physicians should be alerted to a differential risk of the condition depending on certain characteristics, and the method of treatment ; early appropriate actions to prevent or treat lmc in higher - risk patients should be taken. recent trends in diagnosing lmc with mri are highly sensitive although it is not definitive13). a strong adherence of malignant cells to the leptomeninges or the presence of a focal rather than widespread leptomeningeal tumor, can contribute to false - negative cytological results15,37). although the neuroimaging - based diagnosis of lmc without csf cytology is not generally accepted as a definitive diagnosis, a consensus is reached to allow for the treatment of lmc diagnosed by radiographic evidence only in known cancer patients. in our study, 10 patients were diagnosed with lmc based only on an mri. four of them revealed a negative csf finding and in the remaining 6 patients, csf testing was not performed. it is known that certain biological features of breast cancer affect clinical finding including lmc. several reports have suggested a higher risk for brain metastasis in her-2 positive and triple negative (tn) breast subtypes than in hormone receptor (hr) positive subtypes6,19,20). lee.18) reported a different distribution of subtypes of breast cancer between pbm with lmc and pbm without lmc, and a worse prognosis of patients with tn. however, the direct comparison of incidence of lmc according to the subtypes of breast cancer was not made. several studies suggested that mechanical spread of tumor cells to the csf space could contribute to the development of lmc. some studies reported an increased incidence of lmc after posterior fossa tumor surgery compared with supratentorial lesions25,35). other studies observed a higher incidence of lmc after sr compared with non - surgical treatment31 - 33). norris.25) suggested that the more chance of csf exposure in patients receiving posterior fossa tumor removal was responsible for the increased development of lmc. suki.33) reported increased incidence of lmc in patients with pbm, whose tumor was removed in a piecemeal manner than en - bloc excision. thus, authors investigated the incidence of lmc after sr of pbm according to the proximity to csf pathway and suggested the pbm not entirely surrounded by brain parenchyma should be removed in caution not to spill the tumor by piecemeal removal or using cavitron ulatrsonographic aspirator1). in our study, the sr group showed a significantly higher incidence of lmc than the wbrt group, which is in accordance with previous finding. however, the pbm location whether or not the lesions involved posterior fossa, did not affect the risk of development of lmc. this result may reflect not tumor location itself but a chance for csf exposure to be responsible for development of lmc. known prognostic factors for the survival of breast cancer patients include axillary nodal status, tumor size / type / grade, lymphatic / vascular invasion, proliferation markers, ethnicity and patient age at diagnosis. many studies, which evaluated the influence of age on outcome in breast cancer have been small and had conflicting results. two relatively large trials have, however, demonstrated a worse prognosis for patients younger than 35 years of age, even after adjustment for other prognostic factors4,39). a different distribution of molecular phenotypes in the population of young women with breast cancer compared to the general population of women with breast cancer was reported7). young age appears to be an independent risk factor and the median age of patients with brain metastases is about 5 years younger than those without brain metastases6). in our study, an age of younger than 40 years was an independent risk factor for development of lmc in patients with pbm from breast cancer. this propensity is in accordance with the observation of lee.18), where the age of pbm from breast cancer patients with lmc was significantly younger than those without lmc although the variables were not adjusted. however, a recent study reported a favorable outcome showing significantly prolonged survival in patients with pbm from breast cancer who received chemotherapy, compared with the patients without chemotherapy16). these finding leave a possibility that the chemotherapy directly reduce pbm from breast cancer or lmc, in addition to prolonging patient 's survival by decreasing the systemic cancer burden. in our study, the patients in a direction of reduced systemic cancer burden (i.e. patients with stable or ned of systemic cancer and patients with systemic chemotherapy) showed a decreased risk for the development of lmc after treatment for their pbm from breast cancer. the role of wbrt for preventing cns recurrence after sr of pbm was proven in a randomized controlled clinical trial27). however, it is unclear whether or not this " cns recurrence " included lmc, and we could not find any study that compared not the local recurrence rate, but the risk of lmc after sr in patients with or without wbrt9,10). an 11% incidence of lmc in the wbrt only group in our study is the first presented figure indicating the natural risk for the development of lmc without sr. but in this cohort, we did not have a group of patients, who had pbm from breast cancer without wbrt. thus, to evaluate the role of wbrt for preventing the development of lmc, a further well controlled cohort study is needed. this retrospective analysis revealed an increased risk for the development of lmc after sr of pbm from breast cancer compared with wbrt. the young age (< 40) and systemic cancer burden in terms of progressing systemic could be additional risk factors for lmc, whereas continued systemic chemotherapy after the index procedure may reduce the incidence of lmc in patients with pbm from breast cancer. | objectivethe objective of study is to evaluate the incidence of leptomeningeal carcinomatosis (lmc) in breast cancer patients with parenchymal brain metastases (pbm) and clinical risk factors for the development of lmc.methodswe retrospectively analyzed 27 patients who had undergone surgical resection (sr) and 156 patients with whole brain radiation therapy (wbrt) as an initial treatment for their pbm from breast cancer in our institution and compared the difference of incidence of lmc according to clinical factors. the diagnosis of lmc was made by cerebrospinal fluid cytology and/or magnetic resonance imaging.resultsa total of 27 patients (14%) in the study population developed lmc at a median of 6.0 months (range, 1.0 - 50). ten of 27 patients (37%) developed lmc after sr, whereas 17 of 156 (11%) patients who received wbrt were diagnosed with lmc after the index procedure. the incidence of lmc was significantly higher in the sr group compared with the wbrt group and the hazard ratio was 2.95 (95% confidence interval ; 1.33 - 6.54, p<0.01). three additional factors were identified in the multivariable analysis : the younger age group (< 40 years old), the progressing systemic disease showed significantly increased incidence of lmc, whereas the adjuvant chemotherapy reduce the incidence.conclusionthere is an increased risk of lmc after sr for pbm from breast cancer compared with wbrt. the young age (< 40) and systemic burden of cancer in terms of progressing systemic disease without adjuvant chemotherapy could be additional risk factors for the development of lmc. |
subarachnoid - pleural fistula (spf) is an abnormal communication between the subarachnoid and pleural spaces that normally can arise from blunt or penetrating trauma or as a complication of chest operation. this occurs infrequently following cardiothoracic surgery for lung or chest wall resection and closure of patent ductus arteriosus, spine surgery for transthoracic discectomy [3, 4 ], removal of vertebral tumors, or spinal fusion. symptoms develop from the accumulation of pleural fluid, the reduced volume of cerebrospinal fluid (csf), the presence of intracranial air, or the development of meningitis, with outcomes ranging from benign to catastrophic. this complication most frequently occurs in the setting of a tumour that occupies the costovertebral angle requiring extrapleural dissection, excessive rib retraction, or disarticulation of the costotransverse joint leading to nerve root avulsion or dural tear. following confirmation of the spf diagnosis by computed tomography (ct) myelography or radionuclide cisternography, conservative therapies such as lumbar drainage to divert csf flow may be successful, though this is less likely to occur in patients who have undergone previous surgery or received radiotherapy to the affected area. consequently, direct operative repair of the dural defect with fine non - absorbable suture and reinforcing repair with a tissue patch is often necessary for permanent definitive treatment of the established spf. this paper describes the diagnosis and management of two cases of spf encountered by the senior author that illustrate the typical course to presentations of this complication. this motivates a detailed anatomic description of cerebrospinal fluid fistulas to the pleural space, with elaboration of intraoperative techniques to lessen the likelihood of such complication. an 18-year - old woman with a known diagnosis of wilm 's tumor had undergone left nephrectomy and adjuvant chemotherapy at 15 months of age. tumor recurrence was noted six months later in both lungs for which she received additional chemotherapy and bilateral lung radiotherapy. between ages 15 and 17 years, she experienced several bouts of recurrent disease in the right chest treated by stem cell transplant, chemotherapy, and radiotherapy. a severe septic complication developing after the stem cell transplant required 12 weeks of intensive care. thoracic surgery service was consulted for management of disabling persistent right posterior chest wall pain due to multiple pleural - based tumor recurrence in the paravertebral gutter and lung parenchyma (figure 1). right thoracotomy was performed, and complete resection was carried out by extrapleural and intrapericardial pneumonectomy. intraoperatively, a csf leak was recognized and treated with a free muscle plug patch inserted in the aperture of the intervertebral foramen and reinforced with tissel glue (baxter biosurgery, deerfield, il). in the early postoperative period, serial chest radiographs failed to demonstrate the expected mediastinal shift to the right hemithorax (figures 2 and 3). this prompted early investigation with a ct myelogram that confirmed a suspected spf at the right t4/5 intervertebral foramen (figures 4 and 5). she was immediately referred for definitive neurosurgical care involving unilateral t4 and t5 laminectomy that revealed csf leaking from an avulsed nerve root on the lateral aspect of the dural sac. a nonabsorbable, 6 - 0 prolene, continuous suture from caudal to cephalad was used to close this 7 mm defect. the integrity of the repair was confirmed by a valsalva maneuver during which no csf was observed to leak. the patient 's postoperative course was significant only for the development of pneumonia for which she received in - patient treatment. at followup 8 months later, there was no clinical or radiographic evidence of persistent or recurrent spf. a 66-year - old man with chronic liver disease due to hepatitis b developed hepatocellular carcinoma for which a right partial hepatectomy was performed. two years later, a solitary 7 mm metastasis was removed from the lower lobe of the right lung by video - assisted thoracoscopic surgery wedge resection. two years after that, further recurrence was noted in the right lung and in the adjoining parietal pleura at the site of the previous resection. these were removed by wedge resection using open technique at which time concomitant complete parietal pleurectomy was required to remove several other small remote tumor nodules. one year later, tumour recurrence was again noted in the right hemithorax in the para - vertebral gutter. this lesion in the posterior segment of the lower lobe measured 3 cm in size and involved the adjoining 5th rib in close proximity to the intervertebral foramen (figure 6). palliative and prophylactic resection was recommended to relieve persistent chest wall pain and because of the potential of tumor extending centrally into the intervertebral foramen with consequent epidural spinal cord compression or meningeal invasion with csf carcinomatosis. wide en bloc resection was performed with removal of the involved posterior portion of the 5th rib, the t5 transverse process, the adjoining cortex of t5 vertebral body, and the posterior segment of the upper lobe with the necessary extrapleural dissection. during that dissection, a csf leak was observed at the 5th intercostal nerve, with control achieved by application of two large hemoclips after which the nerve was divided. a valsalva maneuver was then performed during which no further csf was observed to leak. a rare complication of chest operation, particularly in the paravertebral gutter region, is the development of fistula between the subarachnoid and pleural spaces resulting from iatrogenic concomitant dural and pleural defects. it is reported to occur in fewer than 1% of patients undergoing en bloc lung resection with the adjoining chest wall, with common anatomic features being involvement of the upper lobe of the lung with chest wall invasion into the region of the costovertebral angle. furthermore, the necrotizing potential of preoperative or intraoperative radiotherapy can impede wound healing of these defects, predisposing patients to fistula formation. the patients presented in this report had chest wall involvement of wilm 's tumor and hepatocellular carcinoma. the first patient had predisposing features of preoperative radiotherapy and an identified intraoperative csf leak. the second patient had extensive chest wall pathology in close proximity to the t5 intervertebral foramen, again with intraoperative observation of csf leakage. requisite for formation of an spf is breach of the dura, arachnoid, and parietal pleura. violation of these membranes can more commonly lead to extradural flow of csf or less commonly intradural accumulation of air with consequent pulmonary and neurological symptomatology. an understanding of the regional anatomy and pathophysiology that underlie the development of spf after thoracic surgical procedures can identify sites that are vulnerable to injury during the dissection and key steps that may be taken to protect against this complication and to facilitate secondary surgical remedy when indicated. the spinal nerve roots are multivesicular structures that are surrounded by merged piaarachnoid enclosure as they emerge through a dural fenestration at each segmental level. the 31 paired spinal nerves, of which 12 are thoracic, are formed by the union of dorsal and ventral roots that carry afferent (sensory) and efferent (motor) fibers respectively. more commonly the dorsal and ventral roots exit through discrete fenestrations in the dura, but an anatomic variant can include the simultaneous emergence of both. the arachnoid also forms a separate sheath for both roots, with the subarachnoid space extending to the proximal dorsal root ganglion providing an anatomical route of csf absorption into arachnoid villi [911 ]. these dural sheaths blend with the epineurium that adheres to the periosteal lining of the lateral intervertebral foramen. the dorsal root ganglion is a collection of sensory cell soma that extends processes centrally to the medulla spinalis and peripherally to the spinal nerve. it normally lies in the medial intervertebral foramen, with the confluence of the dorsal and ventral roots lying just distal to this point. it normally lies just lateral to the edge of the dural reflection, but it may be intradural. ebraheim and coworkers have quantified the anatomic relationship between the thoracic pedicle and the nerve root. among 15 cadavers, they demonstrated that the superoinferior diameter of the nerve root increased from 2.9 mm at t1 to 4.6 mm at t12. no epidural space was seen between the dural sac and the pedicle, and the frontal angle decreased from 120.1 at t1 to 57.1 at t12. similar work by ugur and coworkers demonstrates a decrease in mean root exit angle from 104 at t1 to 60 at t12 with nerve root diameters that increased from between 2.3 mm at t1 to t5 to 3.7 mm at t12. the junction of the two roots forms a very short segment mixed spinal nerve that divides into a large ventral and small dorsal ramus, the former of which becomes the intercostal nerve. the superior and inferior surfaces of adjacent vertebrae are covered with a thin layer of hyaline cartilage and united by a thick fibrocartilaginous intervertebral disc. the cartilaginous endplate covers the cortical vertebral body surfaces and permits longitudinal diffusion of nutrients to the avascular center of the disc. the outer surface of the af is innervated, and peripheral blood vessels also penetrate radially to supply the center of the disc. the central nucleus pulposus is an avascular, viscoelastic gel with predominantly type ii collagen and hydrated proteoglycans. it is normally constrained from cephalocaudal herniation by the endplates and from radial herniation by the competent annulus fibrosus. of more surgical relevance, the anatomy of the costovertebral, costotransverse, and thoracic facet joints must be defined because of their proximity to neural structures of interest. (a) costovertebral jointthe rib head has a facet that articulates in planar joints with a single costal facet on t1, t10, t11, and t12, with all other levels having articulations with the costal facet of their numbered level and additionally the inferior costal demifacet of the vertebral body one level above and with the associated intervertebral disc. the vertebral body costal facets are found dorsally near the root of the pedicle, with more rostral location in the upper thoracic spine and more caudal location in the lower thoracic spine. the ribs that have two articulations have two rib head facets with an intervening interarticular crest that has ligamentous attachments to the intervertebral disc. the superior fibers of the joint capsule extend through the intervertebral foramen to attach to the posterior aspect of the intervertebral disc. the radiate ligaments fan out from the anterior aspect of the rib to the anterior vertebral body superiorly to the vertebra above, laterally to the intervertebral disc, and inferiorly to the corresponding vertebra. costovertebral joints where the rib articulates with two vertebrae also have an extrasynovial, intra - articular ligament that attaches from the crest on the rib head that lies between the two costal demifacets to the intervertebral disc. the rib head has a facet that articulates in planar joints with a single costal facet on t1, t10, t11, and t12, with all other levels having articulations with the costal facet of their numbered level and additionally the inferior costal demifacet of the vertebral body one level above and with the associated intervertebral disc. the vertebral body costal facets are found dorsally near the root of the pedicle, with more rostral location in the upper thoracic spine and more caudal location in the lower thoracic spine. the ribs that have two articulations have two rib head facets with an intervening interarticular crest that has ligamentous attachments to the intervertebral disc. the superior fibers of the joint capsule extend through the intervertebral foramen to attach to the posterior aspect of the intervertebral disc. the posterior capsular fibers are continuous with the fibers of the costotransverse ligament. the radiate ligaments fan out from the anterior aspect of the rib to the anterior vertebral body superiorly to the vertebra above, laterally to the intervertebral disc, and inferiorly to the corresponding vertebra. costovertebral joints where the rib articulates with two vertebrae also have an extrasynovial, intra - articular ligament that attaches from the crest on the rib head that lies between the two costal demifacets to the intervertebral disc. (b) costotransverse jointan articular region on the tubercle of the first ten ribs articulates with a facet on the corresponding transverse process. these synovial joints transition from a convex rib portion in the upper thoracic spine to planar in the lower thoracic spine. the costotransverse, superior costotransverse, and lateral costotransverse ligaments are associated with this joint. the costotransverse ligament attaches the rib to the transverse process, occupying the costotransverse foramen. the superior costotransverse ligament has anterior and posterior layers the anterior layer attaches the crest of the rib neck to the transverse process and is continuous laterally with the internal intercostal membrane, crossed by the intercostal vessels and nerves. the posterior layer attaches the dorsal aspect of the rib to the transverse process and is continuous laterally with the external intercostal muscle. an articular region on the tubercle of the first ten ribs articulates with a facet on the corresponding transverse process. these synovial joints transition from a convex rib portion in the upper thoracic spine to planar in the lower thoracic spine. the costotransverse, superior costotransverse, and lateral costotransverse ligaments are associated with this joint. the costotransverse ligament attaches the rib to the transverse process, occupying the costotransverse foramen. the superior costotransverse ligament has anterior and posterior layers the anterior layer attaches the crest of the rib neck to the transverse process and is continuous laterally with the internal intercostal membrane, crossed by the intercostal vessels and nerves. the posterior layer attaches the dorsal aspect of the rib to the transverse process and is continuous laterally with the external intercostal muscle. (c) facet jointthoracic facet joints consist of apposed articular processes lined with hyaline cartilage extending from adjacent vertebrae. they are synovial joints encased in a fibrous capsule that attaches peripheral to the articular surfaces, with the superior articular facets being slightly convex, oriented 60 from the horizontal plane and 20 from the frontal plane facing posteriorly and laterally [15, 16 ]. the inferior facets are appropriately positioned facing anteriorly and medially to match their partner. thoracic facet joints consist of apposed articular processes lined with hyaline cartilage extending from adjacent vertebrae. they are synovial joints encased in a fibrous capsule that attaches peripheral to the articular surfaces, with the superior articular facets being slightly convex, oriented 60 from the horizontal plane and 20 from the frontal plane facing posteriorly and laterally [15, 16 ]. the thoracic spinal nerve root traverses the radicular canal from the spinal cord to the intervertebral foramen. it is divided into the retrodiscal, parapedicular, and foraminal sections, with the latter of greatest interest to the thoracic surgeon. the borders of the intervertebral foramen include the posterior vertebral body, the intervertebral disc, the lateral posterior longitudinal ligament, and the anterior longitudinal venous plexus (ventral), the superior and inferior articular processes with capsule that merges with ligamentum flavum (dorsal), and the pedicles (cranial and caudal). approaches to the lateral thoracic spine are limited by the rib head that can create a false foramen and obscure the view of the exiting neural elements. the rib head and its facet must be removed to provide visibility to the true intervertebral foramen, to provide access to the pedicle and spinal cord, and to create a suitably flat surface for instrumentation. the foramen are of variable shape : oval (26.6%), auricular (58%), or teardrop (17.4%) and are incompletely filled by the nerve root that are generally apposed to the upper pedicle. the foramen are covered by the fascia that with two distinct perforations for the nerve root and intervertebral vessels, respectively. the trajectory of the thoracic nerve roots at the intervertebral foramen varies by location in the spine, with upper thoracic roots projecting upwards, middle thoracic roots oriented in a horizontal plane, and lower thoracic roots projecting downward. this variation influences the position of the dorsal root ganglion with regard to the spinal cord and the intervertebral foramen, with attendant variability in the extraforaminal extent of the dural sheath. there are various foraminal ligaments closely related to the exiting nerve root that may be present in the thoracic spine intervertebral foramen. the ligamentous structures vary in width and thickness from 2 to 5 mm and substantially decrease the aperture of the intervertebral foramen while no data is reported specific to the thoracic spine, the actual aperture size in thoracolumbar spine among 49 human nonpathological intervertebral foramen was on average 31.5% less than the size of the osseous foramen. the superior and inferior corporopedicular ligaments attach to the superior and inferior pedicle, respectively, and traverse obliquely anteriorly to the posterolateral vertebral body and associated annulus fibrosus. the superior transforaminal ligament attaches from the anterior inferior vertebral notch on the superior pedicle to the articular capsule. the mid - transforaminal ligament attaches from annulus fibrosus and superior and inferior corpopedicular ligaments to the articular capsule. the inferior transforaminal ligament extends from the junction of the annulus fibrosus and the posterior vertebral body to the superior articular facet. lastly, the suspensor radial ligaments extend radially from the nerve roots to the related ligamentous structures. when present, the foraminal ligamentous relationships to the thoracic nerve root are the following : inferior corporopedicular ligament (posterosuperior), superior transforaminal ligament (anterosuperior), ligamentum flavum (posterior), superior corporopedicular ligament (anterior), and mid - transforaminal ligament (inferior). these ligaments can sequester the intervertebral artery and vein away in fatty areolar tissue ventral to the exiting nerve root exiting from the foramen anterior to the superior corpopedicular ligament, inferior to the mid - transforaminal ligament, and superior to the inferior transforaminal ligament. the dura of an emerging thoracic spinal nerve can be injured in the vicinity of the intervertebral foramen during extra - pleural dissection, neural element dissection, excessive rib retraction, or antecedent or inadvertent rib fracture [19, 20 ]. furthermore, traction on a tumor adherent to the nerve can also accidentally tear the dura. chest wall resections commonly breach dural integrity by requiring multiple rhizotomies at costovertebral joints or ligation of the nerve root at the intervertebral foramen. when the procedure requires sacrifice of the nerve root, thoracic surgeons are advised to approach with caution by applying large hemoclips or non - absorbable sutures to the nerve before division. dissection of adjacent and adherent tumor from around the nerve is necessary to allow successful ligation. forceful rib retraction can avulse the nerve root and create a dural defect. at the end of the procedure, patients should be routinely ventilated with increased intrathoracic pressure to raise subarachnoid pressure and identify any leak prior to chest closure. the volume of csf in the adult human cranial vault is approximately 150 ml and it is produced at a rate of 500 ml per day. this formation follows a circadian rhythm and is split nearly between 60% at the choroid plexus tissue and 40% extrachoroidal interstitial fluid movement from the brain parenchyma. absorption of csf through the intracranial and spinal arachnoid villi begins at average csf pressure of 68 mm h2o and rises linearly up to 250 mm h2o, with equivalence of formation and absorption rates occurring at 112 mm h2o. this variability is a homeostatic response to maintain normal intracranial pressure and csf volume. in the presence of concomitant defects in the dura and the parietal pleura, the cyclic changes in the negative intrapleural pressure during respiration become important in directing the flow of csf. this unidirectional movement is from the positive - pressure subarachnoid space to the negative pressure pleural space where the fluid accumulates. the persistence of this pressure gradient has the effect of continuously drawing csf into the pleural space, with the flow impeding spontaneous closure of the fistula. furthermore, while the pleural space can normally remove fluid through its lymphatic stomas at a clearance rate nearly 30 times the fluid formation rate ; excessive csf flowing through the spf along with altered lymphatic drainage by the underlying cancer or the surgical interruption of mediastinal lymphatic vessels can lead to fluid accumulation. spine surgeons are advised, when possible, to preserve the parietal pleura during exposure of the thoracic spine. such iatrogenic spf lesions are more common after anterior approaches to thoracic tumors, probably a consequence of the greater likelihood of the requisite simultaneous damage to both the meninges and the parietal pleura. indeed, hentschel and coworkers describe a retrospective series of 770 patients in whom nine developed a spf, for overall incidences of 2.4% and 0.23% following anterior and posterior approaches, respectively. a csf leak that is identified intraoperatively whenever possible, the surgeon should perform simple ligation with hemoclips to arrest further leakage and to prevent the untoward complications of a persistent spf. there are few other reported techniques of addressing this complication through a thoracotomy, though they can be of great value to the thoracic surgeon instead of changing to a prone position for a laminectomy. while thoracoplasty is possible, it is technically demanding near the intervertebral foramen and may lead to more deleterious bleeding or further dural injury. neurosurgical consultation should be sought to evaluate options of foraminal widening with direct dural closure, or simultaneous or staged posterior laminectomy if necessary to provide definitive treatment. patch grafts of muscle, omentum, and fascia have been described to be successful, secured in place using sutures or fibrin glue [1, 25 ]. during the postoperative recovery, the presentation of such lesions can be through pulmonary morbidity of large transudative pleural effusions or through neurological morbidity of intracranial hypotension, pneumocephaly, or central nervous system infection. chest radiography can suggest the diagnosis of spf when there is presence or rapid accumulation of pleural fluid or mediastinal widening. the pleural cavity is normally able to absorb csf with clinical evidence of this found in ventriculopleural shunts for hydrocephalus [26, 27 ], rendering the incidence of spf to be likely underestimated. accumulation of csf in the thoracic cavity is the most common presentation and can be identified by chest radiography demonstrating unilateral or bilateral pleural effusions with the presence of 2-transferrin in the pleural fluid. this marker occurs nearly exclusively in the csf, arising from the 1-isoform by the action of neuraminidase and is consequently highly sensitive and specific for traumatic or perioperative leak [6, 2931 ]. it has been rarely reported to be falsely negative in surgically - confirmed spf [28, 32 ], and false positives will occur in individuals heterozygous for the transferrin gene. the latter can be avoided by electrophoretic evaluation on both the analyte fluid and a serum control from the same patient. the pleural fluid is otherwise characteristically clear having low nucleated cell count, with total protein consistently less than 1.0 g / dl and a pleural fluid to serum glucose concentration ratio between 0.5 and 1.0. patients with neurological symptoms most commonly present with headache, altered mental status, or declining level of consciousness after recent thoracotomy. headache may occur as a consequence of csf hypovolemia, meningitis, or pneumocephalus ; the latter two are among the most significant neurological complications of spf [5, 7, 35 ]. while plain skull radiographs may demonstrate the presence of intracranial air, such techniques are becoming obsolete in deference to the superior anatomic detail provided by ct scans. accumulation of intracranial air under tension is a neurosurgical emergency and may indicate ventricular drain placement for decompression. the most characteristic physical finding of this condition would be bruit hydroaerique, a splashing sound induced by a rapid change in head position. chadduck and ladehoff describe a rare but morbid complication of remote cerebellar hemorrhage following spinal surgery, with a consensus that downward cerebellar displacement accompanies the relative csf hypovolemia, leading to tension and occlusion on the superior cerebellar bridging veins with consequent venous infarction and hemorrhage. diagnosis of a suspected csf leak after a chest operation is best confirmed by ct myelography or radionuclide cisternography. the anatomic detail provided by ct myelography can aid in planning for surgical correction, but it carries a high false - negative rate [39, 40 ]. nuclear cisternograms with infusion of in - dtpa is more sensitive for detection of spf [5, 41 ], though does not provide anatomic detail and must hence be combined with other imaging for preoperative planning. chest tube drainage of the initial pleural effusion can provide both diagnostic and symptomatic therapeutic benefit. however, it can be deleterious to if suction is applied because it will promote continuous egress of csf into the pleural space and worsen csf hypovolemia, while also maintaining patency of the spf. instead, a water - tight seal should be applied to allow gravity - dependent drainage of pleural fluid accumulation. insertion of a lumbar drain can divert csf flow away from the fistula, though it is rare that such maneuvers are successful, and katz and coworkers report about one patient in whom this leads to pneumonia and meningitis. furthermore, lumbar drainage can not control movement of air into the subarachnoid space in the setting of persistent air leak from the lung parenchyma after lobar or sublobar resection. specific treatment of the fistula is dictated by the defect size and progression of the patient 's symptoms, with larger fistulae often requiring surgical closure, with one or two level posterior laminectomy preferable to reopening of the thoracotomy for securing repair of the defect. reported maneuvers include primary surgical closure with reinforcement by application of omentum, muscle, or fat patches to facilitate repair. other materials that have been used include a methylmethacrylate plug wrapped in pleura, autologous blood patch, or thrombin soaked gelatin. iatrogenic spf following resection of thoracic tumors is a rare complication that introduces significant perioperative morbidity to the affected patient as a consequence of pulmonary or neurological symptoms. the seriousness of this complication should not be underestimated as there may be life - threatening consequences of tension pneumocephalus, cerebellar infarction and bleeding, meningitis, or massive pleural effusion. the thoracic surgeon operating in the vicinity of the intervertebral foramen must be familiar with the regional and applied anatomy at this site, including thorough knowledge of osseous, ligamentous, neural, and vascular structures. this can afford careful avoidance of dural injury and may help identify sites of csf leakage when such injury has occurred to facilitate immediate repair. | subarachnoid - pleural fistula (spf) is a rare complication of chest or spine operations for neoplastic disease. concomitant dural and parietal pleural defects permit flow of cerebrospinal fluid into the pleural cavity or intrapleural air into the subarachnoid space. dural injury recognized intraoperatively permits immediate repair, but unnoticed damage may cause postoperative pleural effusion, intracranial hypotension, meningitis, or pneumocephalus. we review two cases of spf following surgical intervention for chest wall metastatic disease to motivate a detailed review of the anatomy of neural, osseous, and ligamentous structures at the intervertebral foramen. we further provide recommendations for avoidance and detection of such complication. |
thus, the high prevalence of olfactory dysfunction after surgery or head trauma has a great effect on overall quality of life. following head injury with a fracture across the cribriform plate, a minor blow to the occipital area may result in anosmia due to tearing or shearing of olfactory nerve filaments. the olfactory nerve and tracts are also at risk during intradural explorations using the interhemispheric approach or the frontotemporal approach [10, 11 ]. both approaches require some degree of frontal lobe retraction, which may result in temporary or permanent olfactory dysfunction because of nerve avulsion or mechanical compression [3, 4, 6, 12 ]. furthermore, it is well known that bilateral anosmia is highly likely to occur when bilateral olfactory nerves are pulled out from the frontal base. it is not always true that preservation of the anatomical structure will result in preservation of function. however, preservation of anatomical structure may be an important factor in the preservation of olfactory function. based on this background, strength tests of the human olfactory nerve at the frontal skull base were performed using cadavers, and the microsurgical anatomical features of this nerve were reviewed in order to identify operative nuances that may contribute to reducing the rate of postoperative olfactory dysfunction. all procedures were conducted in accordance with the institutional guideline of jikei university and the tokyo medical examiner s office. ninety brains were obtained from autopsies of 68 men and 22 women, aged 2480 years at the time of death. they were within 12 h after death and had no intracranial disease or trauma. the calvaria was opened, and the brain was removed en bloc after the olfactory nerves were severed 1015 mm from the skull base with minimal retraction (figs. 1 and 2a). the calvaria was positioned so that the frontal base was perpendicular to the ground without rotation. then, the cut edges of the olfactory nerves were clipped and connected to a light, small rubber bag by a monofilament string (fig. 2b). in the first set of 30 cases (group 1), each right olfactory nerve was pulled 0 laterally (from the axis of the olfactory nerve) and 0 upward (from the anterior skull base) (1)), and each left olfactory nerve was pulled 0 laterally and 15 upward (2)). in the second set of 30 cases (group 2), each right olfactory nerve was pulled 0 laterally and 15 upward (3)), and each left olfactory nerve was pulled 15 laterally and 15 upward (4)). in the third set of 30 cases (group 3), each right olfactory nerve was pulled 15 laterally and 15 upward (5)), and each left olfactory nerve was pulled 30 laterally and 15 upward (6)). the angle of pulling against the frontal skull base and the axis of the olfactory nerve was regulated by pulling. the olfactory nerves were pulled by a rubber bag through a string while water was poured into the bag at a rate of 1 ml per 10 s until the nerve was pulled out from the cribriform plate. then, the string, clip, and the rubber bag filled with water were weighed, and this value was used as the strength of the olfactory nerve (fig. b the cut edges of the olfactory nerve are clipped and connected to a light, small rubber bag by a monofilament string. whole view (c) and schematic drawing view (d) of this study. the angle of pulling of the olfactory nerve from the frontal skull base and from the axis of the olfactory nerve is regulated by pulling. the olfactory nerve was pulled by a rubber bag through a string during which water was poured into the bag at 1 ml per 10 s until the nerve was pulled out from the cribriform platefig. 3the supine (a) and sagittal (b) schematic drawing views of the anterior skull base. numbers show the direction of pulling of the olfactory nerves in each group schematic drawing view of the technique of olfactory nerve dissection a superior view of the anterior skull base. b the cut edges of the olfactory nerve are clipped and connected to a light, small rubber bag by a monofilament string. whole view (c) and schematic drawing view (d) of this study. the angle of pulling of the olfactory nerve from the frontal skull base and from the axis of the olfactory nerve is regulated by pulling. the olfactory nerve was pulled by a rubber bag through a string during which water was poured into the bag at 1 ml per 10 s until the nerve was pulled out from the cribriform plate the supine (a) and sagittal (b) schematic drawing views of the anterior skull base. numbers show the direction of pulling of the olfactory nerves in each group statistical analysis was performed using the mann whitney u - test for comparisons of the nonparametric variables with a probability value of 0.05 indicating significance. in group 1, the strengths (mean standard deviation [sd ]) of the olfactory nerve for (1) and (2) were 3.681.74 and 3.091.60 g, respectively. the difference between the strengths of (1) and (2) was not significant (p = 0.191, mann whitney u - test for comparisons of the nonparametric variables). in group 2, the strengths for (3) and (4) were 3.141.87 and 4.051.70 g, respectively. the difference between the strengths of (3) and (4) was almost significant (p = 0.068, mann whitney u - test for comparisons of the nonparametric variables). in group 3, the strengths for (5) and (6) were 4.001.44 and 4.201.65 g, respectively. the difference between the strengths for (5) and (6) was not significant (p = 0.684, mann whitney u - test for comparisons of the nonparametric variables) (fig. 4 ; table 1).fig. 4graphs showing the strength of pulling for different directions in each grouptable 1olfactory nerve strength(1) g(2) g(3) g(4) g(5) g(6) gmean3.683.093.144.054.004.20sd1.741.601.871.701.441.65min.1.700.460.120.510.691.41max.10.006.357.466.798.207.72sd standard deviation, min. maximumnumbers in parentheses indicate the strength of the olfactory nerve measured as defined in fig. 3 : (1) pulled 0 laterally (from the axis of the olfactory nerve) and 0 upward (from anterior skull base) ; (2), (3) pulled 0 laterally and 15 upward ; (4), (5) pulled 15 laterally and 15 upward ; (6) pulled 30 laterally and 15 upward graphs showing the strength of pulling for different directions in each group olfactory nerve strength sd standard deviation, min. maximum numbers in parentheses indicate the strength of the olfactory nerve measured as defined in fig. 3 : (1) pulled 0 laterally (from the axis of the olfactory nerve) and 0 upward (from anterior skull base) ; (2), (3) pulled 0 laterally and 15 upward ; (4), (5) pulled 15 laterally and 15 upward ; (6) pulled 30 laterally and 15 upward and in each subgroups of pulling for different directions from (1) to (6), there seemed to be no relationship between age and olfactory strength (fig. 5) moreover, there were no statistical differences of olfactory strength between male and female in each subgroup.fig. 5age (x - axis) and strength of pulling for different directions from (1) to (6) (the y - axis) age (x - axis) and strength of pulling for different directions from (1) to (6) (the y - axis) the results of this study indicated that frontal lobe retraction in a posterior direction from the olfactory bulb, regardless of angle from the skull base posteriorly, is associated with the risk of pulling out the olfactory nerve, while retraction in a lateral direction appears to be relatively safe. this was based on the fact that that there was no significant difference in the strength of the olfactory nerves pulling them in a postero - upward direction between 0 and 15 upward (each, 0 laterally). furthermore, the difference in the strengths of the olfactory nerves when they were pulled in a postero - lateral direction between 0 and 15 laterally (each, 15 upward) was almost significant. it had been thought that only serious injuries of the head with fractures of the anterior cranial fossa resulting in anatomical disruption of the olfactory filaments or tract cause permanent anosmia, but permanent anosmia can result from even trivial injuries. previous studies have reported anosmia after surgical treatment of anterior communicating artery (acoa) aneurysms and suggest that its prevalence depends on the mode of approach to the aneurysm. it has been mentioned that there are three mechanisms of olfactory nerve damage during frontal lobe retraction (depression and elevation) : partial or total avulsion of the olfactory nerve from the cribriform plate [14, 6, 9 ] ; injury during nerve dissection ; and direct pressure damage [2, 9 ]. suggested that maintaining the anatomic integrity of the nerve is important to preserve olfactory function. even a mild retractive pressure on the nerve can lead to temporary or permanent lesions [2, 6 ]. it is said that this is because the microvasculature lying on the dorsal surface of the nerve, which is constituted of microscopic branches of the ethmoidal arteries nourishing the nerve, is fragile to retraction and compression of the frontal lobe. suzuki at al. reported that 42% of patients with bilateral preservation of the olfactory nerves in bifrontal craniotomy for anterior communicating artery aneurysms developed anosmia. they also reported that one - third of the patients who had both olfactory tracts damaged or severed during surgery had normal olfactory function. invisible factors affecting the olfactory nerve, such as heat injury to the nerve by drilling the bone surrounding the nerve or direct pressure damage to the medial frontal lobe (septal area) by retraction, might cause anosmia. furthermore, vries at al. reported no improvement of olfactory nerve function at follow - up after having olfactory nerve damage after the operation, which indicates that the regenerative capacity of the olfactory nerve is limited. thus, in cases in which a mechanical disruption (neurotmesis) of the fila olfactoria has occurred, regeneration would not be expected. on the other hand, rouit and murali noted that traumatic anosmia may recover at any time from a few days up to 5 years. however, in our research, we have focused only on maintaining the anatomic integrity of the olfactory nerve. anatomically, the olfactory bulb is located on the cribriform plate of the olfactory fossa, which is a couple of millimeters lower than the surrounding cranial base. the depth of the olfactory fossa is 4.8 (0.611.7) mm in the anterior part, 5.0 (015.5) mm in the middle part, and 3.2 (010.0) mm in the posterior part. thus, the olfactory filaments are fairly well protected in the cribriform plate against the shearing stresses of the brain that often accompany even minor injuries to the head. from our research, the olfactory nerve could easily be pulled out irrespective of the degrees of pulling from the frontal base towards the posterior direction. however, the olfactory nerve could withstand more force when pulled laterally against the axis of the olfactory nerve. this is because there is only a tiny bone in the posterior wall (planum sphenoidale, frontal edge) compared to the anterior wall of the olfactory fossa. on the other hand, there is a large lateral wall bilaterally along the entire olfactory fossa (fig. thus, it might hold the nerve in the cribriform plate when it is pulled laterally. there is a possibility that the pulling force is absorbed by the lateral wall of the olfactory fossa when the nerve is pulled laterally. however, there was no significant difference in the strength of the olfactory nerve between pulling the nerve 15 and 30 laterally. therefore, with frontal lobe retraction in a posterior direction from the olfactory bulb, the olfactory nerve is at high risk of being pulled out, but in a lateral direction, retraction is relatively safe from the perspective of maintaining the anatomical integrity of the olfactory nerve. however, if the nerve is pulled more laterally, it is pressed harder against the bone of the lateral wall of the olfactory fossa and is twisted against the axis of this nerve. but these data should be considered in realistic surgical settings like bifrontal approach and pterional approach, and one should try to measure the strength of olfactory nerve drawn by spatula pressure and of the own weight of the brain shifting to get a better clinical impact.fig. 6histological study of the olfactory fossa in the coronal view demonstrating that there is a large lateral wall bilaterally at any part of the olfactory fossa. hematoxylin and eosin (h&e) stain, original magnification 20 histological study of the olfactory fossa in the coronal view demonstrating that there is a large lateral wall bilaterally at any part of the olfactory fossa. hematoxylin and eosin (h&e) stain, original magnification 20 several techniques have been reported to prevent perioperative olfactory nerve injury [1, 2, 4, 9, 12 ]. most of them are techniques that minimize the effect on the olfactory nerve from frontal lobe retraction during surgery. nakayama recommended that frontal lobe retraction should be done towards the superficial direction to put less tension on the olfactory nerve. suzuki at al. reported that, to prevent nerve injury on the operated side, dissection of the nerve at the beginning of the microsurgical procedures is recommended, and the key to successful separation of the olfactory tract from the brain is never to apply pressure in a downward direction posteriorly from the olfactory bulb, but to continually apply upward pressure (toward the olfactory bulb) on the frontal lobe to which the tract is adherent. aydin. reported that olfactory nerve function could be preserved at a relatively high rate of 85%. this high rate probably resulted from the microtechnique used during the relatively cautious frontal lobe retraction, which was less than 1.5 cm. in addition, the nerve is pulled out by a strength of only 34 g. the present study had some limitations because, during the actual operation, the nerves are pulled while retracting the brain. thus, these data of strength alone do not provide definitive information that would be useful during operation. if preoperative coronal ct scanning is done, and the surrounding bone structure of the cribriform plate is investigated, that is, the depth of the olfactory fossa from the surrounding anterior skull base in every direction, which direction the olfactory nerve can be pulled to minimize damage to the nerve as much as possible can be identified. in each group, the absolute value of the strength of the olfactory nerves varied widely. this difference may come from not only the direction of pulling the nerve, but also from gender, aging, the length of time after death, and other factors. this study found that frontal lobe retraction in a posterior direction may be dangerous from the point of view of the postoperative prevalence of anosmia, but retraction in a lateral direction may be relatively safe. | olfactory dysfunction may influence the quality of life tremendously. this study investigated the strength of the human olfactory nerve at the frontal skull base using cadavers. a total of 180 olfactory nerves were examined in 90 human cadaveric heads. the cut edges of the olfactory nerves were pulled until they were pulled out from the skull base. in the first set of 30 cases, each right olfactory nerve was pulled 0 laterally and 0 upward, and each left olfactory nerve was pulled 0 laterally and 15 upward. in the second set of 30 cases, each right olfactory nerve was pulled 0 laterally and 15 upward, and each left olfactory nerve was pulled 15 laterally and 15 upward. in the third set of 30 cases, each right olfactory nerve was pulled 15 laterally and 15 upward, and each left olfactory nerve was pulled 30 laterally and 15 upward. the strength of the olfactory nerve was measured when pulled in each direction. there was no significant difference in the strength of the olfactory nerves when pulling them in the postero - upward direction between 0 and 15 upward. the strengths of the olfactory nerves when pulling them in the postero - lateral direction 0 and 15 laterally were 3.141.87 and 4.051.70 g (mean standard deviation [sd ]), respectively ; the difference was almost significant. the olfactory nerve could be pulled more laterally than posteriorly because the retraction force is absorbed by the lateral wall of the olfactory fossa. |
in the absence of telomerase, telomere shortening limits the replicative lifespan of human cells thus providing a stringent tumor suppressive mechanism. however if dna damage checkpoints are compromised, cells can continue to divide until the telomeres lose their end - capping function and are subjected to dna double - strand break repair activity that results in telomere - telomere fusion events. the resulting dicentric chromosomes can initiate cycles of anaphase - bridging, breakage, and fusion that generate the large - scale genomic rearrangements common in human cancer. telomere - driven mutation is therefore a mechanism that generates variation in tumor cell populations, upon which clonal selection can operate and facilitate progression. we have developed high - resolution, single - molecule technologies to study telomere length and fusion in detail ; these methods have provided a level of clarity that was hitherto impossible to achieve in human cells. in colorectal cancer we showed that telomere erosion, dysfunction, and fusion not only precede the adenoma / carcinoma transition, but may also be pre - existent in the normal cells in which the initial mutation occurs. in chronic lymphocytic leukemia (cll) we observed extreme telomere erosion and fusion, consistent with cll b - cells undergoing a telomere - driven crisis. importantly, this was also detected in a subset of patients with early - stage disease prior to clinical progression. our recent large - scale analysis of telomere length and fusion in early - stage cll has allowed definition of the telomere length threshold below which telomere fusion is detected and revealed the prognostic value of stratifying patients according to this parameter. patients with telomeres below the telomere fusion threshold had a significantly shorter overall survival that was even more prognostic in early - stage disease patients (p < 00001, hr = 19.3), and telomere dysfunction was the dominant variable in a multivariate analysis. based on these observations we hypothesize that short dysfunctional telomeres provide a mutator mechanism capable of driving genomic instability and disease progression. it is therefore important to understand the molecular basis of telomere fusion and how this drives mutation and clonal evolution. we have also investigated the process of fusion between short dysfunctional telomeres in human cells by the direct isolation and characterization of the dna sequence of telomere fusion events. irrespective of the tissue or cell model analyzed, we observed a consistent mutational profile with deletion into the subtelomeric dna and microhomology at the fusion junction ; this mutational profile is consistent with alternative non - homologous end - joining (a - nhej) processes. a - nhej involves the coordinated interaction of multiple proteins with nucleating, scaffolding, and resection activity, as well as those ultimately executing dna ligation. we therefore examined the contribution of dna ligase iii (lig3)-dependent a - nhej and dna ligase iv (lig4)-dependent classical - nhej (c - nhej) pathways in mediating fusion between short dysfunctional telomeres. using a dominant negative telomerase (dn - htert) we induced telomere erosion, fusion, and the onset of a telomere crisis in hct116 cells in which the lig3 or lig4 genes had been inactivated using recombinant adeno - associated virus - mediated gene targeting. both wild - type and lig4 clones displayed large - scale genomic rearrangements and telomere fusions, but readily escaped crisis following the re - establishment of telomerase activity. fusions were also detected in lig3 cells but strikingly no clones escaped crisis ; after 2 to 3 months no cells remained in these cultures. all lig3 clones escaped crisis following complementation with a wild - type lig3 cdna, but none escaped following complementation with cdnas containing either a deletion in the lig3 brca1 c - terminal domain or a a874d point mutation, both of which are required for the interaction of lig3 with x - ray repair cross - complementing protein 1 (xrcc1). these data demonstrate an absolute requirement for lig3 in mediating the escape from a telomere - driven crisis. in order to gain some insight into the underlying mechanisms by which lig3 facilitated the escape from crisis, we undertook a detailed molecular characterization of telomere fusion events mediated by lig3 or lig4. sister chromatid telomere fusion events were detected in both lig3 and lig4cells, however there was a marked reduction in interchromosomal events in lig4 cells. sequencing of interchromosomal fusions from lig3 cells revealed a higher incidence of breakpoints within telomere repeats and a reduction in microhomology at the fusion junction. our data demonstrated the involvement of both lig3 and lig4 in the fusion of short dysfunctional telomeres, but also indicated that fusions involving lig3 provide a selective advantage to cells undergoing a telomere - driven crisis that facilitates clonal evolution and escape from crisis. the mechanism by which lig3 facilitates the escape from crisis is not clear ; our hypothesis is that interchromosomal fusions, which predominate in cells that can not escape crisis, are more mutagenic and detrimental to the cells in which they occur ; whereas fusion between sister chromatids resulting in localized amplification and deletion events may confer a selective advantage. we consider that the relative balance between these events dictates the ability of cells to escape crisis and that this is modulated by the activities of a - nhej and c - nhej at short dysfunctional telomeres (fig. 1). further work should elucidate the contributions of other components of the a - nhej pathway in telomere fusion and the escape from crisis, in addition to examining the mutational impact that these pathways have in the context of the evolving cancer genome. figure 1.schematic illustrating our hypothesis for how lig3 may affect the ability of cells to escape a telomere - driven crisis. cells with short dysfunctional telomeres undergo crisis, during which telomere fusion results in the creation of dicentric chromosomes and the initiation of anaphase - bridging, breakage, and fusion cycles ; the ensuing genomic instability leads to large - scale genomic rearrangements. clonal evolution results in cells that escape crisis ; these cells exhibit fewer interchromosomal fusion events compared to fusions between sister chromatids, whereas interchromosomal fusions predominate in cells that can not escape crisis. we consider that this is governed by the relative activities of ligase iii - dependent alternative non - homologous end - joining (a - nhej) compared to ligase iv - dependent classic non - homologous end - joining (c - nhej) pathways. schematic illustrating our hypothesis for how lig3 may affect the ability of cells to escape a telomere - driven crisis. cells with short dysfunctional telomeres undergo crisis, during which telomere fusion results in the creation of dicentric chromosomes and the initiation of anaphase - bridging, breakage, and fusion cycles ; the ensuing genomic instability leads to large - scale genomic rearrangements. clonal evolution results in cells that escape crisis ; these cells exhibit fewer interchromosomal fusion events compared to fusions between sister chromatids, whereas interchromosomal fusions predominate in cells that can not escape crisis. we consider that this is governed by the relative activities of ligase iii - dependent alternative non - homologous end - joining (a - nhej) compared to ligase iv - dependent classic non - homologous end - joining (c - nhej) pathways. telomere fusion during crisis is ultimately a cellular survival mechanism that provides short - term relief from telomere erosion, but importantly also facilitates genome instability that allows for the generation of the genomic rearrangements that can facilitate progression. our work identifies the a - nhej pathway as essential for the ability of cells to clonally evolve and escape a telomere crisis. this indicates that intervention(s) in this pathway may sensitize cells with short dysfunctional telomeres and thus provide a potential therapeutic target in the subsets of tumors in which short dysfunctional telomeres confer such a poor prognosis. moreover, given that telomere dysfunction occurs early in tumorigenesis this may open up the possibility of therapeutic intervention prior to clinical progression. this work was supported by funding to the baird laboratory from cancer research uk, leukemia and lymphoma research and the national institute for social care and health research ; and to the hendrickson laboratory from the united states national institutes of health (gm088351) and the national cancer institute (ca15446). | telomere dysfunction and fusion play key roles in driving genomic instability and clonal evolution in many tumor types. we have recently described a role for dna ligase iii (lig3) in facilitating the escape of cells from crisis induced by telomere dysfunction. our data indicate that lig3-mediated telomere fusion is important in facilitating clonal evolution. |
the renin - angiotensin - aldosterone system (raas) plays an important role in the control of cardiovascular and renal homeostasis by regulating vascular tone, blood pressure (bp), and fluid volume [1, 2 ]. angiotensin ii (ang ii) is a physiologically active component of the raas, produced via an enzymatic cascade that begins with angiotensinogen (agt) cleaving renin (ren) to form angiotensin i (ang i), which is then cleaved by the angiotensin converting enzyme (ace) to form ang ii. ang ii causes vasoconstriction directly by activating ang ii type 1 (at1) receptors on vascular smooth muscle, affects fluid volume via at1 receptor activation in the proximal tubule, resulting in renal sodium and water reabsorption, and plays an important role in the regulation of fluid balance by stimulating aldosterone secretion from the zona glomeruloza of the adrenal glands. ace inhibitors, ang ii receptor antagonists, and aldosterone receptor antagonists have been used as therapeutic interventions to treat hypertension. the genes of the renin - angiotensin have been linked to and/or associated with hypertension in animal models and humans. recently, transgenic rodent models have been developed that over express both human ren and angiotensinogen, which leads to hypertension via chronic overproduction of ang ii. specific examples include the murine double transgenic line (ang 204/1 ren 9), which produces a mean arterial bp 40 mmhg higher than background mice (c57bl/6j) that lack the human genes. in addition, transgenic rats harboring the mouse renin-2 gene developed hypertension, cardiac hypertrophy, and renal damage. the tsukuba hypertensive mice (thm), which express the human ren and angiotensinogen genes, have been proven to develop hypertension. originally, the raas was viewed solely as an endocrine system, in which angiotensinogen of hepatic origin is secreted intothe systemic circulation and cleaved by ren and ace to produce the active peptide ang ii. however, there is increasing evidence that suggests a raas may reside within several organs or tissues, including kidney, lung, heart, and vascular smoothmuscle cells (smc), where it is believed to act in a functionally independent paracrine / autocrine fashion. this hypothesis is further supported by the fact that all components of the raas in the heart, kidney, and lung contain the ace component [3, 6 ]. additionally, high concentrations of ang ii have been demonstrated in the plasma, heart, and kidney of thm [7, 8 ]. in the kidney, prostaglandins (pgs) are important mediators of hemodynamic regulation, salt and water homeostasis, and ren release [9, 10 ]. the main pg in the kidney is pge2, which is synthesized from arachidonic acid (aa) by enzymatic reactions, particularly cyclooxygenases and prostaglandin e synthases (pges). cyclooxygenase (cox) derived pgs have two distinct membrane - anchored isoenzymes, cox-1 and cox-2. cox-1 is constitutively expressed and found in most normal body tissues, while cox-2 is expressed in normal tissues at low levels and is highly induced by proinflammatory mediators in inflammation, injury, and pain settings. the membrane - associated pges-1 (mpges-1) is inducible and functionally linked to cox-2, while mpges-2 is constitutive and coupled to both cox isoforms. it has been suggested that regulation of cox-2 in the kidney is altered by the raas system [9, 12 ]. in thm mice, increased expression of cox-2 in the macula densa has been reported, and an important role for raas in cardiac hypertrophy has been noted. overexpression of cox-2 has also been observed in the aldosterone - treated animals in normotensive and hypertensive rats. in the lung, significant reduction in bp was seen in pg ep1 receptor - deficient mice and was accompanied by increased ren - ang activity. there are currently no published reports on the cellular expression and microanatomic location of cox-1, cox-2, mpges-1, mpges-2 in hypertensive transgenic mice. therefore, using immunohistochemistry, we investigated the microanatomic location and cellular expression of cox-1, cox-2, mpges-1, mpges-2 in kidney, lung, and heart tissues obtained from renin - angiotensingen transgenic mice. this study is the first to report on the pulmonary and cardiorenal microanatomic expression of these molecules in this animal model for human hypertension. 15- to 20-week - old male (n = 15) and female (n = 15) double transgenic mice (h - ang 204/1 h - ren 9) were used in the study. the mice were derived from a founder colony of 5 male mice expressing human angiotensinogen (h - ang 204/1) and 6 females expressing human renin (h - ren6), obtained from dr. curt sigmund at the university of iowa, school of medicine. at charles river laboratories (wilmington, massachusetts), female mice that expressed human ren were bred with angiotensinogen - expressing males to produce the double transgenic line. all the procedures were in compliance with the pfizer ann arbor laboratories animal care and use committee. lung, kidney, and heart tissues were obtained from the mice at the time of necropsy. tissues were fixed in 10% neutral buffered formalin for 24 hours and embedded in paraffin wax. 3m - thick sections were then cut and stained immunohistochemically with antibodies to cox-1, cox-2, mpges-1, and mpges-2. to analyze the expression of cox-1, cox-2, mpges-1, and mpges-2, 3m sections were cut from formalin - fixed, paraffin - embedded blocks, mounted on positively charged glass slides, dried, and then loaded on the automated immunostainer (room temperature using a ventana discovery (ventana medical systems, tucson, az). sections were incubated for 30 minutes with serum free dakocytomation protein blocker (dako corporation, dako, ca) and then rinsed and incubated for 4 minutes with avidin - biotin blocking solution (ventana medical systems). antigen retrieval was completed with a ventana specialty solution (8 = ph) (ventana medical systems). automation included exposure to 100l of primary anti - cox-1 (1:200), cox-2 (1:20), mpges-1 (1:750), or mpges-2 (1:1000) antibody (cayman chemical, ann arbor, mi) diluted with reagent diluent (ventana medical systems) at room temperature for 60 minutes. 100l of the appropriate anti - rabbit biotinylated igg linking solution (vector laboratories) was applied to each section at 1:200 dilution for 60 minutes at room temperature. sections were again rinsed and allowed to react with 100l of diaminobenzidine (dab detection kit) substrate solution (ventana medical systems) for 8 minutes, followed by counterstaining with hematoxylin and then bluing reagent for 4 minutes each, removed from the autostainer, washed in warm water, dehydrated through graded alcohol, cleared in xylene, and cover slipped. control reactions included (1) sections incubated with the omission of primary antibody and processed as mentioned above, and (2) sections incubated with normal rabbit serum instead of the primary antibody and processed as above. the pulmonary and cardiorenal cellular expression and distribution of cox-1, cox-2, mpges-1, and mpges-2 are summarized in tables 1, 2, 3, and 4. staining intensity ranged from negative () to strong (+ + +). in the kidney, strong diffuse cytoplasmic cox-1 expression occurred in the distal convoluted tubules (dct), cortical collecting ducts, and medullary collecting ducts, while proximal convoluted tubules (pct) lacked cox-1 expression (see figure 1(a) and table 1). moderate (+ +) diffuse cox-1 cytoplasmic staining was present in vascular endothelial cells (ec) and smc, cortical interstitial cells (ic), the glomerular visceral epithelium, and the capsular parietal epithelium. mild (+) cox-1 expression was present in medullary ic, glomerular podocytes, and the medullary ascending limb (mal). expression of cox-1 was equivocal in the macula densa. in the lungs and heart, cox-1 was strongly expressed in alveolar macrophages, the bronchial and bronchiolar epithelium, and cardiac vascular endothelial cells, but moderately expressed in alveolar septa, bronchial smooth muscle cells, pulmonary vascular endothelial cells, pulmonary vascular ec, and cardiac vascular smc. cox-1 was not expressed in cardiac myocytes (see figure 1(b) and table 2). mpges-2 was expressed in the kidney at a moderate diffuse level in pct and dct, capsular parietal epithelium, and medullary collecting ducts (see figure 2(a) and table 1). mild expression was present in macula densa, vascular smc and ec, glomerular podocytes and the visceral epithelium, cortical collecting ducts, and the mal. pulmonary expression of mpges-2 included strong diffuse staining in bronchial and bronchiolar epithelial cells (see figure 2(b) and table 2), moderate staining in alveolar macrophages and septa, mild staining in bronchial smc, and pulmonary vascular ec and smc. renal cox-2 expression was strong in the pct and dct (see figure 3(a) and table 3), moderate in macula densa, vascular smc and ec, and medullary collecting ducts, and mild in cortical and medullary interstitial cells, cortical collecting ducts, and the mal. no expression was present in glomeruli or the capsular (parietal) epithelium. in the lung, marked (+ + +) cox-2 expression was present in alveolar macrophages, and the bronchial and bronchiolar epithelium (see figure 3(b) and table 4). cardiac vascular ec had mild cox-2 expression, while cardiac myocytes and cardiac vascular smc were negative. for mpges-1, expression was mild in renal vascular smc and ec, cortical and medullary collecting ducts, and the mal (see figure 4(a) and table 3). expression in the macula densa, glomerular (visceral) epithelium, and capsular (parietal) epithelium was equivocal. mpges-1 was the highest in bronchial and bronchiolar epithelial cells (see figure 4(b) and table 4). mild expression was present in alveolar macrophages and alveolar septa, while expression in pulmonary vascular ec and smc was equivocal. mild mpges-1 expression was present in cardiac vascular ec and cardiac vascular smc, while other cardiac microanatomic locations lacked expression. pgs are modulators of physiological functions and contribute to ren release, regulation of renal microvascular hemodynamics, salt balance, and bp controlvia mechanisms involving the regulation of vascular tone and renal excretory function. the kidney is capable of synthesizing all types of pgs, especially pge2 and pgi2, which influence urinary sodium excretion directly through inhibition of the tubulartransport function and indirectly through the regulation of renin - angiotensin system activity. cox, a rate - limiting enzyme in the pg biosynthesispathway, exists in two major isoforms : the constitutive cox-1 and the inducible cox-2. cox-1 is expressed constitutively at varying levels in the majority of tissues and generally plays a role in tissue homeostasis. in this study, the highest degree of renal cox-1 expression occurred in the dct, cortical collecting ducts, and medullary collecting ducts, while mild - to - moderate cox-1 expression occurred in other microanatomic renal locations. these results are concurrent with cox-1 being the most abundant cox isoform that is constitutively expressed in the kidney and regionally localized in the renal vasculature, collecting ducts, and papillary ic across various species [20, 21 ]. further evidence supporting these results includes previous work in which various species (cow, dog, guinea pig, human, monkey, mouse, rabbit, rat, and sheep) exhibited high levels of cox-1 expression in the collecting ducts [20, 22, 23 ]. since cox-1 is expressed in the collecting ducts of the nephron, an active area in the regulation of sodium excretion in both laboratory animals and humans, it is not surprising that bp increases in cox-1 deficient mice compared with wild - type controls. nonselective nonsteroidal anti - inflammatory drugs (nsaids) may aggravate renin - independent, sodium - sensitive hypertension, possibly in part by inhibition of the cox-1 responsible for sodium excretion. in rats infused with selective cox inhibitors, ns-398 or meloxicam, direct renal interstitial volume expansion significantly increased renal interstitial hydrostatic pressure and fractional excretion of sodium. our finding that most cardiac microanatomic locations had cox-1 expression with the exception of cardiac myocytes is consistent with other studies showing cox-1 expression in both ec and smc of the fetal ductus arteriosus of pigs, rats, sheep, and humans [2831 ]. furthermore, cox-1 was constitutively expressed in the ec of the aorta and microvasculature, fibrous connective tissue of the tricuspid valve, and chordae tendinae of the heart of dog. other examples of cox-1 expression in lungs include human lungs, and the bronchiolar epithelium and smooth muscle, alveolar macrophages, ec, and vascular smc of rat. as a result mpges-2 is constitutively expressed in several tissues, is not induced during inflammation, and has been proposed to mediate pge2 production from both cox isoforms [11, 35 ]. in our study, moderate mpges-2 expression was present in pct and dct, the capsular parietal epithelium, and medullary collecting ducts, while mild or no expression was present in other renal microanatomic locations. in other studies, high expression of mpges was reported in distal tubules, and medullary collecting ducts in normal mouse kidney. other studies using primary cultures of neonatal ventricular myocytes have shown that mpges-2 is constitutively synthesized in myocytes and is not regulated. cox-2 is expressed in variable locations in the kidney across various species. cox-2 deficient mice exhibited severe disruption of renal development and function, suggesting an important role for cox-2 in renal development [38, 39 ]. the normal rodent and canine kidney have prominent constitutive cox-2 expression in the macula densa and thick ascending limb of loop of henle, whereas cox-2 is absent at these sites in the normal primate and human kidney. in our study, cox-2 was present in all microanatomic renal locations, with the exception of the glomeruli and capsular (parietal) epithelium. this finding is consistent with the known regulatory association between cox-2 and ren reported by schnermann. demonstration that indomethacin and sc58236 (a selective cox-2 inhibitor) can decrease bp and suppress plasma ren activity in rats lends further support to this association [41, 42 ]. the marked increase of cox-2 expression in the pulmonary epithelium in our study is consistent with the known role of respiratory epithelia as a first line of defense. pulmonary epithelial cells have an important role in airway homeostasis, perform many important biological functions, and represent the first line of defense against infection. activation of the renin - angiotensin system has also been demonstrated in acute asthma. cardiac vascular ec and smc had cox-2 expression, while no expression was present in cardiac myocytes or smc in our study. an important role for ang ii in the regulation of cox-2 has been reported. mpges-1 was present in renal vascular ec and smc, cortical and medullary collecting ducts, mal, all pulmonary microanatomic locations, and cardiac vascular ec and smc, while no expression was present in other renal or cardiac microanatomic locations. these findings are consistent with increased mpges expression described in patients with hyperprostaglandin e syndrome. mpges-1 was also induced by the proinflammatory cytokine in cardiac myocytes and fibroblasts. in conclusion, in hypertensive mice there are (a) significant microanatomic variations in the pulmonary, renal, and cardiac distribution and cellular localization of mpges-1, mpges-2, cox-1, and cox-2 and (b) no differences in expression between genders. | hypertensive mice that express the human renin and angiotensinogen genes are used as a model for human hypertension because they develop hypertension secondary to increased renin - angiotensin system activity. our study investigated the cellular localization and distribution of cox-1, cox-2, mpges-1, and mpges-2 in organ tissues from a mouse model of human hypertension. male (n = 15) and female (n = 15) double transgenic mice (h - ang 204/1 h - ren 9) were used in the study. lung, kidney, and heart tissues were obtained from mice at necropsy and fixed in 10% neutral buffered formalin followed by embedding in paraffin wax. cut sections were stained immunohistochemically with antibodies to cox-1, cox-2, mpges-1, and mpges-2 and analyzed by light microscopy. renal expression of cox-1 was the highest in the distal convoluted tubules, cortical collecting ducts, and medullary collecting ducts ; while proximal convoluted tubules lacked cox-1 expression. bronchial and bronchiolar epithelial cells, alveolar macrophages, and cardiac vascular endothelial cells also had strong cox-1 expression, with other renal, pulmonary, or cardiac microanatomic locations having mild - to - moderate expression. mpges-2 expression was strong in the bronchial and bronchiolar epithelial cells, mild to moderate in various renal microanatomic locations, and absent in cardiac tissues. cox-2 expression was strong in the proximal and distal convoluted tubules, alveolar macrophages, and bronchial and bronchiolar epithelial cells. marked mpges-1 was present only in bronchial and bronchiolar epithelial cells ; while mild - to - moderate expression was present in other pulmonary, renal, or cardiac microanatomic locations. expression of these molecules was similar between males and females. our work suggests that in hypertensive mice, there are (a) significant microanatomic variations in the pulmonary, renal, and cardiac distribution and cellular localization of cox-1, cox-2, mpges-1, and mpges-2, and (b) no differences in expression between genders. |
extensive surgical resection is often abandoned on identification of wide and deep tumor invasion at the hilar bile duct intraoperatively, because it is difficult to achieve curative resection. on determining that curative resection of the perihilar cholangiocarcinoma is not possible, an intraoperative decision about whether or not to perform palliative bilio - enteric bypass surgery must be made. in patients with complex separation of the hilar bile ducts, it is often difficult to insert multiple biliary stents percutaneously through percutaneous transhepatic biliary drainage (ptbd) or endoscopic retrograde cholangiography approach, as well as maintain them successfully for a prolonged time. a majority of these patients are required to keep one or more ptbd tubes with or without endoscopic retrograde biliary drainage (erbd).123456 in such intractable situations, palliative bilio - enteric bypass to remove ptbd tubes is seriously considered as a measure to improve the quality of life. however, bilio - enteric bypass for multiple separate hilar bile ducts is technically demanding, and its patency is also much shorter than expected because multiple bilio - enteric anastomoses are often performed to the residual tumor tissue itself. biliary reconstruction is difficult in extensive hilar bile duct necrosis resulting from iatrogenic bile duct injury, similar to cases of advanced perihilar cholangiocarcinoma.2 for such difficult biliary reconstruction, we devised a surgical technique termed cluster hepaticojejunostomy (hj) that involves placement of multiple biliary stents and single wide porto - enterostomy on the surrounding connective tissue.7 we have performed the cluster hj technique in more than 20 cases so far, indicative of its clinical applicability. herein, we present the technical details of cluster hj with discussion on further modification. in patients with advanced perihilar cholangiocarcinoma, resectability is usually determined after dissection of the hepatoduodenal ligament. once it is decided to perform palliative bile duct resection (bdr) for bilio - enteric drainage, the infiltrated hilar bile duct mass is transected without further dissection at the level of the hilar plate. retrograde approach toward the hilar bile duct through longitudinal incision of the distal common bile duct often provides wide exposure of the involved hilar bile ducts. at the transected bile duct cut surface, major bile duct openings, which typically include four hepatic ducts each from the right anterior and posterior sections, and left medial and lateral sections, are meticulously identified with the use of the preoperative imaging studies as a road map ; and subsequently, the dilated caudate ducts (usually two or three in number) are identified. it is essential to open all dilated intrahepatic ducts because any missed bile duct can result in late onset of cholangitis. silastic stents of three different sizes (obtained by cutting the silastic t - tubes with outer diameters of 3, 4, and 5 mm) are inserted into each opening after size matching. one or two side holes are made only at the intrahepatic - side ends of the stent tubes, because the tumor can grow into the stent tube through the side holes around the hj site. bleeding from the bile duct cut surface or hilar plate should be meticulously sutured because it can be a source of hemobilia. unification ductoplasty is also attempted at two adjacent bile duct openings, by which three or four duct openings can be unified sequentially. a routine roux - en - y jejunal limb is prepared and pulled up through the retrocolic tunnel. a longitudinal incision is made at the antemesenteric border and posterior wall anastomosis was performed with two or three segmented continuous running sutures with 5 - 0 prolene. since we designed one continuous suture to cover each 1 cm - length of the posterior suture line, three segmented continuous sutures were necessary for a 3 cm - wide bile duct opening. subsequently, each silastic internal stent was inserted and securely transfixed with 5 - 0 prolene. anterior wall anastomosis was done through multiple interrupted sutures at 1.5 mm intervals (25 to 40 sutures). at the start of hj, multiple suture materials with double - arm needles were radially placed to spread the bile duct opening. this radial spreading anchoring traction technique is important to perform cluster hj because it provides a good operative field, as well as facilitates secure full - thickness anastomosis of the anterior wall. very meticulous water - tight anterior wall anastomosis is required because anastomosis to the residual tumor tissue per se can result in anastomotic failure. the intraoperative leak test with diluted methylene blue solution is performed through the pre - existing ptbd tubes. the ptbd tube is not removed during surgery, and kept until follow - up tube cholangiography and/or radioisotope hepatobiliary scintigraphy, usually 2 weeks after surgery. a 72-year - old male patient was referred from other hospital with two ptbd tubes due to perihilar cholangiocarcinoma of bismuth - corlette type iv. thus, the surgical procedure was planned at 3 weeks after resolution of obstructive jaundice and pancreatitis. despite locally advanced tumor, we identified that the left portal vein and left hepatic artery were encased as well as the second - order branches of the right hepatic ducts were deeply involved by the tumor. thus, we abandoned curative resection and decided to perform palliative bdr with cluster hj. the patient had total destruction of hilar bile duct structures, hence the tumor over the right hepatic artery was removed (fig. four intrahepatic bile duct openings (3 from the right liver and 1 from the left liver) were identified and their edges were repaired to prepare them for anastomosis ; subsequently, four size - matched silastic stents were temporarily inserted (fig. the caudate ducts were not markedly dilated, thus small coronary dilators were inserted to identify their course. the anterior wall of the bile duct opening was anchored with multiple 5 - 0 prolene sutures (fig.. the posterior wall of the bile duct opening was continuously sutured with 5 - 0 prolene sutures after dividing into 3 segments with 2 internal intervening sutures (fig. the corner stitches were retracted with rubber vessel loops to make the operative field wide. four silastic stents and one additional stent were firmly inserted into each beaked bile duct opening after forceful mechanical dilatation, and then transfixed with 5 - 0 prolene suture (fig. the anterior wall was finally closed by using interrupted sutures that were previously anchored (fig. after these surgical procedures, a leak test was performed with injection of methylene blue solution through the pre - existing ptbd tube. the patient recovered uneventfully without any noticeable complication, and is currently undergoing adjuvant chemoradiation therapy. due to recent advances in radiological and endoscopic interventions, biliary stenting has become a widely performed technique for treatment of biliary strictures. its advantages are that it is minimally invasive and well tolerated in a majority of patients. application of biliary stents as palliative treatment of biliary malignancies is widely accepted in clinical practice.8 furthermore, retrievable biliary stents are also used for treatment of benign biliary strictures.91011 however, various stent - associated complications, such as stent occlusion and cholangitis, restrict their wide use in benign strictures. although biliary stents can be deployed endoscopically or radiologically with acceptably low risk, their long - term patency is still regarded as suboptimal.12 therefore, surgery has major advantages on anastomotic patency, especially for treatment of benign bile duct diseases. for reconstruction of multiple bile ducts, various surgical techniques with multiple separate anastomoses with or without unification ductoplasty have been introduced. a healthy duct stump is a prerequisite for such surgical techniques, thus it is usually not appropriate to apply them to tumor - infiltrated bile duct stumps. therefore, we developed a reliable surgical technique of cluster hj that would enable secure reconstruction of severely damaged hilar bile ducts. because residual tumors at the hepatic hilum grow progressively after palliative bdr of r2 resection, occlusion is expected in any hj. this sequence suggests that duct - to - mucosa approximation is not necessary because it is not technically possible or reasonable. instead if the internal stent is properly placed, the corresponding intrahepatic bile duct will remain open. for this purpose, we cut a silastic t - tube to a length of 4 - 5 cm and introduced one or two side - holes at its peripheral end only, for an internal stent crossing hj. other types of thin - walled silastic tubes are not recommended because such internal stents need to endure the strong compression pressure caused by tumor progression. secure outer anastomosis is important to prevent anastomotic leak ; thus any connective tissue at the hepatic hilum should be preserved even if the tissue appears to be invaded by the tumor. this surgical procedure of cluster hj is comparable to the kasai procedure, which is a porto - enterostomy performed by suturing a jejunal loop to the hepatic parenchyma that surrounds the transected hepatic ducts.1314 we have sporadically applied cluster hj to patients with iatrogenic bile duct injury prior to our previous report.7 after a phase in our learning curve, we recognized that cluster hj has a better patency rate than other more conventional reconstruction methods. the results of our previous study indicated that cluster hj is a useful surgical technique for the secure reconstruction of severely damaged hilar bile ducts. thus far, we have sporadically performed cluster hj with satisfactory mid - term patency outcomes in more than 20 patients who were indicated. after palliative bdr for unresectable perihilar cholangiocarcinoma, the primary aim is to prevent the insertion of additional ptbd for at least the first 6 months. usually, the life expectancy of patients who require additional ptbd is much shortened ; thus, this practice should be avoided if possible, until it is clearly beneficial for the patient. as the tumor progressed, some intrahepatic ducts began to be dilated according to the status of biliary obstruction although internal stents appeared to be kept in their position. since the silastic t - tube segment has a radio - opaque line, its location can be exactly determined on the liver computed tomography images. proper positioning of the corresponding internal stent on computed tomography images is indicative of a theoretically opened intrahepatic duct, despite considerable degree of possible stricture across the stent. hence, non - absorbable suture material (5 - 0 prolene) was used for transfixation of the internal stents, to ensure that the stents would be not displaced too early. the first is to make the multiple bile duct openings wide and parallel after sequential side - to - side unification. there is no limitation in the width of unified bile duct openings, thus a unified opening of 5 cm in width can be readily acceptable for cluster hj. the second is to radially anchor the traction suture materials at the anterior anastomotic line. we suggest an interval of 1.5 mm, thus a large bile duct opening requires numerous anterior sutures. the third is to make multiple segmented continuous sutures with a suggested interval of 8 - 10 mm ; thus, 2 or 3 intervening sutures are usually necessary for most sizable bile duct openings. secure performance of biliary reconstruction is also important to perform timely, postoperative concurrent chemoradiation therapy. we usually recommend such adjuvant therapy to all patients undergoing r1 or r2 resection regardless of patient age because nearly all patients who have recovered from cluster hj can tolerate such adjuvant anti - tumor therapy. in conclusion,, it will be necessary to perform cluster hj in a larger number of patients including those with benign hilar bile duct injury. | secure reconstruction of multiple hepatic ducts that are severely damaged by tumor invasion or iatrogenic injury is a challenge. failure of percutaneous or endoscopic biliary stenting requires lifelong placement of one or more percutaneous transhepatic biliary drainage (ptbd) tubes. for such difficult situations, we devised a surgical technique termed cluster hepaticojejunostomy (hj), which can be coupled with palliative bile duct resection. the cluster hj technique consisted of applying multiple internal biliary stents and a single wide porto - enterostomy to the surrounding connective tissues. the technique is described in detail in the present case report. performing cluster hj benefits from three technical tips as follows : making the multiple bile duct openings wide and parallel after sequential side - to - side unification ; radially anchoring and traction of the suture materials at the anterior anastomotic suture line ; and making multiple segmented continuous sutures at the posterior anastomotic suture line. thus, cluster hj with radial spreading anchoring traction technique is a useful surgical method for secure reconstruction of severely damaged hilar bile ducts. |
structural genomics has been successful in determining the structures of many unique proteins in a high throughput manner. since 2001, these efforts have resulted in more than 6,772 structure depositions to the pdb, 3,251 of which are from the nih - sponsored protein structure initiative (psi) centers. nevertheless, the number of known protein sequences is much larger than the number of experimentally solved protein structures, and this number is growing at an unprecedented rate due to large - scale genome sequencing and meta - genomics projects. homology (or comparative) modeling methods make use of experimental protein structures to build models for evolutionarily related proteins. this technique predicts the three - dimensional structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). for every experimentally determined structure, often models for hundreds of proteins can be derived using a variety of established methods for comparative protein structure modeling methods, which dramatically increases the structural coverage of protein sequences. structural genomics and homology modeling thereby complement each other in the exploration of protein structure space. the quality of a comparative protein structure model depends on the evolutionary distance between the sequence of the target protein to be modeled and the template structure. in general comparative models sharing more than 40% of sequence identity with the template comparative protein structure models are routinely used in a widespread range of biomedical applications such as the rational design of mutagenesis experiments, the interpretation of disease - related mutations, or structure - based virtual screening studies [46 ]. however, despite this tremendous growth in protein structure data in recent years, structural information is often not used to its full extent in biomedical research projects. one reason is that experimental protein structures are only available for a small subset of all known protein sequences. a significant impediment in using 3d - models effectively is that model information on a specific protein is distributed over different web sites in heterogeneous formats, using incompatible accession code systems. we have developed the protein model portal (pmp, http://www.proteinmodelportal.org/) as part of the psi structural genomics knowledgebase (http://kb.psi-structuralgenomics.org/kb/) in order to provide a single portal which gives access to the various models that can be leveraged from psi targets and other experimental protein structures [7, 8 ]., we describe the challenges that exist for building such a model portal, present the technical implementation, show specific examples for accessing the portal, briefly report on the community workshop held on applications of protein models in biomedical research, and discuss future developments of the project. while information about experimental structures is maintained within a single resource, the world wide pdb, access to protein model information is by far more complicated. the major reasons are listed here : various algorithmic approaches [1016 ] with different strengths and weaknesses have been developed for building 3-dimensional models of proteins. also the quality of individual models highly depends on the evolutionary proximity to the selected structural templates. a consensus view of the results obtained from different modeling resources is very often helpful in identifying the most reliable solution.the content of the pdb database is organized around experimental information (structure centric), whereas model information is based on sequence databases (sequence centric) ; difficulties arise due to the fact that sequence database content and accession codes are often transient and frequently incompatible between different databases.models often cover only fragments of a protein sequence. the different segments modeled for a given target protein are usually based on various alternative alignments, alternative templates, or result from diverse modeling procedures.models are not stable information by itself, but reflect the current status of sequence and structure databases at time of modeling. models have to be revised, as new, more suitable template structures become available. also, changes in the primary sequence of target proteins in the sequence databases necessitate remodeling.models have typically few intrinsic annotations (in comparison to uniprot or pdb databases) which go beyond the alignment to the template structure. changes in the functional annotation of a protein database entry (e.g., uniprot ; interpro) occur independent from and more frequently than changes of the primary amino acid sequence, which would require rebuilding of the model.models are not experimental observations, but the results of theoretical predictions. while standards for describing the reliability and limitations of the most commonly used experimental structure determination techniques have been established, the spectrum of reliability and applicability of current modeling methods is broad and the level of uncertainty is significantly higher [19, 20 ]. therefore, detailed information about each individual model is crucial for assessing its expected accuracy, and thereby determining its scope of applicability.the number of currently available models is orders of magnitudes larger than that of experimental structures (ca. 7 million vs. 50,000) and, therefore, poses technical challenges in efficient data handling.as model information is complex one needs to estimate the expected accuracy and reliability of a specific model or for parts thereof ; users of models are often unsure to which extent a model can be used for a given application. various algorithmic approaches [1016 ] with different strengths and weaknesses have been developed for building 3-dimensional models of proteins. also the quality of individual models highly depends on the evolutionary proximity to the selected structural templates. a consensus view of the results obtained from different modeling resources is very often helpful in identifying the most reliable solution. the content of the pdb database is organized around experimental information (structure centric), whereas model information is based on sequence databases (sequence centric) ; difficulties arise due to the fact that sequence database content and accession codes are often transient and frequently incompatible between different databases. the different segments modeled for a given target protein are usually based on various alternative alignments, alternative templates, or result from diverse modeling procedures. models are not stable information by itself, but reflect the current status of sequence and structure databases at time of modeling. models have to be revised, as new, more suitable template structures become available. also, changes in the primary sequence of target proteins in the sequence databases necessitate remodeling. models have typically few intrinsic annotations (in comparison to uniprot or pdb databases) which go beyond the alignment to the template structure. changes in the functional annotation of a protein database entry (e.g., uniprot ; interpro) occur independent from and more frequently than changes of the primary amino acid sequence, which would require rebuilding of the model. models are not experimental observations, but the results of theoretical predictions. while standards for describing the reliability and limitations of the most commonly used experimental structure determination techniques have been established, the spectrum of reliability and applicability of current modeling methods is broad and the level of uncertainty is significantly higher [19, 20 ]. therefore, detailed information about each individual model is crucial for assessing its expected accuracy, and thereby determining its scope of applicability. the number of currently available models is orders of magnitudes larger than that of experimental structures (ca. 7 million vs. 50,000) and, therefore, poses technical challenges in efficient data handling. as model information is complex one needs to estimate the expected accuracy and reliability of a specific model or for parts thereof ; users of models are often unsure to which extent a model can be used for a given application. to summarize, model information is heterogeneous, highly context dependent, dynamic, and consists of large volumes of data. consequently, a community workshop held at rutgers on archiving structural models of biological macromolecules has recommended no longer accepting models as part of the pdb archive, and to establish a portal for protein model information. following this recommendation, and taking into account the challenges mentioned before, we have developed the pmp as a web portal which federates resources from model providers, experimental protein structures (pdb), and functional annotation databases. the aim of the pmp is to foster the effective usage of molecular model information in biomedical research by providing unified access independent of individual sequence nomenclature and accession code system and by supporting the development of data standards to facilitate exchange of information and algorithms. furthermore, pmp aims to provide a forum for discussions between developers of modeling methods and applied biomedical researchers on best - practices, including methods for quality assessment, guidelines for the publication of theoretical models, and educational resources on usage of models for different biological applications. a common reference system for target proteins is established based on the uniprot database by calculating cryptographic md5 hashes for all full length amino acid sequences. this approach combines database entries with identical amino acid sequences, while proteins sequences which differ by at least one amino acid are kept separate (fig. 1). this reference system is continuously updated with every new uniprot release. the protein modeling portal federates protein model data from different providers (csmp, jcsg, mcsg, nesg, nysgxrc, jcmm, modbase, and swiss - model repository [22, 23 ]) by integrating model meta information : the segment of a target protein for which a model is available, template structure (pdb i d and chain), and sequence identity between the target and the template sequences. searchable indices for matching amino acid sequences, sequence based similarity searches (using blast), and accession code database queries are generated by mapping the model data into the md5-based reference system. the protein model portal database containing this information has been implemented using mysql (fig. 2).fig. 1reference system based on md5 cryptographic hash sums for uniprot full - length target sequences. in this system, identical target protein sequences are grouped together independent from their individual database accession codes (e.g., hemoglobin beta chain from human, chimpanzee, and bonobo), while entries which differ in at least one amino acid position are kept separate (e.g., 7e v variant of human sickle cell anemia hemoglobin)fig. meta information about the available models, i.e., the target protein, template structure, and sequence identity, is retrieved from each partner resource. the uniprot database is used to generate a reference system based on md5 cryptographic hash sums of the full - length primary sequences. searchable indices are generated for all proteins with model information, allowing for accession code - based queries, matching of amino acid sequence fragments, and sequence similarity searches. the portal communicates with all partner resources and the psi structural genomics knowledge base via web services. the three - dimensional coordinates of a model, as well as functional annotation information from uniprot and interpro is retrieved dynamically in real time when required to generate the web page reference system based on md5 cryptographic hash sums for uniprot full - length target sequences. in this system, identical target protein sequences are grouped together independent from their individual database accession codes (e.g., hemoglobin beta chain from human, chimpanzee, and bonobo), while entries which differ in at least one amino acid position are kept separate (e.g., 7e v variant of human sickle cell anemia hemoglobin) schematic flow of data in protein model portal. meta information about the available models, i.e., the target protein, template structure, and sequence identity, is retrieved from each partner resource. the uniprot database is used to generate a reference system based on md5 cryptographic hash sums of the full - length primary sequences. searchable indices are generated for all proteins with model information, allowing for accession code - based queries, matching of amino acid sequence fragments, and sequence similarity searches. the portal communicates with all partner resources and the psi structural genomics knowledge base via web services. the three - dimensional coordinates of a model, as well as functional annotation information from uniprot and interpro is retrieved dynamically in real time when required to generate the web page the pmp is queried from the psi structural genomics knowledgebase using web services based on soap (simple object access protocol) and rest (representational state transfer). users can also access the portal directly using the web - based graphical user interface implemented with php at http://www.proteinmodelportal.org/. functional annotation for individual target proteins is retrieved in real - time from the respective annotation providers using web services : information about individual domains is retrieved from the interpro database using das, whereas sequence annotations from the uniprot database are retrieved using rest from the uniprot server (http://www.uniprot.org). the three - dimensional coordinates of the individual models are stored at the different model providers and retrieved by the portal in real - time when required for the visualization of the model overview page. preview images of the structural model, generated using molscript, render, and raster3d, provide the first quick preview of the protein model. the alignment between the target sequence and the template is inferred dynamically on - the - fly by structural superposition of the final model to the template structure using mammoth. the consequent use of portal technologies allows federating a set of heterogeneous resources into a single portal while at the same time ensuring consistency of the exchanged data. the query form allows the user to search using database accession codes such as uniprot, ipi, genbank, refseq, or entrez identifiers, to search for models built on a specific template structure, or to query the portal database with the amino acid sequence of a protein of interest. for a specific target protein, all models available from the participating resources as well as experimental structures in the pdb are displayed in graphical and tabular form (fig. 3). when a specific model is selected, the model detail page shows information on the template which was used for building the model, provides a graphic preview of the model structure, as well as the target - template sequence alignment (fig. 4). the model can be displayed as ribbon representation inside the webpage using the astex viewer plugin. a download link points to the original home page of the model provider, where additional information about the model building procedure can be found. information about the target primary sequence is retrieved dynamically from the uniprot database, listing all entries that share the same primary sequence. the domain structure of the target sequence is displayed based on pfam domain annotation which is retrieved dynamically from the interpro database. finally, the pmp provides links to services for template selection, model building and quality assessment.fig. information about available models is queried from the model portal database ; information on experimental structures is retrieved from the psi sgkb using web servicesfig. information about the model provider, the segment of the target protein (e.g., mlp - like protein 34 ; arabidopsis thaliana) covered by the model, and the template structure used for model building, are stored in the portal database. all other information required for building the webpage, such as the coordinates of the model, the pfam domain structure, and uniprot annotation of the protein sequence, is retrieved dynamically graphical overview of model and experimental structure information available for a specific protein entry. information about available models is queried from the model portal database ; information on experimental structures is retrieved from the psi sgkb using web services typical view of a model detail page. information about the model provider, the segment of the target protein (e.g., mlp - like protein 34 ; arabidopsis thaliana) covered by the model, and the template structure used for model building, are stored in the portal database. all other information required for building the webpage, such as the coordinates of the model, the pfam domain structure, and uniprot annotation of the protein sequence, is retrieved dynamically the current release of the portal allows searching 7.6 million model structures provided by the different partner sites : csmp, jcsg, mcsg, nesg, nysgxrc, jcmm, modbase, swiss - model repository. at least one model is available for 3.0 million unique sequences out of the 7.1 million distinct sequences of the current uniprot release (14.4). the distribution of chain lengths of the models shows a maximum around 150 residues, indicating that the majority of models consist of single domains. however, more than one quarter of the models have significantly longer chains of more than 300 residues (fig. 5). as model quality is correlated with sequence similarity between target and template, we have analyzed the best available model (i.e., the one with highest sequence identity) for each residue in the model portal database (fig. 6). as expected, for the majority of modeled residues (41%) the templates shared between 20% and 40% sequence identity with the target.fig. the maximum around 150 residues indicates that the majority of models consist of single domains. however, more than one quarter of the models have significantly longer chains of more than 300 residuesfig. 6model quality on residue level. for each residue, the model with the highest sequence identity between target and template is considered. the pie chart shows the percentage of residues which can be modeled at a certain identity level. for the majority of modeled residues (41%) the targets shares between 20% and 40% sequence identity with the templates distribution of chain length. the histogram shows the length distribution of models provided by the model portal. the maximum around 150 residues indicates that the majority of models consist of single domains. however, more than one quarter of the models have significantly longer chains of more than 300 residues model quality on residue level. for each residue, the model with the highest sequence identity between target and template is considered. the pie chart shows the percentage of residues which can be modeled at a certain identity level. for the majority of modeled residues (41%) the targets shares a workshop on applications of protein models in biomedical research was held in san francisco in july 2008, where protein structure modelers explored how models are used in biomedical research, and which requirements and challenges exist for the different applications. the workshop program involved a first day of 16 presentations on topics that ranged from the coverage of protein sequence - structure space to the uses of modeling in medicine. on the second day, the participants discussed the state - of - the - art in applying molecular modeling to biomedical problems, requirements and challenges for various applications, as well as ways to strengthen the collaboration between the modeling and experimental communities. the pmp has been recognized as an important means for increasing the impact of molecular modeling on biology and medicine, and specific recommendations for the further development of the pmp were made. the outcome of the workshop will be made available for the community in the form of a white paper. we have established a single portal that allows users to simultaneously and transparently perform searches against all model information made available by the participating sites, using various protein identifiers or amino acid sequence as the query input. the next step in the development of the pmp will include a common interface for submitting interactive modeling requests to different automated modeling services, tools for comparing and assessing the quality of protein structural models, and the possibility to map a wide variety of functional annotations to the protein models. the consequent usage of web services will not only allow to coordinate information provided by other services within the portal, but also to complement sequence - based resources such as genome browsers, with model - based information. the development of widely accepted standards for data exchange and of tools for quality assessment of models would be one of the challenges in the future, which are expected to be addressed during a second pmp workshop in 2009. | structural genomics has been successful in determining the structures of many unique proteins in a high throughput manner. still, the number of known protein sequences is much larger than the number of experimentally solved protein structures. homology (or comparative) modeling methods make use of experimental protein structures to build models for evolutionary related proteins. thereby, experimental structure determination efforts and homology modeling complement each other in the exploration of the protein structure space. one of the challenges in using model information effectively has been to access all models available for a specific protein in heterogeneous formats at different sites using various incompatible accession code systems. often, structure models for hundreds of proteins can be derived from a given experimentally determined structure, using a variety of established methods. this has been done by all of the psi centers, and by various independent modeling groups. the goal of the protein model portal (pmp) is to provide a single portal which gives access to the various models that can be leveraged from psi targets and other experimental protein structures. a single interface allows all existing pre - computed models across these various sites to be queried simultaneously, and provides links to interactive services for template selection, target - template alignment, model building, and quality assessment. the current release of the portal consists of 7.6 million model structures provided by different partner resources (csmp, jcsg, mcsg, nesg, nysgxrc, jcmm, modbase, swiss - model repository). the pmp is available at http://www.proteinmodelportal.org and from the psi structural genomics knowledgebase. |
polymers are pervasive throughout all aspects of the modern age and, as described by rolf mlhaupt, without polymers, modern life would be impossible because polymers secure the high quality of life and serve as pacemakers for modern technologies. consequently, the urge to find renewable resources to reduce consumption of fossil fuel feedstocks, decrease the use of energy intensive products, and solve recycling and/or degradation issues has led to the investigation of biopolymers. additionally, the increase in fuel costs observed over the past 30 years dictates a transition to polymeric precursors that do not rely expressly on fossil fuels. however, the full life cycle assessment of most bio - based polymers is not well reported, and a focus on the environmental impacts of biopolymers will be necessary. as unrecycled polymers end up in the oceans (estimated 5 billion kg per year), water - degradable polymers such as polycarbonates that degrade into carbon dioxide and diols represent a reasonable class of materials to investigate further. polycarbonates are mainly used where toughness, high optical transparency, and solvent resistance are required. although more stable toward hydrolysis than their polyester counterparts, polycarbonates are able to degrade in water and are, thus, considered as aqueous degradable materials. strategies for the use of renewables in polycarbonates include polycondensation of feedstock products such as aliphatic diols, ring - opening polymerization of 6-membered cyclic carbonates, epoxides and carbon dioxide - based systems, polycondensation of cereal - based products such as 1,4:3,6-isosorbide, and natural phenols to afford copolymers. noteworthy, biorenewable - based polycarbonates have found applications as powder coating agents and as drug delivery vehicles. previous efforts from our lab have led to the synthesis of polycarbonates derived from both carbohydrates and quinic acid. in an effort to broaden the scope to natural products with biological activities, amide)s derived from rather inexpensive ferulic acid and a hydroxyl - amino acid. it was hypothesized that bio - based poly(carbonate amide)s derived from fa and a hydroxyl - containing amino acid would possess interesting mechanical properties arising from the rigid conjugated cinnamic acid core of fa and the h - bonding potential of the amino acid component, while also having the potential to undergo hydrolytic breakdown and lead to biologically beneficial byproducts and carbon dioxide. ferulic acid (4-hydroxy-3-methoxycinnamic acid, fa) is a secondary metabolite of the biosynthesis of lignin, derived from phenylalanine and tyrosine via shikimate pathway. 0.3 wt % in wheat bran), fruits, vegetables, and plant tissues. fa can be naturally found in its free form, as monomers, dimers, or polymers but also as esters formed by condensation with hydroxyl acids, alcohols, or saccharides or as amides formed by condensation with amines. because of its antioxidant properties, fa exhibits a wide range of therapeutic effects such as anticancer, antidiabetic, cardio - protective, neuro - protective, and anti - inflammatory activities. it has also found use as a food preservative and has shown antibacterial activity against gram - negative and -positive bacteria, including the anthrax agent b. subtilis. moreover, fa has been incorporated into a biodegradable polymer as a pendant group to enhance antioxidant properties, specifically for tissue engineering applications. ester), poly(ether ester)s, a methacrylic fa copolymer, and a polyamide. to the best of our knowledge, all chemicals and reagents were used as received from sigma - aldrich co or vwr international. caution : special precautions should be taken when working with phosgene precursors, including diphosgene and triphosgene. they are highly toxic by inhalation and ingestion ; use of personal protective equipment, including a respiratory mask, is recommended. tetrahydrofuran (thf) and dichloromethane were purified by passage through solvent purification system (jc meyer solvent systems) and used as dried solvents. column chromatography was performed on a combiflash rf4x (teledyne isco) with redisep rf column (teledyne isco). ir spectra were recorded on a shimadzu ir prestige attenuated total reflectance fourier - transform infrared spectrometer (atr - ftir) and analyzed using irsolution v. 1.40 software. size exclusion chromatography (sec) measurements were performed on a waters chromatography inc. (milford, ma) system equipped with an isocratic pump model 1515, a differential refractometer model 2414, and a four - column set of 5 m guard (50 7.5 mm), styragel hr 4 5 m dmf (300 7.5 mm), styragel hr 4e 5 m dmf (300 7.5 mm), and styragel hr 2 5 m dmf (300 7.5 mm) using dmf (0.05 m libr) as the eluent (1.00 ml / min) at 70 c. polymer solutions were prepared at a concentration of about 5 mg / ml and an injection volume of 200 l was used. data collection and analysis were performed with empower 2 v. 6.10.01.00 software (waters, inc.). the system was calibrated with poly(ethylene oxide) standards (polymer laboratories, amherst, ma) ranging from 106 to 174 000 da, and an additional internal calibration based on the oligomeric fraction was also realized (supporting information, methods to monitor the polymerizations). glass transition temperatures (tg) and melting points (mp) were measured by differential scanning calorimetry (dsc) on a mettler - toledo dsc822 (mettler - toledo, inc., measurements of tg were recorded with a heating rate of 15 c / min, and those for mp were recorded with a heating rate of 10 c / min. thermogravimetric analysis (tga) was performed under an ar atmosphere using a mettler - toledo model tga / dsc 1, with a heating rate of 10 c / min. all steady - state emission, excitation, and 3 d spectra were obtained with a horiba fluoromax4 with automatic polarizers. measurements were performed in dmf in matched quartz cuvettes with path lenghts of 1 cm. to a solution of tyrosine ethyl ester hydrochloride (1.57 g, 6.39 mmol) in ch2cl2 (23 ml) were added ferulic acid (1.97 g, 6.39 mmol) and hobt (863.3 mg, 6.39 mmol). the reaction was cooled down at 0 c, and et3n (2.7 ml, 19.17 mmol) and edci (992 mg, 6.39 mmol) were added. was added a saturated aqueous solution of nahco3, and the crude product was extracted with ch2cl2, dried over na2so4, and filtered. the solvent was removed, and the crude product was purified by flash chromatography 0% to 100% of acoet in hexane over 40 min (80 g silica cartridge). the solvent was removed to give the desired product as a white foam (1.65 g, 4.28 mmol, 67%). ftir (atr) max (cm) : 36003150, 1726, 1667, 1514. h nmr spectrum (500 mhz, cdcl3) : 7.54 (d, j = 15.5 hz, 1 h), 7.28 (d, j = 0.9 hz, 1 h), 7.046.93 (m, 4 h), 6.89 (dd, j = 8.1, 0.9 hz, 1 h), 6.786.73 (m, 2 h), 6.296.24 (m, 2 h), 6.07 (br s, 1 h), 4.99 (dtd, j = 6.7, 5.7, 1.0 hz, 1 h), 4.21 (q, j = 7.2, 2 h), 3.88 (s, 3 h), 3.16 (dd, j = 14.1, 5.7 hz, 1 h), 3.08 (dd, j = 14.1, 5.7 hz, 1 h), 1.29 (t, j = 7.2, 3 h) ppm. c nmr spectrum (125 mhz, cdcl3) : 172.0 (c), 166.0 (c), 155.3 (c), 147.6 (c), 146.7 (c), 142.1 (ch), 130.4 (2 ch), 127.3 (c), 127.0 (c), 122.5 (ch), 117.2 (ch), 115.5 (2 ch), 114.7 (ch), 109.6 (ch), 61.7 (ch2), 55.9 (ch3), 53.5 (ch), 37.2 (ch2), 14.2 (ch3) ppm. ms (esi) m / z (%) 386.2 (100, [m + h ]). esihrms calcd for c21h24no6 (m+h) 386.1604 ; found 386.1609 ; mp = 70 c. to a solution of l - tyrosine ethyl ester chlorohydrate (5.4 g, 22.0 mmol) in thf (110 ml) were added dmap (536.8 mg, 4.4 mmol), et3n (9.2 ml, 65.9 mmol), and tbdmscl (8.3 g, 54.9 mmol). the reaction was stirred at reflux for 3 h. after cooling down at room temperature, the mixture was filtered and the filtrate was purified by flash chromatography 0% to 100% of acoet in hexane over 40 min (80 g silica cartridge). the solvent was removed to give the desired product as a colorless oil (5.8 g, 17.9 mmol, 81%). ftir (atr) max (cm) 1732, 1510. h nmr spectrum (500 mhz, cdcl3) : 7.04 (d, j = 8.4 hz, 2 h), 6.76 (d, j = 8.4 hz, 2 h), 4.15 (q, j = 7.1 hz, 2 h), 3.66 (dd, j = 7.7, 5.5 hz, 1 h), 3.00 (dd, j = 13.7, 5.5 hz, 1 h), 2.81 (dd, j = 13.7, 7.7 hz, 1 h), 1.24 (t, j = 7.1 hz, 3 h), 0.97 (s, 9 h), 0.18 (s, 6 h) ppm. c nmr spectrum (125 mhz, cdcl3) : 175.1 (c), 154.5 (c), 130.2 (2 ch), 129.8 (c), 120.1 (2 ch), 60.9 (ch2), 55.9 (ch), 40.4 (ch2), 25.7 (3 ch3), 18.2 (c), 14.2 (ch3), 4.4 (2 ch3) ppm. ms (esi) m / z (%) 324.2 (100, [m + h ]). esihrms calcd for c17h30no3si (m+h) 324.1995 ; found 324.2004. to a solution of 10 (5 g, 15.5 mmol) in ch2cl2 (77 ml) were added ferulic acid (5 g, 15.5 mmol) and hobt (2.1 g, 15.5 mmol). the reaction was cooled down at 0 c and et3n (4.3 ml, 31.0 mmol) and edci (2.4 g, 15.5 mmol). was added a saturated aqueous solution of nahco3 and the crude product was extracted with ch2cl2, dried over na2so4, and filtered. the solvent was removed under vacuum, and the crude product was purified by flash chromatography 0% to 50% of acoet in hexane over 50 min (120 g silica cartridge). the solvent was removed to give the desired product as a yellow foam with 10% of 10 (6.39 g, 12.8 mmol, 83%). ftir (atr) max (cm) 35003150, 1732, 1657, 1593. h nmr spectrum (500 mhz, cdcl3) h : 7.53 (d, j = 15.6 hz, 1 h), 7.056.94 (m, 5 h), 6.88 (d, j = 8.4 hz, 1 h), 6.74 (d, j = 8.4 hz, 2 h), 6.25 (d, j = 15.6 hz, 1 h), 6.19 (d, j = 7.8 hz, 1 h), 4.96 (dt, j = 7.8, 5.7 hz, 1 h), 4.17 (qd, j = 7.0, 1.8 hz, 2 h), 3.88 (s, 3 h), 3.163.08 (m, 2 h), 1.25 (t, j = 7.1 hz, 3 h), 0.96 (s, 9 h), 0.17 (s, 6 h) ppm. c nmr spectrum (125 mhz, cdcl3) : 171.8 (c), 165.6 (c), 154.6 (c), 147.6 (c), 146.8 (c), 141.7 (ch), 130.3 (2 ch), 128.5 (c), 127.0 (c), 122.4 (ch), 120.0 (2 ch), 117.4 (ch), 114.8 (ch), 109.5 (ch), 61.5 (ch2), 55.8 (ch3), 53.4 (ch), 37.1 (ch2), 25.6 (3 ch3), 18.1 (c), 14.1 (ch3), 4.5 (2 ch3) ppm. ms (esi) m / z (%) 500.2 (100, [m + h ]) ; esihrms calcd for c27h38no6si (m+h) 500.2468 ; found 500.2453 ; mp = 47 c. a solution of 11 (2.07 g, 4.14 mmol) and pyridine (3.1 ml, 6.2 mmol) in ch2cl2 (12.5 ml) was added dropwise to a solution of p - nitrophenyl chloroformate (1.69 g, 8.28 mmol) in ch2cl2 (25.1 ml). the reaction was stirred at room temperature for 7 h. water was added, and the crude was extracted with ch2cl2, dried over na2so4, and filtered. the residue was purified by flash chromatography on a silica cartridge (80 g, hexane / acoet 0 to 50% over 30 min). the desired product was obtained as a yellow foam (1.95 g, 2.93 mmol, 71%). h nmr spectrum (500 mhz, cdcl3) : 8.30 (d, j = 9.2 hz, 2 h), 7.57 (d, j = 15.5 hz, 1 h), 7.48 (d, j = 9.2 hz, 2 h), 7.21 (d, j = 8.7 hz, 1 h), 7.157.09 (m, 2 h), 6.98 (d, j = 8.4 hz, 2 h), 6.75 (d, j = 8.4 hz, 2 h), 6.37 (d, j = 15.5 hz, 1 h), 6.22 (d, j = 7.8 hz, 1 h), 4.96 (dt, j = 7.8, 5.6 hz, 1 h), 4.19 (qd, j = 7.1, 2.6 hz, 2 h), 3.93 (s, 3 h), 3.213.05 (m, 2 h), 1.27 (t, j = 7.1 hz, 3 h), 0.96 (s, 9 h), 0.17 (s, 6 h) ppm. c nmr spectrum (125 mhz, cdcl3) : 171.7 (c), 164.8 (c), 155.4 (c), 154.7 (c), 151.0 (c), 150.2 (c), 145.5 (c), 140.55 (c), 140.50 (ch), 134.5 (c), 130.3 (2 ch), 128.4 (c), 125.4 (2 ch), 122.3 (ch), 121.6 (2 ch), 121.0 (ch), 120.8 (ch), 120.1 (2 ch), 111.5 (ch), 61.6 (ch2), 56.1 (ch3), 53.4 (ch), 37.1 (ch2), 25.6 (3 ch3), 18.1 (c), 14.2 (ch3), 4.5 (2 ch3) ppm ; ms (esi) m / z (%) 665.3 (100, [m + h ]). esihrms calcd for c34h41n2o10si (m + h) 665.2530 ; found 665.2519 ; mp = 56 c. to a solution of 12 (1.74 g, 2.74 mmol) in ch2cl2 (13.7 ml) the reaction was stirred at room temperature for 23 h. a saturated solution of nahco3 was added, and the crude was extracted with ch2cl2, dried over na2so4, and filtered. the residue was purified by flash chromatography on a silica cartridge (120 g, hexane / acoet 0 to 100% over 40 min). the desired product was obtained as a white foam (1.28 g, 2.33 mmol, 85%). ftir (atr) max (cm) 33003150, 1782, 1732, 1661, 1514. h nmr spectrum (500 mhz, cdcl3) : 8.27 (d, j = 9.2 hz, 2 h), 7.52 (d, j = 15.6 hz, 1 h), 7.45 (d, j = 9.2 hz, 2 h), 7.15 (d, j = 8.7 hz, 1 h), 7.11 (br s, 1 h), 7.037.02 (m, 2 h), 6.95 (d, j = 8.4 hz, 2 h), 6.72 (d, j = 8.4 hz, 2 h), 6.51 (d, j = 7.9 hz, 1 h), 6.37 (d, j = 15.6 hz, 1 h), 4.96 (dt, j = 8.0, 5.7 hz, 1 h), 4.234.16 (m, 2 h), 3.86 (s, 3 h), 3.13 (dd, j = 14.1, 5.7 hz, 1 h), 3.03 (dd, j = 14.1, 6.2 hz, 1 h), 1.26 (t, j = 7.1 hz, 3 h) ppm. c nmr spectrum (125 mhz, cdcl3) : 171.9 (c), 165.5 (c), 155.5 (c), 155.3 (c), 150.9 (c),150.3 (c), 145.5 (c), 140.9 (c), 140.5 (ch), 134.3 (c), 130.3 (2 ch), 127.0 (c), 125.3 (2 ch), 122.3 (ch), 121.6 (2 ch), 120.74 (ch), 120.67 (ch), 115.5 (2 ch), 111.6 (ch), 61.8 (ch2), 56.0 (ch3), 53.7 (ch), 37.0 (ch2), 14.1 (ch3) ppm. ms (esi) m / z (%) 551.2 (100, [m + h ]). esihrms calcd for c28h27n2o10 (m + h) 551.1666 ; found 551.1654. to a solution of 13 (702 mg, 1.27 mmol) in pyridine (0.42 ml) was added dropwise triethylamine (0.36 ml, 2.55 mmol). the mixture was stirred at room temperature for 5 h and was quenched with a saturated solution of nahco3. the product was extracted with ch2cl2 and dried over na2so4, and the solvent was removed. the mixture was solubilized in ch2cl2, precipitated into meoh three times, and dried under vacuum to give the desired product as a yellowish solid which follows aaaa, aaaa, aaaa patterns in 22/15/63 proportions (389.5 mg, 0.94 mmol, 74%). ftir (atr) max (cm) 34003200, 1778, 1736, 1661, 1624, 1508. h nmr spectrum (500 mhz, cdcl3) : 7.617.51 (m, 14 h), 7.237.20 (m, 98 h), 6.436.23 (m, 28 h), 5.00 (br s, 14 h), 4.234.14 (m, 28 h), 3.87 (br s, 42 h), 3.273.13 (m, 28 h), 1.321.10 (m, 42 h) ppm. c nmr spectrum (125 mhz, cdcl3) : 171.5 (14 c), 165.1 (14 c), 152.0 (3.1 c(o)aaaa), 151.3 (8.8 caaaa), 151.22 (5.2 caaaa), 151.16 (8.8 c(o)aaaa), 150.7 (2.1 c(o)aaaa), 150.2 (8.8 caaaa), 150.0 (5.2 caaaa) 141.1, 141.0, 140.9 (14 ch + 14 c), 134.2, 134.1, 134.0 (28 c), 130.52, 130.49 (28 ch), 122.6 (8.8 chaaaa), 122.5 (5.2 chaaaa), 120.94, 120.89, 120.8 (28 ch), 120.6 (14 ch), 111.5 (5.2 chaaaa), 111.4 (8.2 chaaaa), 61.7 (14 ch2), 56.1 (14 ch3), 53.3 (14 ch), 37.2 (14 ch2), 14.2 (14 ch3) ppm. tg = 134 c ; tp = 350 c. to a solution of 11 (532.5 mg, 1.07 mmol) in ch2cl2 (12 ml) were added diphosgene (79 l, 0.66 mmol) and a catalytic amount of activated charcoal. the solution was cooled to 45 c, and n, n - dimethylaniline (0.24 ml, 1.92 mmol) was added dropwise. the reaction was allowed to stir at 45 c and then warmed slowly to room temperature and reacted overnight. the salts and activated charcoal were removed by filtration, and the solvent was removed under vacuum. the product was purified by flash chromatography (24 g, 0% to 50% of acoet in hexane over 20 min) to lead to a white foam (492.8 mg, 0.88 mmol, 82%). ftir (atr) max (cm) : 1790, 1738, 1659, 1632, 1510. h nmr spectrum (500 mhz, cdcl3) : 7.57 (d, j = 15.6 hz, 1 h), 7.15 (d, j = 8.7 hz, 1 h), 7.137.06 (m, 2 h), 6.97 (d, j = 8.4 hz, 2 h), 6.75 (d, j = 8.4 hz, 2 h), 6.36 (d, j = 15.6 hz, 1 h), 6.12 (d, j = 7.7 hz, 1 h), 4.96 (dt, j = 7.7, 5.6 hz, 1 h), 4.20 (q, j = 7.2 hz, 2 h), 3.91 (s, 3 h), 3.14 (dd, j = 5.6, 2.6 hz, 2 h), 1.28 (t, j = 7.1 hz, 3 h), 0.97 (s, 9 h), 0.17 (s, 6 h) ppm. c nmr spectrum (125 mhz, cdcl3) : 171.6 (c), 164.7 (c), 154.8 (c), 150.7 (c), 148.9 (c), 141.5 (c), 140.4 (c), 135.0 (ch), 130.4 (2 ch), 128.3 (c), 122.1 (ch), 121.3 (ch), 120.7 (ch), 120.1 (2 ch), 111.5 (ch), 61.6 (ch2), 56.1 (ch3), 53.4 (ch), 37.1 (ch2), 25.6 (3 ch3), 18.2 (c), 14.2 (ch3), 4.4 (2 ch3) ppm. in the glovebox, a mixture of 14 (198 mg, 0.35 mmol) and agf (89.4 mg, 0.70 mmol) was prepared. then outside of the glovebox, acetonitrile (0.14 ml) and pyridine (0.56 ml) were added. the mixture was ventilated with a cartridge containing naoh to quench fumes of phosgene. the reaction mixture was stirred at room temperature for 15 min and was quenched with a saturated solution of nahco3. the mixture was solubilized in dmf, filtered through a pad of celite, then precipitated out into meoh three times, and dried under vacuum to give the desired product as a white solid which follows aaaa, aaaa, aaaa patterns in 5/5/90 proportions (80.9 mg, 0.20 mmol, 57%). ftir (atr) max (cm) : 33003200, 1789, 1737, 1659, 1622, 1510. h nmr spectrum (500 mhz, d6-dmso) : 8.678.47 (m, 20 h), 7.577.12 (m, 160 h), 6.72 (d, j = 15.9 hz, 20 h), 4.684.54 (m, 20 h), 4.183.98 (m, 40 h), 3.89 (br s, 60 h), 3.202.86 (m, 40 h), 1.350.94 (m, 60 h) ppm. c nmr spectrum (125 mhz, d6-dmso) : 171.5 (20 c), 164.9 (20 c), 151.7 (2 c(o)aaaa), 150.94 (16 caaaa), 150.88 (4 caaaa), 150.8 (16 c(o)aaaa), 150.4 (2 caaaa), 149.4 (20 c), 140.1 (20 c), 138.8 (20 ch), 135.5 (16 caaaa), 135.4 (4 caaaa), 134.5 (16 caaaa), 134.4 (4 caaaa), 130.4 (32 chaaaa), 130.3 (8 chaaaa), 122.7 (20 ch), 122.1 (20 ch), 121.0 (8 chaaaa), 120.8 (32 chaaaa), 120.3 (20 ch), 111.8 (20 ch), 60.6 (20 ch2), 56.05 (20 ch3), 53.8 (20 ch), 36.1 (20 ch2), 14.0 (20 ch3) ppm. tg = 129 c, tp = 337 c. to a solution of 11 (1.1 g, 2.2 mmol) and 4-nitrophenyl chloroformate (225.2 mg, 1.1 mmol) in ch2cl2 (2.2 ml), at room temperature was added et3n (0.46 ml, 3.3 mmol). the residue was purified by flash chromatography on a silica cartridge (40 g, hexane / acoet 0 to 60% over 30 min). the desired product was obtained as a white foam (657.8 mg, 0.64 mmol, 58%, brm : 84%). ftir (atr) max (cm) : 1782, 1732, 1667, 1508. h nmr spectrum (500 mhz, cdcl3) : 7.54 (d, j = 15.6 hz, 2 h), 7.20 (d, j = 8.7 hz, 2 h), 7.087.05 (m, 4 h), 6.99 (d, j = 8.4 hz, 4 h), 6.75 (d, j = 8.4 hz, 4 h), 6.37 (d, j = 7.1 hz, 2 h), 6.36 (d, j = 15.6 hz, 2 h), 4.95 (dt, j = 7.9, 5.8 hz, 2 h), 4.18 (qd, j = 7.1, 2.3 hz, 4 h), 3.90 (s, 6 h), 3.183.01 (m, 4 h), 1.26 (t, j = 7.1 hz, 6 h), 0.96 (s, 18 h), 0.16 (s, 12 h) ppm. c nmr spectrum (125 mhz, cdcl3) : 171.4 (2 c), 154.7 (2 c), 151.2 (2 c), 150.7 (c), 141.0 (2 c), 140.7 (2 ch), 134.1 (2 c), 130.3 (4 ch), 128.4 (2 c), 122.5 (2 ch), 120.7 (2 ch), 120.6 (2 ch), 120.0 (4 ch), 111.5 (2 ch), 61.5 (2 ch2), 56.0 (2 ch3), 53.5 (2 ch), 37.0 (2 ch2), 25.6 (6 ch3), 18.1 (2 c), 14.1 (2 ch3), 4.5 (6 ch3) ppm. ms (esi / maldi) m / z (%) not detected. to a solution of 15 (4.76 g, 4.93 mmol) in ch2cl2 (25 ml) was added dropwise bf3oet2 (2.5 ml, 19.7 mmol). the reaction was stirred at room temperature for 23 h. a saturated solution of nahco3 was added, and the crude was extracted with ch2cl2, dried over na2so4, and filtered. the residue was purified by flash chromatography on a silica cartridge (4 g, hexane / acoet 0 to 100% over 15 min). the desired product was obtained as a white foam (2.55 g, 3.2 mmol, 65%). ftir (atr) max (cm) : 36003100, 1784, 1738, 1661, 1614, 1512. h nmr spectrum (500 mhz, d8-thf) : 8.18 (s, 2 h), 7.53 (d, j = 15.6 hz, 2 h), 7.46 (d, j = 8.0 hz, 2 h), 7.26 (d, j = 1.8 hz, 2 h), 7.17 (d, j = 8.2 hz, 2 h), 7.12 (dd, j = 8.2, 1.8 hz, 2 h), 6.97 (d, j = 8.4 hz, 4 h), 6.666.59 (m, 6 h), 4.83 (dt, j = 8.0, 6.3 hz, 2 h), 4.11 (q, j = 7.1 hz, 4 h), 3.88 (s, 6 h), 3.05 (dd, j = 13.9, 6.3 hz, 2 h), 2.96 (dd, j = 13.9, 6.7 hz, 2 h), 1.20 (t, j = 7.1 hz, 6 h) ppm. c nmr spectrum (125 mhz, d8-thf) : 172.2 (2 c), 165.3 (2 c), 157.3 (2 c), 152.3 (2 c), 151.1 (c),141.8 (2 c), 140.0 (2 ch), 135.6 (2 c), 130.8 (4 ch), 127.9 (2 c), 123.0 (2 ch), 122.4 (2 ch), 120.7 (2 ch), 115.7 (4 ch), 112.4 (2 ch), 61.3 (2 ch2), 56.01 (2 ch3), 54.5 (2 ch), 37.7 (2 ch2), 14.3 (2 ch3) ppm. h nmr spectrum (500 mhz, d6-dmso) : 9.24 (s, 2 h), 8.48 (d, j = 7.7 hz, 2 h), 7.447.37 (m, 4 h), 7.34 (d, j = 8.2 hz, 2 h), 7.21 (dd, j = 8.4, 1.8 hz, 2 h), 7.02 (d, j = 8.5 hz, 4 h), 6.74 (d, j = 15.8 hz, 2 h), 6.66 (d, j = 8.5 hz, 4 h), 4.52 (ddd, j = 8.8, 7.7, 5.8 hz, 2 h), 4.06 (q, j = 7.1 hz, 4 h), 3.91 (s, 6 h), 2.95 (dd, j = 13.9, 5.8 hz, 2 h), 2.86 (dd, j = 13.9, 8.8 hz, 2 h), 1.13 (t, j = 7.1 hz, 6 h) ppm. c nmr spectrum (125 mhz, d6-dmso) : 171.7 (2 c), 164.8 (2 c), 156.0 (2 c), 150.9 (2 c), 150.4 (c(o)aaaa),140.1 (2 c), 138.6 (2 ch), 134.5 (2 c), 130.0 (4 ch), 127.0 (2 c), 122.7 (2 ch), 122.3 (2 ch), 120.3 (2 ch), 115.1 (4 ch), 111.9 (2 ch), 60.5 (2 ch2), 56.1 (2 ch3), 54.2 (2 ch), 36.2 (2 ch2), 14.0 (2 ch3) ppm. ms (esi / apci / maldi) m / z not detected. to a solution of 16 (122.1 mg, 0.15 mmol) in pyridine (0.60 ml) at 0 c was added dropwise diphosgene (21 l, 0.17 mmol). the mixture was ventilated with a cartridge containing naoh to quench fumes of phosgene. the reaction mixture was stirred at room temperature for 2 h and was quenched with a saturated solution of nahco3. the product was an orange gel which was solubilized in warm dmf / dmso mixture and precipitated out into meoh three times and dried under vacuum to give the desired product as a white / yellowish solid which follows an aaaa pattern (91.3 mg, 0.11 mmol, 73%). ftir (atr) max (cm) : 34003200, 1776, 1736, 1667, 1625, 1601, 1504. h nmr spectrum (500 mhz, d6-dmso) : 8.60 (d, j = 7.6 hz, 45 h), 7.42 (d, j = 15.6 hz, 45 h), 7.417.24 (m, 315 h), 7.247.17 (m, 45 h), 6.72 (d, j = 15.8 hz, 45 h), 4.61 (q, j = 7.8 hz, 45 h), 4.06 (q, j = 6.8 hz, 90 h), 3.90 (s, 135 h), 3.142.95 (m, 90 h), 1.11 (t, j = 7.1 hz, 135 h) ppm. c nmr spectrum (125 mhz, d6-dmso) : 171.5 (45 c), 164.9 (45 c), 151.7 (23 c(o)aaaa), 150.9 (45 c), 150.4 (22 c(o)aaaa), 149.4 (45 c), 140.1 (45 c), 138.8 (45 ch), 135.4 (45 c), 134.5 (45 c), 130.3 (90 ch), 122.7 (45 ch), 122.1 (45 ch), 121.0 (90 ch), 120.3 (45 ch), 112.0 (45 ch), 60.6 (45 ch2), 56.08, 56.06 (45 ch3), 53.8 (45 ch), 36.1 (45 ch2), 14.0 (45 ch3) ppm. tg = 130 c, tp = 343 c. to a solution of 8 (70.3 mg, 0.18 mmol) in pyridine (0.34 ml) at 0 c was added dropwise diphosgene (12 l, 0.10 mmol). the mixture was ventilated with a cartridge containing naoh to quench fumes of phosgene. the reaction mixture was stirred at room temperature for 15 h and was quenched with a saturated solution of nahco3. the product was extracted with ch2cl2 and dried over na2so4, and the solvent was removed under vacuum. the brown film formed was solubilized in ch2cl2, precipitated into meoh three times, and dried under vacuum to give the desired product as a brownish solid which follows aaaa, aaaa, aaaa patterns in 37/23/40 proportions (35.4 mg, 0.086 mmol, 48%). during the reaction, a yellow solid part was formed, insoluble in ch2cl2, chcl3, pyridine, dmf, dmso, acetone, and h2o even after sonication and heating. this insoluble fraction could not be analyzed and consequently may explain the lower yield observed. ftir (atr) max (cm) : 34003200, 1778, 1732, 1666, 1628, 1504. h nmr spectrum (500 mhz, cdcl3) : 7.53 (br d, j = 15.0 hz, 36 h), 7.226.93 (m, 238 h), 6.476.22 (m, 72 h), 5.044.93 (m, 36 h), 4.18 (br q, j = 7.2, 6.5 hz, 72 h), 3.86 (br s, 108 h), 3.283.11 (m, 72 h), 1.42- 1.02 (m, 108 h) ppm. h nmr spectrum (500 mhz, d6-dmso) : 8.60 (br d, j = 7.7 hz, 36 h), 7.477.15 (m, 252 h), 6.766.65 (m, 72 h), 4.644.57 (m, 36 h), 4.104.01 (m,72 h), 3.89 (br s, 108 h), 3.172.93 (m, 72 h), 1.11 (br t, j = 7.1 hz, 108 h) ppm. c nmr spectrum (125 mhz, cdcl3) : 171.5 (36 c), 165.1 (36 c), 152.0 (8.3 c(o)aaaa), 151.3 (14.4 caaaa), 151.1 (21.6 caaaa), 150.7 (14.4 c(o)aaaa), 150.2 (13.3 c(o)aaaa), 150.0 (36 c), 141.1, 141.0, 140.9 (36 ch + 36 c), 134.1, 134.0 (72 c), 130.52, 130.49 (72 ch), 122.6 (14.4 chaaaa), 122.5 (21.6 chaaaa), 120.94, 120.89, 120.8 (72 ch), 120.6 (36 ch), 111.4 (21.6 chaaaa), 61.8 (36 ch2), 56.1 (21.6 ch3aaaa), 56.0 (14.4 ch3aaaa), 53.3 (36 ch), 37.2 (36 ch2), 14.2 (36 ch3) ppm. c nmr spectrum (125 mhz, d6-dmso) : 171.4 (34 c), 165.9 (34 c), 151.6 (8.3 c(o)aaaa), 150.93 (14.4 caaaa), 150.87 (21.6 caaaa), 150.8 (14.4 c(o)aaaa), 150.3 (13.3 c(o)aaaa), 149.4 (36 c), 140.1 (36 c), 138.7 (36 c), 135.5 (14.4 caaaa), 135.4 (21.6 caaaa), 134.44 (14.4 caaaa), 134.38 (21.6 caaaa), 130.4 (28.8 chaaaa), 130.3 (43.2 chaaaa), 122.6 (36 ch), 122.1 (36 ch), 121.0 (43.2 chaaaa), 120.8 (28.8 chaaaa), 120.3 (36 ch), 111.9 (21.6 chaaaa), 111.8 (14.4 chaaaa), 60.6 (36 ch2), 56.1 (36 ch3), 53.8 (36 ch), 36.1 (36 ch2), 14.0 (36 ch3) ppm. we envisioned that the poly(carbonate amide)s could be synthesized readily either by copolycondensation of a diol with a phosgene derivative or by homopolycondensation of an ab monomer obtained by prior conversion of one of the alcohol groups to an activated carbonate or chloroformate. the required diol monomer would first be obtained by coupling fa to a hydroxyl - containing amino acid by means of a peptidic bond (scheme 1). this approach would enable degradation to occur via two distinct pathways : aqueous hydrolysis of the carbonate bond and enzymatic cleavage of the amide bond. among the naturally occurring amino acids, serine, with its more reactive primary alcohol, was expected to be the most attractive coupling partner, followed by threonine, which contains a less reactive secondary alcohol, and finally tyrosine, which features a phenolic functionality that could lead to solubility issues for high molecular weight polymers. an ab monomer activated as a p - nitrophenyl carbonate 4 was initially designed, as the polymerization of similar species has been reported in the literature. this monomer was preferred to its more reactive chloroformate analogue because the synthesis of the latter requires the manipulation of phosgene derivatives. the monomer synthesis began from the commercially available l - serine, which was protected as its ethyl ester. this protection step was necessary to selectively convert the hydroxyl group to a silyl ether derivative and also for the later selective amidation with ferulic acid (scheme 2). the tbdms protecting group is cleavable under mild acidic conditions and was chosen for its stability under basic conditions used in the following steps. a peptidic coupling of this intermediate with ferulic acid generated 2 in two steps (51%). the structure was confirmed by the presence of a signal attributed to the quaternary carbon peak of the amide by c nmr (165.8 ppm) and by ftir (1658 cm) spectroscopies. the p - nitrophenyl carbonate 3 was synthesized in 84% yield by reaction of the phenol with p - nitrophenyl chloroformate in the presence of pyridine. subsequent deprotection by boron trifluoride provided the activated ab monomer 4 (88%). formation of the carbonate link in 3 and 4 was confirmed by both c nmr (150.3 ppm) and ftir (1778, 1784 cm) spectroscopies. a doublet of triplets at 2.49 ppm by h nmr spectroscopy, as well as a broad band around 32003400 cm by ftir spectroscopy, confirmed the presence of a free alcohol in 4. unfortunately, rather than undergoing polymerization, 4 led to an elimination product 5 upon treatment with base. no reaction occurred during our first attempt at polymerizing monomer 4 in the presence of pyridine, and the starting material was recovered unchanged. the use of a stronger base, triethylamine, did not lead to a polymer either. further details confirming the structure of the former compound are presented in the supporting information. alternatively, the introduction of the activated p - nitrophenyl carbonate on the primary alcohol of the serine residue was not attempted. we reasoned that the presence of such an electron - withdrawing group would most likely enhance the acidity of the proton on the -carbon of the amino acid residue, favoring the elimination pathway. tyrosine was selected over threonine in order to avoid any possible side reaction on the -carbon of the amino acid residue in the activated monomer. in an attempt to minimize the toxicity of the products potentially released during degradation of the polymers, the commercially available l - tyrosine ethyl ester, which would generate ethanol upon hydrolysis, was used as a starting building block (rather than use of a methyl ester that would generate methanol). interestingly, the natural amides (e)-n-(feruloyl)-l - tyrosine methyl ester 6 and tert - butyl ester 7 have already been synthesized and tested biologically (figure 1). a stronger antioxidant effect was revealed for the more sterically hindered tert - butyl ester when compared with the methyl ester, and both surpassed ferulic acid in lipid peroxidation assays. the anticipated hydrolytic degradation product of a fa tyrosine - based poly(carbonate amide) system, (e)-n-(feruloyl)-l - tyrosine ethyl ester 8 (figure 1), may, therefore, present a bioactivity similar to its methyl and tert - butyl counterparts. based upon this strategy, a series of four monomers were synthesized, having functionalities installed that would allow for condensation polymerization via carbonate formation and including regiochemistries that would produce either head - to - tail, tail - to - head - to - head - to - tail, or random sequences in the resulting poly(carbonate amide)s. a monoactivated ba monomer was initially designed in order to synthesize a regioregular aaaa (head - to - tail) polymer, where a is the phenol of fa, a is the phenol of tyrosine, and b is an activated carbonate or chloroformate derivative of the fa phenol. the p - nitrophenyl carbonate ba monomer 13 was targeted first using the same synthetic pathway as described above for 4. the phenol group of the tyrosine ethyl ester hydrochloride 9 was protected with tbdmscl, resulting in 10 (81%). the free alcohol of 11 was activated to a p - nitrophenyl carbonate 12 in the presence of pyridine (71%). finally, the deprotection of 12 under mild conditions afforded the activated ba monomer 13 in high yield (85%). c nmr and ftir spectroscopies confirmed both the formation of the carbonate and the deprotection of the phenol group (scheme 3). polymerizations were conducted in the presence of a base to generate the nucleophilic phenoxide group. optimization of the experimental conditions was performed through variation of the solvents (ch2cl2 and pyridine), the initial monomer concentration (1 m, 3 m), the base (et3n, pyridine, dipea, dmap or a mixture), the reaction times (5 to 72 h), and temperatures (rt, 50 c, 95 c). the outcomes of the reactions were evaluated in terms of the final degree of polymerization (dpn), as determined by sec. the reliability of the number - average molecular weights (mn) determined against peo calibration was confirmed by comparison with the mn obtained from a relative calibration performed from the oligomeric fraction contained in the crude polymerization mixture (table s1, see details in supporting information). also, chain - end functionalization of the polymers with reactive capping agents (allylchloroformate and furfuryl isocyanate) and their subsequent analysis by h nmr spectroscopy gave further support to the dpn values that are reported and also revealed that the primary component of the system is an acyclic polymer (see details in supporting information). despite parametrization, all the reactions resulted in polymers with dpn values limited to a range of 1520, according to sec and h nmr spectroscopic measurements. one of the samples was further characterized by c nmr spectroscopy to obtain insight into the regioselectivity of the polymerizations. surprisingly, the analysis of the spectrum showed evidence of the formation of all three possible regioisomers (head - to - tail, head - to - head, tail - to - tail) in the isolated polymer (vide infra), instead of the expected aaaa (head - to - tail) regiospecificity as implied by monomer design. the poor regioselectivity and the limited dpn achieved can plausibly be explained by the release of a p - nitrophenolate, which may act as an additional nucleophile able to attack the polymeric carbonyl - based backbone and promote exchange reactions or by the insufficient difference in the leaving group ability of p - nitrophenol and the fa phenol. determined by sec (dmf, 0.05 m libr) using oligomer calibration. determined by c nmr spectroscopy. in an attempt to minimize regiorandomness and achieve higher dpn, the alcohol 11 was converted to the more reactive chloroformate 14 by reaction with either diphosgene, which gave the best yield (82%), or triphosgene (70%) (scheme 3). despite the lower yield, triphosgene was preferred because of its ease of use. the presence of a chloroformate functionality in 14 was confirmed by c nmr (148.9 ppm) and ftir (1790 cm) spectroscopies. interestingly, this new ba monomer, designed to generate a polymer with higher regioregularity and molecular weight, was obtained in only three synthetic steps. the polymerization was then triggered by the deprotection of the phenol silyl ether, which generated the propagating nucleophile in a single sequence. several sources of fluoride such as tetrabutylammonium fluoride, potassium fluoride, cesium fluoride, and silver fluoride were evaluated for the deprotection of the silyl ether to the phenolate. among them, silver fluoride, which drove the reaction by precipitation of silver chloride, was the most promising and afforded oligomers. the screening of solvents (pyridine, pyridine / ch3cn, thf / ch3cn, or ch3cn) revealed the necessity of both ch3cn (agf solubility) and pyridine (polymer solubility) to achieve polymerization. the use of 2 equiv of agf proved necessary to obtain high dpn values, whereas higher loadings complicated the purification process. although no effect of the reaction temperature (rt vs 60 c) was observed, a marked effect of the initial monomer concentration was noticed. in dilute media (0.1 m) only small oligomers were formed (supporting information, table s3, entry 1) while some precipitate was noticeable at 0.5 m (entry 3), and the reaction mixture almost instantaneously crashed out of solution at even higher concentration (0.75 m, entry 4). a kinetic study was performed at an intermediate concentration of 0.5 m (entries 58). after 1.5 h, a polymer of mn = 11.1 kg mol (= 1.92) was extracted with a low yield (14%) from the heterogeneous reaction mixture. reduction of the reaction time down to 15 min facilitated the work - up and led to a polymer of mn = 8.3 kg mol (= 2.08, dpn = 20) with a decent yield (56%). the corresponding polymers exhibited a high degree of aaaa (head - to - tail) regioselectivity as inferred from c nmr spectroscopic measurements. with the aaaa polymer in our hands, we then sought to synthesize the aaaa polymer with a high regioselectivity by polycondensation of the dimeric monomer 16. the aaaa monomer 16 was obtained from 11 by dimerization, using p - nitrophenyl chloroformate as an activating reagent, followed by deprotection of the phenol groups under mild conditions (scheme 4). the initial formation of the carbonate bond between the two fa subunits in 16 was performed because of the potentially lower reactivity of the corresponding phenols during the polymerization process (steric hindrance of the o - methoxy group). also, this strategy allowed for ready attribution of the peak at 150.4 ppm to the carbonate in an aaaa sequence in the c nmr spectrum, which is necessary to quantify the relative ratio of regioisomers in the polymers. the desired aaaa polymer was obtained by polycondensation of the diphenol 16 with a phosgene derivative. a first set of experiments performed in pyridine, which acted as a base and solvent, revealed that neat diphosgene was more effective at promoting polycondensation than was triphosgene. optimization of the reaction conditions, such as the initial monomer concentration, the diphosgene feed ratio, and the reaction time, led to the isolation of a high molecular weight aaaa polymer of mn = 18.0 kg mol (= 2.06, dpn = 44) in 2 h with 74% yield (supporting information, table s4). detailed c nmr spectroscopic analyses revealed the presence of only two signals attributed to carbonate groups (150.4 and 151.7 ppm for the fa fa carbonate of the monomer aaaa and the newly established tyrosine tyrosine carbonate aaaa sequences, respectively) which confirmed the high regioselectivity of the aaaa polymer (figure 2). c nmr attribution of carbonyl carbons corresponding to the carbonate functionalities of the fa fa coupling aaaa in monomer 16 (black), the newly established tyrosine tyrosine couplings aaaa of the resulting polymer (blue), the predominance of fa tyrosine couplings aaaa from the polymerization of 14 and all three types of carbonate carbonyls from the random couplings of fa and tyrosine groups during the polymerization of 8. in order to fully realize a fa - co - tyrosine strategy for the generation of bio - derived poly(carbonate the so - called aa monomer 8 was obtained in only one step (67%) by peptidic coupling of commercially available fa and l - tyrosine ethyl ester hydrochloride (scheme 5). the formation of the amide linkage in 8 was confirmed by spectroscopic characterization. polymerization conditions were based on those employed during the polymerization of monomer 16 ; modifying the diphosgene equivalency, the global concentration and the reaction time, a polymer of mn = 14 kg mol (: 2.57, dpn = 34) was obtained (47%) (supporting information, table s5). the relative ratio of the three types of carbonates, having tyrosine tyrosine, fa fa, and tyrosine fa couplings, aaaa, aaaa, and aaaa sequences, respectively, present in the polymer was found to be 37:23:40, as determined by the integration of the c nmr signals of the carbonate carbonyl carbon (figure 2). it is clear that the aaaa and aaaa sequences are more prevalent than the aaaa sequence in the regiorandom polymer sample. this lack of true regiorandomness could be rationalized by a lower reactivity of the phenolic group from the fa subunit vs the one from the tyrosine residue during polycondensation because of a higher steric hindrance. a summary of the properties of this regiorandom polymer is provided table 1, entry 4. the thermal stability of all four polymers has been assessed by thermogravimetric analysis (tga) and differential scanning calorimetry (dsc) (table 1). tg < 135 c) and the first derivative tga peak (337 c < tp < 351 c) for each of the polymers under investigation reveals that the thermal properties were not influenced by the regiochemistry. the thermal stability of a representative polymer sample (aaaa polymer from 13) was compared to the monomer (8)/multimer (16) and the starting materials (figure 3). as expected, the aa and aaaa monomers (8 and 16, respectively) were less thermally stable than the polymer (318 and 328 c, respectively) and more stable than the l - tyrosine ethyl ester (224, 276, and 375 c) and fa (243 c). high residual masses were observed at 500 c for all polymers as well as for monomer 8 and multimer 16. this stability may plausibly be explained by the formation of highly conjugated structures. thermogravimetric analysis of aaaa polymer from 13, aa monomer 8, aaaa monomer 16, fa, and tyrosine. fluorescent properties of fa and tyrosine have been investigated in the past, and these compounds have been used as probes for assessing intermolecular interactions as well as fa proportion and disposition in food. a recent study of tyrosine s three - dimensional emission spectra provided a way to distinguish tyrosine from tryptophan, which can find applications in the analysis of deep sea chemistries. because of these fluorescent properties in the starting materials (supporting information, figures s4 and s5), a comparison of absorbance, excitation, and emission spectra of the aa monomer 8 and the random polymer was obtained, revealing a shift in the maximum wavelength in the emission spectra from 394 nm (8) to 427 nm (random polymer from 8) (figure 4). these initial results displayed a variation in the fluorescence properties of the monomer and the polymer. this red - shift to a material that is suited to 350 nm excitation and 430 nm emission regimes makes the emissivity of the random polymers viable for their immediate use as imaging agents, employing one of the most common imaging modalities in microscopy (dapi filter set). absorbance, excitation, and emission spectra for (a) monomer 8 and (b) the corresponding regiorandom polymer. the design and synthesis of a poly(carbonate amide) based on fa, a renewable and biocompatible resource present in fruits and vegetables, has been reported. in order to generate a poly(carbonate amide) by polycondensation, fa was coupled to a hydroxyl - amino acid via an amide link. l - serine couple was synthesized but failed to polymerize. moving to an l - tyrosine partner led to the generation of an array of new fa a good overall control of the regioselectivity of the resulting polymers was achieved by careful design of the monomers and led to the synthesis of poly(carbonate amide)s possessing head - to - head, tail - to - tail, and head - to - tail sequences in a nearly regiospecific fashion. although the glass transition and thermal degradation properties of the fa tyrosine - based poly(carbonate - amide)s (tg and tp) were not significantly influenced by the regioselectivity or the chain length, preliminary results on the fluorescent properties showed a shift in the maximum of the excitation and emission spectra between the aa monomer 8 and the corresponding random polymer. further fluorescence studies are underway to probe the imaging promise of this new class of bio - derived poly(carbonate amide)s. amide)s derived from fa and a hydroxyl - containing amino acid could potentially undergo hydrolytic breakdown and lead to biologically beneficial byproducts and carbon dioxide. moreover, the rigidity of both fa and tyrosine subunits as well as the polar, hydrogen - bonding amide groups may lead to materials with enhanced mechanical properties. specifically, applications would include engineering plastics, biomedical components, and other applications where mechanical strength and degradation are both desired. | ferulic acid (fa), a bio - based resource found in fruits and vegetables, was coupled with a hydroxyl - amino acid to generate a new class of monomers to afford poly(carbonate amide)s with potential to degrade into natural products. l - serine was first selected as the hydroxyl - amino partner for fa, from which the activated p - nitrophenyl carbonate monomer was synthesized. unfortunately, polymerizations were unsuccessful, and the elimination product was systematically obtained. to avoid elimination, we revised our strategy and used l - tyrosine ethyl ester, which lacks an acidic proton on the position of the ethyl ester. four new monomers were synthesized and converted into the corresponding poly(carbonate amide)s with specific regioselectivities. the polymers were fully characterized through thermal and spectroscopic analyses. preliminary fluorescent studies revealed interesting photophysical properties for the monomers and their corresponding poly(carbonate amide)s, beyond the fluorescence characteristics of l - tyrosine and fa, making these materials potentially viable for sensing and/or imaging applications, in addition to their attractiveness as engineering materials derived from renewable resources. |
electromagnetic interference (emi) is a transient disruption or alteration in a devices normal function caused by an external signal. in implantable cardiac rhythm devices, the lead creates a loop antenna allowing a magnetic field to induce a current between the lead and the generator. the power of the external force, distance the external force is from the device, and duration and frequency of the signal will determine the degree (if any) of interference. implantable cardioverter defibrillators (icds) are designed in controlled situations to communicate with specific programmers using radiofrequency (rf) signals and are designed to respond to an appropriate static magnetic field by suspending delivery of therapeutic shocks. icds incorporate various magnetic sensors that allow programming of the device by applying an external magnet (magnet mode). unplanned static magnetic fields can potentially prevent therapeutic shocks, while induced current from rf signal sources may be incorrectly sensed as an arrhythmia and treated inappropriately. currently, research has been conducted for potential emi interference caused by rf from cell phones, digital music players, and ipods. additionally, in a study by lee and colleagues, emi from magnets in portable headphones created magnet mode response in 38% of patients with icds when placed within 2 cm from the device. with the advancements in shielding, filters, and detection algorithms, emi of medical devices by emitting rf is relatively uncommon. however, with newer electronic device technology, strong magnets are used to activate features or provide fixation for attachments such as covers. one example is the apple ipad2 and later models that incorporate strong magnets along its frame to attach the smart cover. the smart cover also incorporates magnets to assist fixation and to turn the ipad on and off. ipad devices are often used in close proximity to a person 's body, ie, when reading sitting up or laying down. no research to determine emi in the ipad2 or later models was found in the literature and because of their magnets, we theorized that the ipad2 could cause emi in patients with icds. the purpose of this study was to determine whether the ipad2 would cause emi in patients with icds and, if so, at what distance. this study was approved by the institutional review board of dignity health system. prior to patient enrollment, to determine the number of magnets present on an ipad2, we utilized magnetic visualization film and placed it over the face of the device without the smart cover in place. additionally, using a magnet detector created by 2 reed switches placed perpendicular to each other, we determined a safe distance between the ipad2 and an icd utilizing a buzzer and a battery. this magnetic detector system responded to a magnetic field of 0.007 microtesla (7 gauss), whereas icds respond to 0.01 microtesla (10 gauss). the reed switches ' perpendicular alignment allows them to respond to all magnetic fields that may activate magnet mode in an icd. patients over 18 years old with icds were then enrolled in the study after obtaining informed consent. a baseline interrogation of the icd was performed to note settings and ensure proper function. a doughnut magnet was placed on each device as a control to make sure the programmer was picking up the magnetic field correctly. the subject then held the ipad2 with its smart cover attached but open to expose the screen as if he / she was browsing the internet. the distance that varied between 12 and 18 inches from the implanted device was recorded. a programmer was used to check for emi and interrogate the device for 1 minute. this was done with both the 3 g (cellular data) on and again with 3 g off. the subject then reclined and laid the ipad2 across his / her chest directly over his / her icd, as if he / she fell asleep while reading. the ipad2 was slowly moved around the entire surface of the implanted device only by the investigators to ensure all potential sites for magnet mode trigger were tested. the participant 's device was interrogated again before the end of the office visit to ensure proper functioning. prior to the start of subject recruitment, we determined there were 6 rectangular hinge magnets on one side of the ipad2 frame and 4 magnets on the opposite frame edge for latching the cover in place. the smart cover had 6 rectangular hinge magnets that coincided with the hinge magnets on the ipad2 frame and 14 on the opposite side that coincided with the frame to latch the cover. there was one circular magnet for the purpose of switching the device on or off. no noise or oversensing was seen in any devices, but magnet mode was triggered in 9 (33%) of the devices. the magnet mode trigger in the 9 devices only occurred when the ipad2 was laid directly on top of the implanted icd. figure 2 shows a printout from a programmer indicating the trigger of magnet response by an ipad2. additionally, in 2 of the positive magnet mode triggers of boston scientific patients, the icd was programmed off indefinitely when the ipad2 was placed on the patients ' chest over their device. in this case, the icd did not turn back on when the ipad2 was removed. jude medical, the icd was interrogated after an ipad2 was applied directly on top. we hypothesized that body mass index (bmi) may have been associated with a positive magnet mode response in our patient population. however, on average, the subjects bmi who had a positive magnet mode trigger (m=29.70 kg / m, se=1.55) compared with those that did not have a magnet mode trigger (m=29.74 kg / m, se=1.14) did not show a significant difference t(24)=.03, p=0.96. results from our magnetic detector determined a magnetic field of < 0.007 microtesla (7 gauss) occurred at 2 cm away from the ipad2. the strongest magnets appear to be on the hinges between the ipad2 and the smart cover. all positive magnet mode triggers occurred around the areas where the magnets were imbedded in the ipad2 and when it was directly on the body and over the device. in those patients that had a magnet mode trigger, all were in their baseline rhythms and were unaffected. this study showed that the ipad2 can cause emi in patients with icds when they are used in close proximity to the device. all positive magnet mode triggers occurred when the ipad2 was laid directly on the chest and over the device of the patient. there are 10 magnets embedded in the ipad2 frame and 20 on the smart cover. it is not certain why only some patients had a positive magnet mode trigger ; however, it is known that the magnetic field weakens greatly with distance and the magnetic sensor in the icd is rather small. because magnet mode was only triggered when the ipad2 was placed directly over the device, it was presumed that the magnets in the ipad2 were in close proximity to the magnetic sensor in the icd. the location of the magnetic sensor varies depending on the manufacturer and model of the icd. it may be possible that certain locations of the magnetic sensor made the icd more susceptible to the magnets in the ipad2. the ipad2 in patients with a negative response were thoroughly checked ; all borders and surface area of the icd were checked for an extended time to attempt to illicit a positive response. it was originally hypothesized that a person with a lower bmi would have a higher chance of testing positive. theoretically a person with a lower bmi has less fat / muscle, and so the distance between his or her icd and skin might be smaller. however, the difference in bmi in our study between the positive and negative groups was not significant. it may be possible that bmi is not an accurate measurement of the thickness of the tissue over a patient 's icd since fat distribution may differ depending on the patient 's body type. in older boston scientific icd models such as the prizm 1, prizm 2, and vitality 1, the device has a feature called change tachy mode with magnet in which the icd 's antitachycardia feature will be programmed off when a magnet is applied to the implanted device for at least 30 seconds. this feature will not resume until a magnet is once again reapplied for at least 30 seconds. this feature was intended for use in operating rooms to prevent inappropriate shocks to patients with icds from emi from surgical equipment. this feature is no longer available in any current icd models ; once a magnet is removed, all programmed features resume. in our study, 2 patients had an older boston scientific vitality 1 icd. both patients ' devices were programmed off because we analyzed each location with the ipad2 for approximately 1 minute. therefore, as shown in this study, patients with older devices that develop a magnet mode trigger with an ipad2 are at risk for untreated tachyarrhythmias, since their icds may remain off for an extended period of time. newer icd models with positive magnet mode trigger only turn off their tachy mode while the ipad2 is placed directly on the icd device and will resume programming once the ipad2 is removed. other tablets were not tested in this study however ; the microsoft surface also utilizes magnets in the cover and frame. these magnets are stronger than the ipad2 and should be considered a risk to patients with icds (david liang, personal communication, february 4, 2014). other manufacturers of tablets that utilize magnets in their covers and frames could potentially be a risk factor for magnet mode trigger in patients with implanted icds. jude medical is the company most often used in our area, therefore was the most often studied device. a larger sample size and other companies such as biotronik and sorin these device companies have a small market share in the united states and are not utilized in the geographical area where this study was conducted. though our sample size was small, we feel that this study can still make a conclusion that the ipad2 can trigger magnet mode in patients with icds when applied directly on top of the device such as in the sleeping position. jude medical is the company most often used in our area, therefore was the most often studied device. a larger sample size and other companies such as biotronik and sorin these device companies have a small market share in the united states and are not utilized in the geographical area where this study was conducted. though our sample size was small, we feel that this study can still make a conclusion that the ipad2 can trigger magnet mode in patients with icds when applied directly on top of the device such as in the sleeping position. future studies could exam larger numbers of patients and other manufacturing device companies for magnet mode triggers in the presence of the ipad2. currently available only in europe, there are icds compatible with magnetic resonance imaging (mri). research to determine if magnet interference with these newer icd devices from small magnets such as those in the ipad2 could be important. in our study there are also magnets imbedded in the frame of the ipad2 and later models ; determining if magnet mode could be triggered without the cover may add important safety information. additionally, magnet sensor location on the implanted icd generator may have an association with emi response and determining location may show beneficial results ; in our study there were both positive and negative emi in patients with identical models however, implantation position was not determined. finally, the depth of the implanted device to the skin surface may have a correlation with magnet mode response. on the basis of this study, we recommend the following concerning ipad2 or later models and their use in patients with icds : patients should avoid placing the ipad2 directly on top of their icds.for certain boston scientific models, such as the prizm 1, prizm 2, and the vitality 1, change tachy mode with magnet this will prevent the antitachycardia feature of the icd from remaining permanently disabled should magnet mode be activated.upon implanting icds, doctors should turn on magnet mode trigger monitoring so that when the patient 's device is interrogated, the programmer will be able to tell if the icd has encountered any magnet mode triggers in the past. patients should avoid placing the ipad2 directly on top of their icds. for certain boston scientific models, such as the prizm 1, prizm 2, and the vitality 1, change tachy mode with magnet this will prevent the antitachycardia feature of the icd from remaining permanently disabled should magnet mode be activated. upon implanting icds, doctors should turn on magnet mode trigger monitoring so that when the patient 's device is interrogated, the programmer will be able to tell if the icd has encountered any magnet mode triggers in the past. with the aging population, it has been projected that there will be an increase in icd placement. among individuals who choose to use electronic devices, the ipad2 and later models with the smart cover have become an integral part of their daily lives. as new electronic products that utilize magnets are produced, patients and healthcare providers should be educated regarding the risk of potential icd malfunction with magnet exposure. | backgroundover 140 million ipads have been sold worldwide. the ipad2, with magnets embedded in its frame and smart cover and 3 g cellular data capability, can potentially cause electromagnetic interference in implantable cardioverter defibrillators. this can lead to potentially lifethreatening situations in patients. the goal of this study was to determine whether the ipad2 can cause electromagnetic interference in patients with implantable cardioverter defibrillators.methods and resultstwentyseven patients with implantable cardioverter defibrillators were studied. the ipad2 was held at reading distance and placed directly over the device with cellular data capability activated and deactivated. the manufacturers / models of devices and the patients ' body mass index were noted. the presence of electromagnetic interference was detected by using a programmer supplied by each manufacturer. magnet mode with suspension of antitachycardia therapy was triggered in 9 (33%) patients. all occurred when the ipad2 was placed directly over the device. the cellular data status did not cause interference and no noise or oversensing was noted. there was no significant difference between the mean body mass index of the groups with or without interference.conclusionsthe ipad2 can trigger magnet mode in implantable cardioverter defibrillators when laid directly over the device. this is potentially dangerous if patients should develop lifethreatening arrhythmias at the same time. as new electronic products that use magnets are produced, the potential risk to patients with implantable defibrillators needs to be addressed. |
the content of each peptide flask was dissolved by addition of hepes buffer 100 mm, ph 7.4 (a128) or ultrapure milli - q water (a116). the concentration of a was then measured by uv - visible spectroscopy (276 nm (tyr10)=1410 m cm). uv - visible spectra were recorded on a biochrom libra s50 or a specord 205 spectrophotometer (analytik jena, germany). fluorescence spectra were recorded on a flsp920 spectrometer (edinburgh instruments ltd, uk), with bandwidth for excitation and emission=2 nm. a complex was first prepared by mixing equimolar amounts of a128 and cucl2 in hepes buffer 50 mm, ph 7.4. the metalation of a was monitored by the decrease of fluorescence (see ref.). a solution of ligand 1 or cq in dmso was then added (1 or 2 mol equiv, respectively), and the reaction was monitored by uv - visible spectroscopy. final concentrations were [a128]=[cu]==20 m, [cq]=40 m ; dmso / hepes buffer=5:95 v / v. the 50 % decrease of fluorescence of a upon metalation by copper was confirmed in buffered mixture containing up to 10 vol % of an organic solvent, namely ch3cn (ref.), or dmso (present report, data not shown). x - band (9.525 ghz) esr spectra were recorded in quartz tubes at 120 k, using a bruker elexsys - ii e500 spectrometer. for experiments with ligand 1, the solvent was hepes buffer 100 mm, ph 7.4, containing 13 vol % of dmso. [a116]=185 m ; a116/cu molar ratio=1:1 (figure 3 a), a116/cu/1=1:1:0.5 (figure 3 b), a116/cu/1=1:1:1 (figure 3 c), cu/1=1:1 (figure 3 d). the addition of 16 vol % of dmso in hepes did not induce modification of the spectrum of cu the solvent was dmso / hepes buffer 100 mm, ph 7.4, 90:10 v / v. a116/cu molar ratio=1:1 (figure 5 a), a116/cu / cq=1:1:1 (figure 5 b), a116/cu / cq=1:1:2 (figure 5 c), cu / cq=1:2 (figure 5 d). the sample solutions were injected (7.5 l) using a mobile phase ch3oh / h2o (90:10 v / v), flow rate=0.15 ml min. the cone voltage was 15 v, and spectra were acquired in the positive ion mode, in the m / z range 1002500. the mixture of a116/cucl2/cq (1:1:1 mol ratio) was prepared in ultrapure milli - q water (ph 5.8)/meoh (1:1 v / v). final concentration was 100 m, injected volume was 7.5 l. the series of multicharged patterns at m / z=1161.9, 774.9, and 581.4 was not detected in the absence of cu. as a service to our authors and readers, this journal provides supporting information supplied by the authors. such materials are peer reviewed and may be re - organized for online delivery, but are not copy - edited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors. | the accumulation of redox - active metal ions, in particular copper, in amyloid plaques is considered to the cause of the intensive oxidation damage to the brain of patients with alzheimers disease (ad). drug candidates based on a bis(8-aminoquinoline) tetradentate ligand are able to efficiently extract cu2 + from copper - loaded amyloids (cu a). contrarily, in the presence of a bidentate hydroxyquinoline, such as clioquinol, the copper is not released from a, but remains sequestrated within a acu clioquinol ternary complex that has been characterized by mass spectrometry. facile extraction of copper(ii) at a low amyloid / ligand ratio is essential for the re - introduction of copper in regular metal circulation in the brain. as, upon reduction, the cu+ is easily released from the bis(8-aminoquinoline) ligand unable to accommodate cui, it should be taken by proteins with an affinity for copper. so, the tetradentate bis(8-aminoquinoline) described here might act as a regulator of copper homeostasis. |
as obesity is becoming increasingly more prevalent in the united states, weight loss to reduce adipose tissue mass is strongly promoted as a means to decrease the disease risk associated with excess adiposity (5, 57). unfortunately, the majority of individuals who lose weight are unlikely to maintain the reduced weight for an extended period of time (15, 51, 59). repeated periods of weight loss and regain form a pattern known as weight cycling. hill (2004) indicates that popular and lay literature have asserted that weight cycling (i.e. yo - yo dieting) may increase the risk of developing cardiovascular disease or type ii diabetes to a greater extent than remaining weight stable at an obese body mass index (bmi ; 30 kg / m)(27). because there is no universally - accepted definition of weight cycling, differences in experimental design may have contributed to discrepancies in scientific outcomes. weight gain has significant implications concerning disease risk, which is believed to be mediated by an elevated level of systemic inflammation. low - grade systemic inflammation is associated with obesity and it may serve as a link between adiposity and the development of cardiovascular disease and type 2 diabetes (66). to our knowledge, discerning a difference in disease risk between maintenance of obesity and weight cycling is important and may provide insight concerning individual differences in disease progression. if weight cycling is associated with an increased disease risk, continually recommending weight loss to those unable to maintain reduced weights may be a major public health issue. this review has two aims : 1) to compare studies that both support or refute the theory that weight cycling is independently associated with increases in disease risk2) to discuss the possibility that weight cycling impacts pro - inflammatory biomarkers. it has been estimated 24% of american men and 38% of women are currently attempting to lose weight (35, 53, 64). when individuals with an obese bmi are considered, 65% of men and 68% of women are trying to lose weight, which is a fivefold increase compared to those within the normal bmi (1824.9 while successful weight loss is achieved, researchers have indicated that long - term maintenance of a reduced weight appears to be rare. the probability of weight regain increases in the time following initial weight loss (43). researchers believe this is due to the energy gap created during caloric restriction where decreased energy expenditure is paired with an increased drive to eat (43). rodent studies have demonstrated that this gap persists regardless of the duration of weight reduction, which increases the probability of weight regain.(41). this drive to eat causes a hyperphagic response when free access to food is allowed and when paired with suppressed lipid utilization, weight regain is often rapid and efficient (41, 43). while this finding was elucidated through use of a rodent model, human weight regain data supports this concept. one year after a modest weight loss (14.5% of body weight), votruba. (2002) reported that within a year of weight loss, 16 out of 28 women regained weight and had a 19% increase in body weight and a 26% increase in percent fat mass (59). weiss reported that by one year after a modest weight loss (10% of body weight), 33% of adult subjects regained all lost weight. furthermore, they concluded that the odds of regaining were positively associated with the percentage of initial weight lost (63). field. reported that approximately 55% of overweight and obese women who lost 10% of their body weight regained all lost weight within 4 years (15). in support of this finding, within 9 years of the initial weight loss (5% of body weight), 95% of women and 93% of men were unable to maintain the reduced body weight(51). collectively, these studies suggest that while initial weight loss is possible, long - term maintenance is problematic, especially when large amounts of weight are lost or an individual is overweight or obese. repeated bouts of weight loss followed by regain forms a pattern known as weight cycling. survey data collected by williamson and colleagues (64) indicated that 25% of men and 27% of women trying to lose weight have made long - term attempts (classified as trying for over 1 year or always trying to lose weight). it has also been shown that 7% of men and 10% of women can be classified as severe weight cyclers (intentionally lost at least 5 kg and regained at least three different times), while 11% of men and 19% of women are mild weight cyclers (lost and regained at least 5 kg on one or two occasions) (36). while these results were generated from a group of adults in finland, the conclusion that 18% of men and 27% of women weight cycle is comparable to the prevalence described by williamson. these numbers are likely a conservative estimate of the prevalence of weight cycling, which may be even greater in the united states. the physiological changes associated with weight cycling, such as energy expenditure, metabolism and fuel utilization, have been documented using a rat model. maclean and colleagues (2004) have documented the physiological alterations occurring in obesity - prone rats that contribute to the rapid, efficient regain during relapse following weight loss and maintenance. their focus has been on the energy gap created during a period of caloric restriction that is characterized by decreased energy expenditure and an increased drive to eat (42). they found that in addition to changes in energy intake, alterations in metabolic efficiency and fuel utilization (favoring carbohydrate oxidation) may significantly affect the propensity to regain weight (43). for instance, in the 16 weeks following moderate weight loss (14%), food efficiency was increased 10-fold upon the first day of a 56-day re - feeding in weight cycle rats compared to rats with established obesity. while this dramatic rise was reduced within several days, food efficiency remained elevated above levels in obese mice for the first 4 weeks of relapse (41). the most dramatic changes occurred during the first week of relapse, a time when nearly 40% of lost weight, which was primarily fat mass, was regained (41, 42). researchers also noted that as the length of maintenance increased, the amount of weight regained upon relapse also increased. furthermore, regain was accompanied by a 30% increase in adipocyte concentration per fat pad. based on the above literature, it is clear that weight gain during relapse appears to induce more rapid adipose tissue growth and hyperplasia due to metabolic shifts favoring lipid storage. because adipose tissue is a metabolically active tissue, responsible for production of leptin, cytokines and adiponectin as well as responding to traditional hormone systems(32), it is possible that the consequences of weight gain during relapse may also differ from that of initial weight gain. in recent years, lay literature has asserted that weight cycling may be more detrimental to health than simply remaining overweight or obese (27, 30). researchers have found associations between weight cycling and an overshoot of lipogenic enzyme, triglyceride and cholesterol levels in animals and increased risk of heart attack and stroke in humans (4, 16, 34, 52). however, other researchers noted no long term adverse effects on body composition, blood pressure, lipid profile or risk of developing type ii diabetes (13, 22, 37, 49, 56). due to limited research in the area of weight cycling all of the negative consequences may not be known. existing studies differ considerably in their research design, subject population used, duration of treatment, incorporation of exercise, magnitude and frequency of weight cycles. the lack of a universal definition of weight cycling is perhaps a great contributor to the variability within experimental design. this variability is discussed in greater detail in the following sections. according to published research, weight cycling has been evaluated in one of two main ways : using a cross - sectional survey model in humans or a longitudinal endpoint model in rodents. the next two sections highlight research that either support or refute the theory that weight cycling contributes to detriments in health, demonstrating the no definite conclusions can be reached at this time. utilization of animal experimental models allows for more control of subject treatment than a human experimental model. such designs are useful for examining mechanisms underlying weight cycling ; however, care should be taken when translating these findings to humans. an increase in internal validity, resulting from more control of subject treatments may explain why reported conclusions regarding weight cycling in animals is a bit more consistent than humans. the most likely explanation for inconsistent findings is due to the manner in which the weight cycling response is elicited. in agreement with the series of studies completed by maclean., rapid regain occurs during relapse in rats that have been on caloric restriction diets (6, 17, 26, 28). weight cycling was associated with increased food efficiency (6, 50) and increased caloric consumption (26, 52). found that despite being at lower weights than control rats, weight cycled rats were significantly fatter(50). reported that weight cycled rats had a lower percentage of fat free mass than their free fed (high fat diet) counterparts and that amount of weight lost during period of restriction decreased with each successive cycle (28). during re - feeding periods in weight cycled rats, researchers have consistently reported overshoots in lipoprotein lipase, serum triglycerides, and serum cholesterol above what has been observed in rats that are free - fed a high fat diet (17, 34, 52). it has been speculated that body weight overshoot may create a state of hyperlipogenesis that may persist for several days after re - feeding. kim. demonstrated that fasting serum leptin was significantly increased in weight cycled rats compared to lean and pair - fed controls despite similar quantities of fat mass(33). since leptin concentration is highly correlated to the degree of adiposity (11, 39), further interpretation of this finding suggests that weight cycling may induce a physiological change in adipocyte release of leptin. in contrast, brownell. did not observe alterations in body composition, but this could be contributed to an experimental design that only allowed weight cycled rats to regain weight until they matched their obese controls, even though weight gain was still persisting at that time (6). also, cleary. indicated that weight loss and weight regain were linear in nature, opposing the energy gap theory proposed by maclean. however, rats in the cleary model were fed a purified diet rather than a high fat diet ; the purified diet contained 20.5% high nitrogen casein, 50% cornstarch, 5% sucrose and 5% corn oil with 9% celufil as a filler (9). demonstrated that re - feeding rats a moderate fat (22%) diet yielded blunted responses compared to those fed a high fat (45%) diet (52). furthermore, given a choice, weight cycled rats have been shown to self - select diets with high fat content during re - feeding(50), so perhaps a purified diet was not an appropriate variable for re - creating the weight cycling experience. gray. stated that the rate of each weight loss was not hindered by weight cycling(23) ; however, in this study, the first restriction was marked by a 50% reduction of intake and the second restriction was a 75% reduction, so perhaps weight loss would not have been similar between weight cycles had the second restriction not have been so severe. kim.(33) were not able to demonstrate a hyperphagic response, but their use of a 24hr fast plus 24hr relapse for 21 cycles may not have been a design that could show effects of weight cycling seen in previously mentioned studies ; every - other - day restriction has lead to other positive adaptations (less weight gain, increased insulin sensitivity) elsewhere (1). this may indicate that larger weight cycles (increased weight loss frequency / magnitude per cycle) have differing effects as suggested by weight cycling studies in humans. some existing scientific literature supports the theory that weight cycling increases disease risk (directly or indirectly) in humans. wallner and colleagues found that a history of weight cycling was associated with a more pronounced android fat distribution in women compared to those who were normal - weight or overweight without a history of weight cycling (60). it is possible that women who are prone to the accumulation of abdominal adiposity may be more likely to weight cycle for a more aesthetically desirable figure (48). regardless of whether weight cycling causes the accumulation of android adiposity or vice versa, other researchers have found that a history of weight cycling was independently associated with an increased risk of developing hypertension (24) and clinically significant decreases in hdl - cholesterol in women (47). french (16) and vergnaud.(58) demonstrated associations between weight cycling and risk for heart attack and stroke, as well as the development of metabolic syndrome(16, 58). studied men enrolled in the multiple risk factor intervention trial who were at elevated risk for coronary heart disease due to smoking, hypertension and hypercholesterolemia, finding that greater weight variability over 4 years of follow up was associated to increase all - cause mortality(4). in contrast to the preceding reports, several other researchers reported that weight cycling has no independent impact on health status. prentice. found that weight cycling did not significantly alter body composition (49). however, unlike wallner.(60), who asked for 4 years worth of weight history, this study was completed in only 18 weeks. it may be possible that any deleterious effects of weight cycling do not manifest immediately or that the magnitude of the weight loss was not sufficient to induce long - term change. studied obese patients, in a multi - disciplinary weight loss program, who had relapsed and re - entered. multiple attempts at weight loss over 12 years showed no effect on the rate at which weight could be lost each time or on blood pressure or lipid profile ; in fact, these measurements at baseline were significantly lower at the time of re - entry compared to the initial start for men and women (37). initial blood pressure in men (134/88 mmhg) and women (126/82 mmhg) was recorded at the restart baseline at 129/85 mmhg and 121/78 mmhg, respectively. while no subjects were hypertensive mmol / l between initial and restart baselines and cholesterol was reduced by 0.1 and 0.5 women s values were all within the normal / low risk range and men s values remained in the borderline high range. cholesterol values for both genders were all in the borderline high risk range. while deemed statistically significant, the differences between baselines may not be physiologically relevant as disease risk did not appear to change. even though this study was longitudinal in nature, perhaps the use of regular exercise as part of the program acted as a confounding factor, as aerobic exercise is independently and positively correlated with decreases in blood pressure and cholesterol (19, 25). a similar exercise effect was reported by field. where mild and severe weight cycling was strongly associated with weight gain and hypertension, controlling the statistical analysis for weight and weight gain greatly attenuated this correlation(13) ; however, the questionnaire data also revealed that severe weight cyclers exercised significantly more that non weight cyclers. (2004) noted that weight cycling had no effect on cardiovascular disease risk factors ; weight cycling throughout adulthood was not associated with changes in body composition, fat distribution blood pressure or insulin levels(22). one major difference in this study, compared to those with competing findings, was that graci.(22) used morbidly obese subjects (bmi up to 69 kg / m) and perhaps there is a less - pronounced response to weight cycling in this population because the subjects already have an elevated disease risk. (65) and jeffery.(31) may have been effected by the short duration of measurement period (2.5 years) or the failure to use appropriate blood pressure cuffs for obese patients(24). field. concluded that 4 years of weight cycling, prior to diagnosis of type 2 diabetes, was not predictive of disease development while wannamethee found that weight fluctuation does not directly increase risk of death (14, 61). when differences in experimental design are accounted for, a significant gap in scientific knowledge exists concerning the exact role of weight cycling in the progression of chronic diseases that are normally attributed to excess adiposity. il-6 stimulates hepatic release of acute phase proteins, including c - reactive protein (crp) (3, 66). free fatty acids and tnf- act in concert to exacerbate systemic inflammation (54). il-8, released from adipocytes, monocytes and macrophages, has been thought to induce chemotaxis helping to form atherosclerotic plaques(21). il-6 and tnf- can act in an autocrine or paracrine manner, impairing insulin receptor activity and glucose sensitivity in adipocytes and muscle tissue (2, 3, 45, 66). il-6 and crp in circulation target and damage arterial endothelial lining ; this damage helps to initiate or progress atherosclerosis (3, 46, 66). several mechanisms are responsible for the pro - inflammatory response in adipose tissue due to hypertrophy. cytotoxic stressors, such as oxidative stress and hypoxia, induced by hypertrophy in adipose tissue have been reported to trigger subsequent pro - inflammatory events (18, 55). as cellular stress persists, adipocytes secrete il-6, tnf- and leptin (38, 55) and unless revascularization is adequate, cells may become necrotic (44). the level of necrotic adipocyte death is positively correlated with increased adiposity and concentration of resident macrophages (8). leptin aids in the transmigration of blood monocytes into adipose tissue compartments, where they mature into macrophages (12). also, leptin stimulates pre - adipocyte stem cells to mature into adipocytes or macrophages. thus, an adiposity driven increase in adipose tissue macrophages concentration is a result of monocyte influx and directed pre - adipocyte transformation (7). evidence exists that macrophages may be retained longer in adipose tissue from obese compared to lean subjects (40). in lean individuals, the actions of leptin and granulocyte macrophage colony stimulating factor (gm - csf) are opposed by ghrelin ; however, reduced ghrelin in obese individuals causes deregulation of macrophage development in adipose tissue (20). macrophage accumulation has significant implications for inflammatory disease risk because they are a significant source of il-6, tnf-, and il-8 (62, 67). to our knowledge, there is only one published study that examined the effect weight variability has on pro - inflammatory or related factors. reported that japanese men with a history of weight variability had an independently increased odds ratio of elevated crp (68). one limitation of this study was that it was a cross - sectional design thus it was not possible to evaluate cause and effect, no information on intentionality of weight change and the cross - sectional design with a majority of subjects having final bmis less than 25 kg / m. this lack of literature suggests that in order to fully understand the possible effects of weight cycling, we must include examination of pro - inflammatory responses to this pattern. in recent years, lay and popular literature has claimed that weight cycling may be more detrimental that simply remaining overweight or obese. scientific research has yielded mixed results, but this may be due to differences in population used, experimental design and method of weight cycling. a current gap in research is the pro - inflammatory effect of weight cycling.. since obesity is so prevalent, weight loss is almost universally recommended as treatment to reduce disease risk. but because research indicated that relapse is likely, it is important to understand if weight cycling adds to the current disease risk of obesity. because weight cycling has a distinct effect on adipose tissue and adipose tissue is a source of inflammatory cytokines, elucidating any increases in inflammation beyond that measured in sustained obesity may help us to understand the independent disease risk that can be associated with weight cycling. | research indicates that weight cycling, or yo - yo dieting is a common occurrence in overweight and obese populations. the long term negative health consequences of weight cycling are debated and it is unclear whether or not this weight change pattern poses a greater disease risk compared to obesity maintenance. this review discusses the prevalence of weight cycling and physiological alterations occurring during weight loss that promotes weight regain. we also discuss the effect weight regain has upon adipose tissue in terms of rate and type of accumulation. also within this review are discussions surrounding the previously published literature based upon human and rodent research. we focus on previous limitations and difference in experimental design that have perhaps resulted in mixed findings concerning independent effects of weight cycling on health parameters. the final purpose of this review is to discuss future directions in evaluating the pro - inflammatory response to weight cycling in order to compare the disease risk compared to obesity maintenance. |
complications of foot ulcers are the major cause of hospitalization and amputation in diabetic patients and lead to significant health care costs as evidenced by the fact that 2040% of health care resources are spent on diabetes - related diabetic foot [1, 2 ]. diabetic foot syndrome (dfs) is defined, according to the world health organization, as ulceration of the foot (distally from the ankle and including the ankle) associated with neuropathy and different grades of ischemia and infection. it represents a serious long - term complication of diabetes mellitus leading to amputations, disability, and reduced quality of life. approximately 82000 lower extremity amputations directly related to diabetes are performed in the usa annually. of these amputations, the majority (80%) have been preceded by foot ulceration. the presence of foot ulceration is considered an important risk for morbidity, mortality, and disability as supported by the fact that about 80% of nontraumatic amputations are caused by the presence of diabetes and 85% of these amputations are preceded by a foot ulceration. an estimated 15% of patients with diabetes will develop a lower extremity ulcer during the course of their disease. several population - based studies indicate a 0,5% to 3% annual cumulative incidence of diabetic foot ulcers. the prevalence of foot ulcers reported for a variety of populations ranges from 2% to 10%. in a retrospective us cohort study of 8.905 patients with type 1 and type 2 diabetes, the incidence of dfs was 5,8% over an observational period of 3 years. in 15,6% of patients with dfs, lower limb amputations were necessary and survival was significantly shortened and the cost for 4065-year - old male with new foot ulcers was $ 27987 for the two years after diagnosis. pathogenesis of diabetic foot ulcers is complex and multifactorial and it is well known that these lesions rarely result from a single pathology. the most common components of this detrimental pathway which lead to foot ulcerations include peripheral neuropathy, foot deformity, abnormal foot pressures, limited joint mobility, external trauma, peripheral vascular disease, and peripheral edema (see figures 5 and 6). a frequently diabetes - related complication is neuropathy representing the most important contributory cause in the pathway to ulceration. diabetic peripheral neuropathy (dpn) is an impairment of normal activities of the nerves throughout the body and can alter autonomic, motor, and sensory functions. in sensory neuropathy, the lack of protective sensation makes the foot vulnerable to unattended minor injuries caused by an excess of pressure and mechanical or thermal injury. according to an important prospective multicenter study, sensory neuropathy was the most frequent component in the causal sequence to ulceration in diabetic patients. motor neuropathy alters the biomechanics and, gradually, the foot anatomy causing foot deformities, limited joint mobility, and altered loading of the foot. these disarrangements may also alter the distribution of forces during walking and cause reactive thickening of skin (callus) at sites of abnormal load. furthermore, ischaemic necrosis of tissues beneath the callus leads to breakdown of skin and subcutaneous tissue, resulting in a neuropathic ulcer. autonomic neuropathy often results in changes to the texture and turgor of the skin, such as dryness and fissuring, creating a portal of entry for bacteria. autosympathectomy with consequent sympathetic failure, arteriovenous shunting, and microvascular thermoregulatory disfunction impairs normal tissue perfusion and microvascular responses to injury. another disorder contributing to the development of foot ulcers is peripheral vascular disease that affects the blood vessels of small and large sizes. both macro- and microvascular diseases could contribute to the consequences of peripheral vascular disease, resulting in the inability of the dysvascular limb to heal itself properly. the incidence and prevalence of peripheral arterial disease (pad) increase with age in both diabetic and nondiabetic subjects and, in those with diabetes, increase with the duration of diabetes. hypertension, smoking, and hyperlipidemia, which are frequently present in patients with diabetes, contribute additional risk for vascular disease. studies have shown that peripheral vascular disease develops at a younger age among patients with diabetes as compared to the general population. measurement of the ankle - brachial index (abi), which represents the systolic blood pressure at posterior tibial or dorsalis pedis level compared with brachial blood pressure, can be used to define clinically occlusive pad. in patients with an abi 130 mg / dl, hypertriglyceridemia, and microalbuminuria / proteinuria in diabetic foot patients compared with diabetic patients without foot complications and this finding is consistent with the hypothesis that diabetic foot syndrome in diabetic subjects could represent a possible marker of cardiovascular risk. they also reported that patients with diabetic foot were more likely to have a cerebrovascular event (tia and ischemic stroke) both on a retrospective evaluation (previous tia and ischemic stroke) and on a prospective evaluation (new onset tia and stroke on a 5-year follow - up). the most prevalent subtypes of stroke were lacunar and laas subtype with a slight higher prevalence of the lacunar subtype and this finding could suggest a role of both microvascular disease and atherosclerotic macroangiopathies, a determinant of vascular morbidity in patients with diabetic foot. the higher cardiovascular risk associated with diabetic foot may be related to a cumulative effect of the single risk factor linked to neuropathy and pad, which are two well - known medical conditions recently associated with increased cardiovascular morbidity [18, 19 ], but another explanation could be recognized in the role of microangiopathy as a determinant of overall vascular risk. nevertheless, because the groups evaluated in this study did not start with balanced cv risk factors and cv disease, authors can not exactly correct for so many other powerful indicators especially with small numbers and relatively few events ; so our findings can only warn clinicians that diabetic foot has to trigger a vital search for treatable cardiovascular risk factors and diseases. the diabetic foot syndrome is the most frequent cause of hospitalization of diabetic patients and one of the economically most demanding complications of diabetes. people with diabetes have been shown to have higher mortality than people without diabetes, but the cerebrovascular risk profile of these patients is not fully evaluated. another study has been conducted to evaluate the possible role of diabetic foot as a cerebrovascular risk marker in type 2 diabetic patients. authors enrolled 102 type 2 diabetes patients with diabetic foot and 123 diabetic patients without diabetic foot. statistically significant differences were found in the distribution of the main cardiovascular risk factors with exception of hypertension. they observed a higher prevalence of previous cerebrovascular events (transient ischemic attack, ischemic stroke) and of incidence of new onset cerebrovascular events at a 5-year follow - up. regarding clinical subtype of ischemic stroke classified according to trial of org 10172 in acute stroke treatment (toast) classification on a retrospective and prospective basis, we observed a higher prevalence of both the lacunar and large artery atherosclerosis subtype with a slight higher prevalence of lacunar subtype in patients with diabetic foot. these findings showed a worse cerebrovascular risk profile in diabetic patients with diabetic foot than in diabetic subjects without foot ulceration with a higher prevalence of cardiovascular risk factors and of anamnestic cerebrovascular events and incidence of new cerebrovascular events at a 5-year follow - up. microalbuminuria is defined by the detection of urinary albumin excretion rates of 30 to 300 mg in a 24 h urine collection. it is still the only anomaly of early diabetic kidney that has prognostic value statements. in fact, the appearance of microalbuminuria in diabetic patients is a very important index for progression to the most serious kidney disease. it represents a cardiovascular risk indicator in diabetic populations and also in hypertensive and general populations. several studies and experimental data show in fact that microalbuminuria is associated with an increased risk for all - cause and cardiovascular mortality, cardiac abnormalities, cerebrovascular disease, and possibly pad. in a recent study conducted by our group, we have showed the prevalence of microalbuminuria in patients with diabetic foot was higher than in patients without diabetic foot. in addition, we also showed a significant positive correlation between some clinical and laboratory variables, including microalbuminuria and the levels of interleukin 6 (il-6) and resistin which are adipocytokines that may contribute to insulin resistance and to the development of inflammatory responses. hypertension is defined, according to the 1993 world health organization criteria, like a systolic blood pressure 140 mm hg and/or diastolic blood pressure 90 mm hg in subjects who are not taking antihypertensive medication. diabetes mellitus and hypertension are both common diseases and they represent two powerful independent risk factors for cardiovascular, renal, and atherosclerotic disease. diabetic nephropathy is considered to be the main factor that contributes to the development of hypertension in patients with diabetes mellitus type 1. in the case of type 2 diabetes mellitus, hypertension is more often essential and it is part of a plurimetabolic syndrome in a context of insulin resistance. in all cases, hypertension worsens the prognosis of patients, increasing the risk of both macrovascular and microvascular complications. in fact, in the framework of diabetes and hypertension accelerates the development of diabetic retinopathy, nephropathy, and peripheral vascular disease. in diabetic patients with hypertension, therefore, it is necessary and appropriate treatment of hypertension in diabetic patients, which should include nonpharmacological interventions, drug therapy, regular monitoring of blood pressure, and educational endeavors. a study conducted by pinto. showed that the prevalence of hypertension was similar in both groups of diabetic patients with and without diabetic foot. in addition, we also showed a significant positive correlation between some clinical and laboratory variables, including hypertension and the levels of il-6 and resistin which are adipocytokines that may contribute to insulin resistance and to the development of inflammatory responses. there are numerous cardiovascular diseases that occur in patients with diabetes, both type 1 or type 2. the defects in the synthesis and clearance of plasma lipoproteins are among the most commonly metabolic abnormalities that accompany diabetes. the diabetic dyslipidemia, a characteristic pattern characterized by the presence of low levels of high - density lipoprotein (hdl) cholesterol, hypertriglyceridemia, and postprandial lipemia, which is observed more frequently in type 2 diabetes, is one of several factors that contribute to accelerating macrovascular disease in diabetic patients. among the different factors involved in developing of diabetic dyslipidemia insulin effects on liver apoprotein production, regulation of lipoprotein lipase (lpl), actions of cholesteryl ester transfer protein (cetp), and peripheral actions of insulin on adipose and muscle should be considered. the acknowledgment and treatment of dyslipidemia are therefore two important elements in the framework of a multidisciplinary approach aimed at the prevention of coronary heart disease. according to current guidelines for the prevention of coronary heart disease in diabetic patients elevated, ldl - c is the primary target of lipid - lowering therapy and statins however, considering the complexity of the profiles of dyslipidemia in diabetic patients, multiple drugs are often required to achieve therapeutic targets. in addition, the other risk factors usually associated with diabetes mellitus, such as hypertension, hyperglycemia, and obesity, should be effectively managed to reinforce the effects of lipid - lowering therapy. in our recent study, we showed a higher prevalence of dyslipidemia in patients with diabetic foot ulcers than in those without diabetic foot. in addition, we also showed a significant positive correlation between some clinical and laboratory variables, including dyslipidemia and the levels of il-6 and resistin which are adipocytokines that may contribute to insulin resistance and to the development of inflammatory responses. in diabetes, there is a complex interplay of several variables with inflammatory metabolic disorders and their effect on the cardiovascular system. simplified explanation may be that inflammation increases insulin resistance, which in turn leads to obesity, while perpetuates diabetes, high blood pressure, prothrombotic state, and dyslipidemia. some studies [2325 ] suggest an interaction between hormones, cytokines, and resistin. nevertheless, the circulating levels of adiponectin, which is the most abundant adipocytokine, are reduced in conditions such as obesity, type 2 diabetes, and coronary heart disease (chd) [2628 ]. in this context, hypoadiponectinemia was associated with low hdl - cholesterol (hdl - c) concentrations, decreased ldl particle size, and increased markers of systemic inflammation. jeffcoate. suggested that in the diabetic foot there is an inflammatory cascade through an increased expression of proinflammatory cytokines, including tumor necrosis factor alpha (tnf - a) and interleukin-1b. therefore, it is very suggestive that diabetic foot is characterized by a pronounced inflammatory reaction. subclinical inflammation represents a risk factor for both type 2 diabetes and several diabetes - related complications, but data on diabetic neuropathies are scarce. thus, some authors investigated whether circulating concentrations of acute - phase proteins, cytokines, and chemokines differ among diabetic patients with or without diabetic polyneuropathy. they measured 10 markers of subclinical inflammation in 227 type 2 diabetic patients with diabetic polyneuropathy who participated in the population - based monica / kora survey f3. after adjustment for multiple confounders, high levels of c - reactive protein and il-6 were most consistently associated with diabetic polyneuropathy, high mnsi score, and specific neuropathic deficits, whereas some inverse associations were seen for il-18. this study shows that subclinical inflammation is associated with diabetic polyneuropathy and neuropathic impairments and this association appears rather specific because only certain immune mediators and neuropathic impairments are involved. nevertheless, few data exist on the role of systemic inflammation in patients with diabetic foot syndrome although a low - grade immune activation represents an important risk factor not only for the development of type 2 diabetes but also for several vascular complications of diabetes such as macrovascular (myocardial infarction and stroke) and microvascular ones (neuropathy and nephropathy). the status of the immune system may be relevant at several stages in the development of chronic wounds. immune activation may precede the incidence of a diabetic foot ulcer in the same way that it precedes the manifestation of type 2 diabetes and chd. since pro- and anti - inflammatory processes are crucial in the different phases of wound healing, it is well understood how the disturbances of the immune system interfere with tissue homeostasis and wound healing after the manifestation of ulcers leading to the chronic, nonhealing wounds that are characteristic of dfs. evaluated the association between foot ulcers and immune status in a cross - sectional study in diabetic patients with and without foot ulcers by measuring a range of immune mediators (acute - phase proteins, cytokines, and chemokines) representing different aspects of the immune system. these authors conducted this study to compare circulating levels of these immune mediators between both groups, to use multivariate regression models to identify potential confounders of these associations, and to investigate whether systemic immune activation was associated with the severity of the foot ulcer. circulating levels of acute - phase proteins, cytokines, and chemokines were measured in diabetic patients with an ulcer and without an ulcer. patients with an acute foot ulcer had higher levels of c - reactive protein (crp), fibrinogen, il-6, macrophage migration inhibitory factor, macrophage inflammatory protein-1, and interferon--inducible protein-10 as well as lower levels of rantes (regulated on activation normal t - cell expressed and secreted), whereas no differences were found for il-8, il-18, and monocyte chemoattractant protein-1. in multivariate models, size of ulcer, according to the university of texas classification, but not the grade of infection, was independently associated with three markers of subclinical inflammation (crp, il-6, and fibrinogen). however, no study, to our knowledge, has evaluated the role of adiponectin, resistin, and immune - inflammatory biomarkers such as proinflammatory cytokines in patients with diabetic foot compared with those without foot complications. on this basis, tuttolomondo. conducted a study with the aim to evaluate plasma levels of adiponectin, resistin, and il-6 in subjects with diabetic foot in comparison with subjects without foot complications. authors recruited 34 subjects with type 2 diabetes mellitus and foot ulceration hospitalized for every condition related to diabetic disease, but not for new vascular events (group a). as for controls, 37 patients with type 2 diabetes mellitus without foot ulceration have been recruited (group b), all hospitalized for every condition related to diabetic disease but not for new vascular events. this study demonstrated that diabetic subjects with diabetic foot showed in comparison with diabetics without diabetic foot higher il-6 and resistin plasma levels and lower adiponectin plasma levels. resistin, although postulated to contribute to insulin resistance, may also contribute to inflammatory responses. early investigations into the role of resistin as an inflammatory factor demonstrated that lps (lipopolysaccharide) upregulated resistin expression in rat wat, 3t3-l1 adipocytes, and human monocytes. although some initial studies in animal models have produced discrepancies about the fact that proinflammatory cytokines may regulate resistin, several recent human studies have supported the concept of inflammatory cytokine mediation of resistin [34, 35 ]. moreover, osawa. reported that elevated serum resistin concentration appears to be an independent risk factor for ischemic stroke, especially lacunar and atherothrombotic infarction in the general japanese population. in particular, in this study, authors showed that the combination of high resistin and the presence of either diabetes or hypertension increased the risk of ischemic stroke. this is consistent with our findings regarding the higher plasma levels of both il-6 and resistin in diabetic subjects with foot ulceration in comparison with diabetics without foot complications. a recent study by reilly and coworkers suggested resistin as a metabolic link between inflammation and atherosclerosis. in contrast with resistin, adiponectin that is known to enhance insulin sensitivity and reduce atherosclerotic plaques, suppressed a resistin - mediated rise in vcam-1 and icam-1. adiponectin levels can be assessed by either of three variables : total adiponectin, hmwa, and the sa index. recently, almeda - valdes. showed that total adiponectin, hmwa, and the sa index had similar utility for the identification of the metabolic abnormalities. this finding may stimulate the use of adiponectin in clinical and epidemiological settings because the measurement of total adiponectin is better standardized, cheaper, and more accessible than the other two approaches. hypoadiponectinemia can be viewed as an early sign of a complex cardiovascular risk factor predisposing to the atherosclerosis process as well as a contributing factor accelerating the progress of the atherosclerotic plaque. adiponectin also exhibits anti - inflammatory and atheroprotective actions in various tissues by suppressing the expression of vascular adhesion molecules and scavenger receptors, reducing the expression of the inflammatory cytokine tnf - a, raising no production, and suppressing the proliferation and migration of smooth muscle cells. furthermore, two receptors that mediate adiponectin 's actions in fatty - acid oxidation and glucose uptake, namely, adipor1 and adipor2, have been identified. very recently, halvatsiotis. have demonstrated for the first time that a sequence variant in the intron 5 of the adipor2, rs767870 among the eight studied, is associated with cardiovascular disease in a population of greek individuals. recent findings by tuttolomondo. of lower median plasma levels of adiponectin in subjects with diabetic foot could confirm this issue. furthermore, the same authors observed a significant negative correlation between adiponectin plasma levels and some cardiovascular risk factors such as hypertension, dyslipidemia, and clinical variables indicating previous cardiovascular morbidity such as previous tia / stroke and incident vascular morbidity such as neuropathy, microalbuminuria, and pad and these findings further suggest a possible role of hypoadiponectinemia as a putative marker of cardiovascular morbidity both prevalent and incident. axis may contribute to the increased risk of cardiovascular events in patients with type 2 diabetes. in patients with diabetic foot, this expression in lower plasma levels of adiponectin and higher plasma levels of il-6 could be linked to foot ulcers pathogenesis by microvascular and inflammatory mechanisms. some authors evaluated adipocyte volume and its relationship with tnf - a, il-6, adiponectin, and high - sensitivity c - reactive protein (hs - crp) levels. patients were divided into 4 groups : lean healthy controls [body mass index (bmi) : 24.2 1.4 kg / m ], nondiabetic obese patients (30.2 2.9), obese (30.1 3.2), and nonobese (22.2 1.5) type 2 diabetic patients. tnf - a, hs - crp, adiponectin, and il-6 levels were measured preoperatively and sc fat specimens were obtained during operation. in this study, mean adipocyte volumes were higher in obese diabetic patients than in other groups. mean tnf - a, hs - crp, and il-6 levels were higher in obese diabetic patients than in control subjects, obese nondiabetic, and nonobese diabetic patients. mean tnf - a levels of nondiabetic obese patients were higher than the control group. mean adiponectin levels of control subjects were higher than nondiabetic obese, nonobese diabetic, and obese - diabetic subjects. mean hs - crp levels were higher in diabetic patients whether they were obese or not. authors observed a positive correlation between adipocyte size and tnf - a, il-6, and hs - crp levels and negative correlation between adipocyte size and adiponectin levels. these findings furtherly confirm the existence of an adipose - inflammatory vascular axis strictly involved in diabetic complications such as dfs owing to the fact that adiposity and its related conditions, such as diabetes, are at the same time pro - inflammatory and inflammatory conditions. indeed, recent studies suggest that adiponectin may play a role in the modulation of inflammatory vascular response by inhibiting the expression of adhesion molecules on endothelial cells, inhibiting endothelial cell nf-b signaling, and suppressing macrophage function [47, 48 ]. other studies showed that adiponectin suppressed the tnf - a stimulated expression of e - selectin, vcam-1, and icam-1 in human endothelial cells [40, 46 ]. this suggests further that adiponectin may be vasoprotective as reported by other authors [47, 48 ] and negatively modulate the atherogenic processes and that adiponectin has an interaction with an important inflammatory cytokine such as tnf - a. to evaluate the association of adipokines with the macrovascular complications of type 1 diabetes mellitus, a study has been conducted, evaluating serum adiponectin, leptin, and resistin levels in type 1 diabetic patients and analyzing their relationship with carotid intima media thickness (cimt). the authors showed that adiponectin levels in diabetics were higher and leptin levels were lower than controls. adiponectin was also negatively correlated with cimt, age, bmi, and waist - to - hip ratio and positively with creatinine. leptin levels were correlated with total cholesterol and high - density lipoprotein (hdl). authors concluded that increased adiponectin correlates negatively and resistin positively with cimt in type 1 diabetic patients, thus representing possible candidate markers of adipose - inflammatory dysfunction also in diabetic type 1 subjects and possible markers of cardiovascular risk. the pathophysiology of insulin resistance and atherosclerosis share a common inflammatory basis. on this basis, some authors to test this hypothesis evaluated 40 patients with a myocardial infarction (mi). endothelium - dependent (fmd (flow - mediated dilation)) and independent (ntg (nitroglycerine)) vasodilatation (determined by ultrasound), s(i) (insulin sensitivity index ; determined by isoglycemic - hyperinsulinaemic clamp) and serum levels of crp (c - reactive protein), tnf - a, il-6, resistin, and adiponectin (determined by elisa) were measured. fmd, s(i), and adiponectin levels resulted significantly lower in patients with type 2 diabetes mellitus, whereas tnf - a and il-6 levels have been observed as significantly higher in the same diabetic patients. authors also reported that tnf - a concentrations and brachial artery diameter were negatively correlated with fmd whereas s(i) was positively correlated with fmd. these results indicate how endothelium is negatively impacted in multiple ways by the diabetic state after an mi also suggesting endothelium as the main organ - target of adipose - inflammatory dysfunction of diabetes. recently, zietz. reported that low levels of adiponectin are associated with low levels of hdl - cholesterol and might represent an independent cardiovascular risk factor, whereas high levels of adiponectin are associated with high levels of hdl - cholesterol indicating a protective risk profile. furthermore, tuttolomondo. observed significant either positive (for il-6 and resistin) and negative (for adiponectin) correlations in subjects with diabetic foot between these immunoinflammatory and metabolic markers and some clinical and laboratory variables and these correlation further underline the relationships with inflammatory background. recently, pinto. underlined the role of dfs to predict cardiovascular morbidity in diabetic patients, even after correction for other well - known cardiovascular risk factors. in our study, both univariate and multivariate analyses showed the predictive positive role of resistin and il-6 plasma levels and a negative one of adiponectin towards diabetic foot presence. previous studies have shown the relationship between inflammatory cytokines and cardiovascular morbidity in diabetic patients. tuttle. showed that both il-6 and tnf - a are chronically increased in diabetic women with and without cvd compared to nondiabetic women. the additive concentration of cytokines in diabetes and cvd suggests a common inflammatory state in both diabetes and cvd. thus, most recent evidences suggest that diabetic atherosclerosis is not only a disease of hyperlipidemia simply, but is also an inflammatory disorder. a study has been conducted to evaluate the prevalence of inflammatory markers such as high - sensitivity c - reactive protein (hscrp), adiponectin, and nuclear factor-b (nf-b) expression, in peripheral blood mononuclear cells in indian patients with type 2 diabetes mellitus with and without macrovascular disease. authors enrolled 29 consecutive cases of type 2 diabetic patients with proven mvd (group a), 28 matched cases without mvd (group b), and 14 healthy controls (group c) were evaluated for the clinical parameters fasting blood glucose (fbg), 2 h postprandial blood glucose (ppbg), glycosylated hemoglobin (hba1c), lipid profile, and the above - mentioned inflammatory markers. the authors reported that subjects with type 2 diabetes showed higher hs - crp and nf-b expression and lower values of adiponectin compared to healthy controls. group a had significantly higher serum hs - crp than group b (p = 0.0001) despite comparable values of bmi, fbg, 2-h ppbg, hba1c, and lipid parameters. group a had significantly higher serum hs - crp and nf-b expression and significantly lower levels of adiponectin than group c. in group a, serum adiponectin negatively correlated with nf-b expression. in group b, adiponectin values correlated negatively with both fbg and 2-h ppbg. these finding further suggest that patients with type 2 diabetes were in a proinflammatory state strictly related to subsequent vascular complications such as diabetic foot syndrome. several diabetic foot problems including ulcerations, infections, and gangrene represent the most common cause of hospitalization among diabetic patients and their management, cost millions of euro every year, and place a tremendous burden on the health care system. peripheral sensory neuropathy, deformity, and trauma are the most common features underlying diabetic foot ulcers although other risk factors such as calluses, edema, and peripheral vascular disease have also been identified as etiological factors contributing to the development of diabetic foot ulcers. although the pathogenesis of peripheral sensory neuropathy is still poorly understood, there seem to be multiple mechanisms involved, including the formation of advanced glycosylated end products and diacylglycerol, oxidative stress, and activation of protein kinase c. the data linking glycemic control and neuropathy are not as clear cut as those for retinopathy because of the difficulty in identifying objective measures to assess the many stages of neuropathy over time and because the symptoms, or lack thereof, of neuropathy may be misleading if assessed only through patient questionnaires. finally, the differential diagnosis of peripheral neuropathy is quite large, and patients may have other etiologies as well. the diabetic foot can be classified into the neuropathic foot, characterized by the neuropathic ulcer, the charcot joint, and neuropathic oedema associated with a good circulation, in which neuropathy predominates, and the ischaemic foot in which atherosclerosis is the dominant factor leading to a reduction in blood flow with absent pulses. in the neuropathic foot, blood flow is increased, the vessels are still and dilated as a result of medial wall calcification and there is evidence for arteriovenous shunting. the neuropathic ulcer characteristically develops on the plantar surface following inflammatory autolysis and haematoma formation under neglected callosities. chiropody is therefore the mainstay of treatment and recurrence is prevented by redistribution of weight bearing forces by moulded insoles in special footwear. charcot osteoarthropathy is often preceded by fracture which is a further a complication of diabetic neuropathy and precipitates the rapid bone and joint destruction of the charcot joint. medical management can be successful in up to 72%, with the remainder needing arteriography to assess suitability for arterial reconstruction or angioplasty. in the diabetic leg, atherosclerosis is predominant in the branches of the popliteal artery making arterial reconstruction difficult. diabetes and its vascular complication have also a clear inflammatory pathogenesis, but few studies evaluated immunoinflammatory background of diabetic foot syndrome (dfs). only few previous studies [17, 21, 22 ] evaluated inflammatory markers such as cytokine and adipokines in patient with diabetic foot. hypoadiponectinemia can be viewed as an early sign of a complex cardiovascular risk factor predisposing to the atherosclerosis process and a contributing factor accelerating the progress of the atherosclerotic plaque. adiponectin exhibits anti - inflammatory and atheroprotective actions in various tissues by suppressing the expression of vascular adhesion molecules and scavenger receptors, reducing the expression of the inflammatory cytokine tnf - a, raising no production, and suppressing the proliferation and migration of smooth muscle cells. these data are consistent with the findings reported by tuttolomondo. of lower median plasma levels of adiponectin in subjects with diabetic foot. furthermore, we observed a significant negative correlation between adiponectin plasma levels and some cardiovascular risk factors such as hypertension, dyslipidemia, and clinical variables indicating previous cardiovascular morbidity such as previous tia / stroke and incident vascular morbidity such as neuropathy, microalbuminuria, and pad and these findings further suggest a possible role of hypoadiponectinemia as a putative marker of cardiovascular morbidity both prevalent and incident. axis may contribute to the increased risk of cardiovascular events in patients with type 2 diabetes. in patients with diabetic foot expression in lower plasma levels of adiponectin and higher plasma levels of il-6 could be linked to foot ulcers pathogenesis by microvascular and inflammatory mechanisms. these findings further underline the importance of inflammatory and metabolic milieu such as cytokines and adipose hormones in foot complications in diabetics as already reported for other vascular complications of diabetes [45, 5161 ]. | diabetic foot ulcerations have been extensively reported as vascular complications of diabetes mellitus associated with a high degree of morbidity and mortality ; in fact, some authors showed a higher prevalence of major, previous and new - onset, cardiovascular, and cerebrovascular events in diabetic patients with foot ulcers than in those without these complications. this is consistent with the fact that in diabetes there is a complex interplay of several variables with inflammatory metabolic disorders and their effect on the cardiovascular system that could explain previous reports of high morbidity and mortality rates in diabetic patients with amputations. involvement of inflammatory markers such as il-6 plasma levels and resistin in diabetic subjects confirmed the pathogenetic issue of the adipovascular axis that may contribute to cardiovascular risk in patients with type 2 diabetes. in patients with diabetic foot, this adipovascular axis expression in lower plasma levels of adiponectin and higher plasma levels of il-6 could be linked to foot ulcers pathogenesis by microvascular and inflammatory mechanisms. the purpose of this review is to focus on the immune inflammatory features of dfs and its possible role as a marker of cardiovascular risk in diabetes patients. |
down syndrome (ds) is the most common chromosomal aneuploidy in live births and a leading cause of mental retardation. prenatal detection of chromosomal anomalies, such as trisomy 21, in cases with ds relies on invasive practices such as amniocentesis or chorionic villi sampling. these procedures are associated with a risk of fetal loss or adverse pregnancy outcome. alternative strategies are sought, such as the noninvasive prenatal diagnosis of fetal genetic anomalies via the analysis of rare trafficking fetal cells or cell - free fetal nucleic acids in maternal blood. as this long sought goal has not yet reached clinical implementation, current clinical practice relies on a series of screening steps aimed at detecting at - risk pregnancies. in many centres the screening procedure for ds is carried out in the late first and early second trimester (1114 weeks of gestation) and involves a combination of ultrasound and serum marker analysis. although the efficacy of this method has improved considerably, and is more accurate than previous second trimester screening approaches, it is hampered by the skill and precision of the ultrasonographer and external factors such as ethnicity or other factors, such as assisted reproductive technologies. hence, it is obvious that new tools are needed to improve current state of the art. the rationale for this strategy is that placentae from fetuses with ds, especially the villi, are morphologically distinct from the placentae of euploid fetuses, a feature attributed to altered aberrant protein expression. as the placenta, a large organ with rapid cell turn - over is in direct contact with the maternal circulation, proteins released by it should be detectable in maternal plasma, and could consequently serve as promising markers for abnormal placentation involved in a number of pregnancy related disorders. in this regard, preliminary proteomic investigations have shown that quantitative alterations in protein expression are found to occur in the amniotic fluid [7, 8 ] and maternal serum of pregnancies with fetuses affected with down syndrome, and that these could serve as new potential biomarkers. unfortunately none of these studies used quantitative approaches of the type that permit precise assessment of the extent of up- or down - regulation of the proposed biomarkers. as such, it will be difficult to validate these in blinded studies of clinical serum or plasma samples, a crucial facet in determining their specificity [911 ]. these studies are further complicated by the complex nature of the plasma / serum proteome, whereby the majority of low abundance proteins are masked by the preponderance of a few highly abundant proteins. this high dynamic range effectively precludes the use of more conventional proteomic strategies, such as those employing gel electrophoresis with or without or fluorescent labelling, for example, difference gel electrophoresis (dige). this deficit renders the identification of potential biomarkers in plasma / serum by conventional comparative proteomic approaches very challenging. for this reason an approach using isobaric tags for absolute and relative quantitation (itraq) has been proposed for the discovery of plasma biomarkers. this chemical labelling method involves the stable incorporation of isotopes into an amine tagging reagent, which can be reliably detected by mass spectrometry, thereby permitting comparative quantitation in a multiplex manner. currently 4-plex and 8-plex reagents are commercially available, which are used to label the protein samples of interest following trypsin digestion. the use of different isobaric tags implies that up to 4 or 8 different samples, one of which serves as a reference, can be examined simultaneously in a single mass spectrometric analysis. it is for this reason that the itraq approach has been suggested to be suitable for the discovery of biomarkers in a wide range of body fluids and tissues, including plasma. in order to examine whether this approach would be suitable for the detection of potential biomarkers for down syndrome, we performed a proof - of - principle experiment, in which we examined samples from 6 cases with down syndrome in comparison to 6 matching controls. in our study we have used a 4-plex itraq labelling in conjunction with a 4800 maldi tof / tof approach to examine plasma samples obtained in first trimester pregnancies. our results indicate that quantitative differences can be detected between aneuploid samples and samples from euploid pregnancies and that such alteration may reflect upon changes known to occur in down syndrome. blood samples for this case - control proteome study were collected from six pregnant women carrying a ds fetus (1113 weeks of gestation) and six pregnant women with normal euploid pregnancies. this study was undertaken with the approval of the institutional ethical board of the university hospital, basel, switzerland and written informed consent was required in all instances. 9 ml blood was drawn into bd p100 tubes (bd diagnostics, franklin lake, ny, usa), which are specially designed for proteomics experiments, in that the ethylenediaminetetraacetic acid (edta) and protease inhibitor present in the tube prevent coagulation and stabilize the plasma proteome. following phlebotomy the samples were centrifuged at 3000 g for 30 minutes at 10c, whereby the plasma was separated from the cellular fraction by aid of a mechanical separator. 100 l aliquots were stored at 80c until further use. for an overview of the work - flow used in this analysis refer to figure 1. highly abundant plasma proteins were depleted using proteominer protein enrichment kit (bio - rad laboratories, inc.), as per the manufacturer 's instructions. 1 ml of plasma was used for the depletion and after the whole procedure, 300 l was eluted in elution reagent. after depletion protein concentration was measured by using rc - dc protein assay kit (bio - rad laboratories, inc.) equal amounts (100 g) of depleted plasma protein from six of the ds cases and controls were pooled separately in duplicate for the itraq labelling. these samples were denatured with 2% sds in 500 mm triethylammonium bicarbonate (teab) (sigma - aldrich) for 15 minutes at room temperature, following which they were reduced with 2 l of 50 mm tri-(2-carboxyethyl) phosphine (tcep) (sigma - aldrich) at 60c for 1 hour and were then alkylated with 10 mm s - methylmethanethisulfonate (mmts) for 10 minutes in room temperature. after alkylation, the proteins were digested overnight at 37c with 1 u/l trypsin (tpck treated) (applied biosystems, foster city, ca 94404, usa). peptides were labelled with one unit of itraq reagent multi - plex kit (applied biosystems, foster city, ca 94404, usa) that was reconstituted in 70 l of ethanol. itraq labels 114, 116 were used separately for labelling the pooled duplicated control sample and 115, 117 were used separately to label the pooled duplicate down syndrome samples. the itraq labelling reagent solution was added to the digest and incubated for 1 hour at room temperature. to assess the accuracy of the ratiometric quantitation of itraq reagent a split in signal was performed and the data were corrected as described in detail by unwin and colleague. strong cation exchange chromatography was preformed to remove the excess itraq reagent and interfering substances for the mass analysis. dried peptides were resuspended in 200 l of buffer a and were loaded on poly sulfoethyl a column (200 mm length 4.6 i d, 5 m particle size, 200 pore size) on a biolc hplc unit (dionex). buffer a consisted of 10 mm khpo4 and 25% acetonitrile, 500 mm kcl, ph 3.0, and buffer b consisted of 10 mm kh2po4, 25% acetonitrile, and 500 mm kcl ph 3.0. the 60 minutes gradient comprised of 100% buffer a for 5 minutes, 5%30% buffer b for 40 minutes, 30%100% buffer b for 5 minutes, 100% buffer b for 5 minutes, and finally 100% buffer a for 5 minutes. subsequently, these fractions were pooled according to the chromatogram profile based on the peak intensity and the products dried in a vacuum concentrator, after which they were stored at 20c prior to mass spectrometric analysis. the dried scx itraq - labeled peptides were dissolved in buffer a which consist of 95% h2o, 5% acetonitrile, 0.1% tfa and were loaded on c18 trap column (1 mm 300 m i.d. column) at 30 l / minutes and separated on an analytical column (150 mm 100 m i.d. the peptides were separated using a linearly increasing concentration of acetonitrile in buffer b from 5% to 30% in 120 minutes, and from 30% to 60% in 40 minutes. the elute was mixed with matrix (2 mg / ml alpha - cyano-4-hydroxycinnamic acid in 80% acetonitrile, and 0.1% tfa) at a flow rate of 800 nl / min and deposited on an opti - tof lc / maldi (applied biosystems) plate in 10s fractions, using an automatic robot (probot, dionex). the mass spectrometer 4800 plus maldi tof / tof analyzer (applied biosystems) was set to perform data acquisition in positive ion mode. monoisotopic precursor selection for ms / ms was done by automatic precursor selection using an interpretation method using the 12 most intense peaks per spot with an ms / ms mode condition of 4000 laser shots per spectrum. msms was done with a gas pressure of 2 10 bar in the collision cell. protein identification and quantification was done by using the proteinpiolt software v2.0 (applied biosystems ; mds - sciex). the search was performed against the human database of uniprotkb / swiss - prot (version 3.50) from the ebi website (http://www.ebi.ac.uk./ipi/ipihelp.html) and concatenated target - decoy database search strategy was used to check the false positive rate in our case it was found to be 0%, which boosted the reliability of our data. the paragon algorithm [17, 18 ] in proteinpiolt v2.0 software was used as the default search program with digestion enzyme set as trypsin and methyl methanethiosulfonate as cysteine modification. the search also included the possibility of more than eighty biological modifications and amino acid substitution of up to two substitutions per peptide using the blosum 62 matrix. data were normalized for loading error by bias correction calculated with progroup algorithm identified proteins with at least 95% confidence and with a protscore of 1.3, were reported. the results obtained from proteinpiolt software v2.0 software were exported to microsoft excel for the further analysis. the study was performed in double duplex manner, where ds samples were labelled with 115 and 117, control were labelled with 114 and 116. peptides were selected based on the criteria defined in the protein pilot software, which means all the peptides were included for quantitation with an exception for those without an itraq modification or reporter ion, an area count less than 40 and peptides with p value less than.001 were excluded. as described by gan and colleague in their study on estimation of relative quantitative ratio from itraq experiments, we also used only peptides above or equal to 70% confidence level for the estimation of relative quantitation. the panther database was used to elucidate the molecular function, biological process and signaling pathway associated with each individual protein (http://panther.appliedbiosystems.com/). samples were obtained from 6 cases with a confirmed ds fetus and 6 samples from normal euploid singleton pregnancies. care was taken to match both maternal and gestational age, to rule out any confounding influence of these two parameters. this was accomplished through the use of a large, highly diverse bead - based library of combinatorial peptide ligands. when plasma was applied to the beads, a small fraction of the high abundance proteins saturated their high affinity ligands and the excess high abundance proteins were washed away. in addition a very small amount of high abundance proteins and low abundance proteins were concentrated on their specific affinity ligands. the samples were pooled separately in and duplicate in order to have more precise analytical replicate measurements. the itraq analysis was done in double duplex style, the ds samples were labelled with itraq 115 and 117 and the control samples with itraq 114 and 116, using the work - flow illustrated in figure 1. following tandem ms ms, and by focussing on itraq reporter ions in low molecular mass range (114117 da) for quantification, we identified 235 proteins with 95% confidence. however, after manually rechecking the ms / ms data thoroughly peak by peak, only 187 out of 235 proteins (78.5%) had a relative quantitation derived from the analysis of two or more peptides, while for 45 proteins, the quantitation was based on single peptide. for 3 proteins no quantitation could be ascertained by analysis using protein pilot. figure 2 shows the msms spectrum of the precursor ([m + h ] +, m / z 1527.7 da). in low - mass region the reporter ions, are seen while area under the curve was used for quantification. as we did the experiment in double duplex manner, ds (115 and 117) and control (114 and 116), it was possible to estimate the cutoff point for differentially expressed protein in our sample [19, 20 ]. based on 187 relative abundant protein ratios from ds and control sample, an average variation of 4.4% (0.04) was measured. if the cutoff was set at 5% average variance then only 72% of the proteins would fall with in this variation range, but. that means any relative change in protein ratio below or above 1.2 fold was considered as differentially under or over expressed. the functional distribution of these proteins is illustrated in figure 4. for this interpretation, an analysis of 235 proteins was performed using the panther classification system, which sorts the proteins into respective classes based on their biological process. it is of interest that two of the major groups involve cell adhesion molecules (13%) and extra cellular matrix proteins (18%) and member of the protease family, factors that are known to be aberrant in down syndrome. further more large groups were found to involve signalling molecules (13%) and 18% defense / immunity proteins. of the large group of significantly up - regulated proteins, it is highly noteworthy that this includes hcg (beta human chorionic gonadotropin), a subunit of the human chorionic gonadotropin protein known to be elevated in pregnancies with ds fetuses, and which forms one of the backbone of current screening programs in the first trimester. this finding, therefore strongly suggests, that the itraq method we have chosen appears to be able to identify relevant proteins (table 2). in this group, elevations were also recorded for pregnancy zone protein (pzp) (p20742), thereby indicating that our assay is indeed detecting proteins of placental origin. elevation of two members of the amyloid family (serum amyloid p - component and amyloid beta a4) was also observed, which could be a significant finding, as these proteins have been suggested to play a role in the dementia found to occur in adult ds patients. an elevation in the inflammatory response marker, c - reactive protein and members of the immunoglobulin family like ig lambda chain c region was also noted, which could be indicative of an increased inflammatory response in ds pregnancies. phosphatidylinositol - glycan (p80108), insulin - like growth factor (p35858) is involved in protein - protein interactions that result in protein complexes and hepatocyte growth factor (p26927) was also prominent amongst the list of the up - regulated proteins. amongst the group of down - regulated proteins (table 3) were a number of molecules involved in cell adhesion and extracellular matrix including titin (q8wz42), basement membrane specific heparan sulfate proteoglycan (p98160), actin, cytoplasmic 2 (gamma - actin) (p63261)and fibrinogen alpha chain (p02671). this observation could be important as changes in tissue elasticity are a hallmark of ds cases, and play significant role in their detection by ultrasound via the presence of an increased neck fold (nuchal translucency). peroxiredoxin 2 (prdx2), an antioxidant enzyme, is under - expressed in ds fetal brain and dynein heavy chain 9, was also noted in our list of down regulated proteins. the panther database was used for the pathway analysis on the proteins which were shown to be over or under expressed in our study. it was interesting to note that 13.3% proteins identified in the ds sample correspond to proteins found in the alzheimer disease - amyloid secretase and alzheimer disease - presenilin pathways illustrated in figure 5. more than the 40% of the protein we identified in ds samples correspond to proteins in the integrin signalling pathway. this might be due to the fact that cell adhesion molecules and extra cellular matrix protein represent 13% and 18% of the total proteins identified, respectively as illustrated in figure 4. quantitation of serum or plasma proteins via itraq analysis has recently been suggested to be suitable for the detection of biomarkers, as the method is highly reproducible, with little run - to - run variation. this aspect, which is optimal to embark on such a fishing - expedition, is actually quite surprising, granted the large number of individual steps in the work - flow. this conclusion was, however, derived at after a lengthy comparison involving three pools of case and control samples, as well as a number of individual samples, where a coefficient of variation of 11.7% was noted. in a preliminary proof - of - principle experiment, we have now assessed whether this method could be suitable for the detection of biomarkers useful for ds screening. from our small - scale preliminary evaluation we identified over 200 proteins whose concentration was altered in the plasma of pregnancies with a ds fetus when compared to those with euploid fetuses. it is of interest that hcg is detected in the pool of proteins found to be elevated. this glycoprotein, which is produced early in pregnancy by the developing embryo and subsequently by the syncytiotrophoblast, forms the backbone of 1st and 2nd trimester screening strategies. in the 1st trimester, pregnancies at - risk of carrying a fetus affected by ds are identified on the basis of an almost 2 fold mom (multiples of the median) elevation in hcg. the presence of this important screening marker in our pool of elevated plasma proteins suggests that the strategy we have chosen for the identification of new biomarkers is functional and worthy of further pursuit. in this context it is worth noting that this marker was not detected in two previous studies [9, 11 ] using proteomic technologies for the identification of protein markers for ds in maternal plasma or serum. this could be due to limitations in detection sensitivity of the 2d gel approach these studies had used. that our assay is indeed detecting proteins of placental origin is illustrated by presence of pregnancy zone protein which is a glycoprotein and a proteinase inhibitor which derive its importance in pregnancy by playing potential role in preventing the attack from maternal immune system on the developing fetus which can be seen as foreign allograft. this protein was amongst our list of up - regulated proteins, which is similar to alpha 2-macroglobulin, is quantitatively the most important pregnancy - associated plasma protein. amongst the pool of up - regulated proteins were serum amyloid p - component and amyloid beta a4, which is encouraging as members of this family have found to be elevated in previous studies on pregnancies with ds fetuses, which may be a reflection of an altered gene expression pattern associated with ds - related dementia. it is also important to note that the amyloid precursor protein (app) gene is located in ds region of chromosome 21. teller and colleagues have shown that a relatively minor from of amyloid beta protein was present in the brain of ds affected pregnancy as early as in late 2nd trimester. elevations in a number of inflammatory molecules, most of which are probably of maternal origin, may be a reflection of the elevated release of placental debris which has been suggested to occur in pregnancies with ds fetuses. phosphatidylinositol - glycan is expressed in the placental tissue, also involved in many cellular process and plays important role in several signal transduction pathways. of the down - regulated proteins like actin, gelsolin, heparan sulfate proteoglycan, and fibrinogen alpha chain, it is noteworthy that all these protein are involved in cell adhesion, extracellular matrix, cell structure or muscle contraction, since ds fetuses are known to exhibit connective tissue abnormalities, the most pronounced of which is the increased neck fold (nuchal translucency). since adult ds patients are prone to skeletal muscle deficiency, our finding concerning reduced titin plasma levels is intriguing, as this very large protein plays an important role in muscle contraction. in one of recent study du and colleague have shown trophoblast expression of titin in first trimester placentae. only a limited number of studies have attempted to use proteomic approaches for the discovery of new biomarkers for pregnancies at - risk of carrying a fetus with ds, two of these used 2-de approaches [9, 11 ], while a further used a seldi method. study of the former studies that by nagalla and colleagues is the largest, having examined serum samples from 56 pregnant women. this study used samples collected in both the 1st and 2nd trimester of pregnancy, which were recruited as part of the nih funded faster study, and largely made use of the fluorescent 2d - dige process. in their study, 18 proteins were found to be elevated in 1st trimester samples, which included members of the apoliprotein family, clusterin and proteins involved in skeletal development (tetranectin). the study by kolialexi and colleagues used traditional 2de stained with coomassie blue on 20 maternal plasma samples (8 cases, 12 controls, 1618 weeks of pregnancy), by which means 8 candidate proteins were detected. in contrast to the study by nagalla. a down regulation for clusterin was noted. in the study by busch and colleagues using a seldi approach, traces were noted which differed between ds cases and controls. however, no attempt was made to discern what proteins were responsible for these altered patterns, nor was any detailed description provided of how they could be reproduced. other than common elevation in serum amyloid and complement component families, little commonality exists between our study and these studies. this may be due to a number of factors including, limited study size, time of sampling collection, sample processing and storage, as well as use of very different technical approaches. in our follow - up studies we would like to validate these putative biomarkers using immunoblot and enzyme linked immunosorbent assay (elisa). more recently selected reaction monitoring (srm) has evolved as a method of choice for validation of biomarkers using mass spectroscopy. the increasing popularity of the itraq approach due to its reproducibility and robustness, including studies for cancer or inflammatory autoimmune disorder specific biomarkers suggests that it will become the method of choice for future studies, until it is surpassed by a new technical development. as pregnancy represents a unique constellation, whereby a foreign being is supported and nourished by the host, it may serve as an ideal model for proteomic analyses, as any unique markers should ideally disappear post delivery. furthermore, as very few specific biomarkers exist to assist with the screening of a number of pregnancy - related disorders, especially preeclampsia or preterm labour, it is likely that this will become the focus of considerable research attention in the near future. in this report we conclude that isobaric labelling technique is a suitable approach for the quantitative detection of new screening biomarkers in the plasma of pregnancies with a ds fetus compared to those with euploid fetuses. in this preliminary proof - of - principle study, we were able to detect quantitatively under- or over - expressed proteins. in the future additional studies, using larger sample sizes will be required to identify a panel of biomarkers which can be used in screening for ds pregnancies. | currently no specific biomarkers exist for the screening of pregnancies at risk for down syndrome (ds). since a quantitative proteomic approach with isobaric labelling (itraq) has recently been suggested to be highly suitable for the discovery of novel plasma biomarkers, we have now used this method to examine for potential quantitative changes in the plasma proteome of the pregnancies bearing ds fetuses in comparison to normal healthy babies. in our study, we used plasma from six women with ds pregnancies and six with uncomplicated pregnancies care were taken to match cases and controls for gestational and maternal age, as these could be a confounder. in our quantitative proteomics analysis we were able to detect 178 proteins using itraq labelling in conjunction with 4800 maldi tof / tof. amongst these we observed changes in hcg, a known screening marker for ds, indicating that our assay was functional. we found a number of elevated proteins ig lambda chain c region, serum amyloid p - component, amyloid beta a4, and under expressed proteins like gamma - actin and titin in ds pregnancies. these proteins are also found in the sera of patients with alzheimer disease, which share similar pathologies of ds. our study therefore indicates that the itraq labelling approach may be indeed useful for the detection of novel biomarkers. |
nonsteroidal anti - inflammatory drugs (nsaids) are one of the most commonly prescribed classes of medication, and they are routinely employed for their analgesic, antipyretic, and antiinflammatory properties. because they are potent inhibitors of cyclooxygenase (cox) enzymes, they reduce the synthesis of pro - inflammatory prostaglandins (pgs). nsaids have been widely used systemically for many decades and have more recently become available in the form of topical ophthalmic formulations. in ophthalmology, topical nsaids are mostly used to stabilize pupillary dilation during intraocular surgery, to control postoperative pain and inflammation (particularly after refractive surgery), and to treat allergic conjunctivitis and pseudophakic cystoid macular edema (cme) [2, 3 ]. a growing body of evidence suggests that nsaids may also be beneficial in diabetic retinopathy (dr), ocular tumors, and age - related macular degeneration [1, 48 ]. they catalyze the biosynthesis of eicosanoids from arachidonic acid to produce 5 classes of pgs : pge2, pgd2, pgf2, pgi2, and thromboxane a2. pge2 increases the iop by local vasodilation and increased permeability of blood aqueous barrier. on the other hand pgf2 thirdly, pgs cause vasodilation and increase the vascular permeability with the disruption of the blood - ocular barrier with leukocyte migration and therefore edema formation. by definition, nsaids lack a steroid nucleus. cox-1 and cox-2 are the main cox isoforms, although there is a third isoform, cox-3. cox-3 is an acetaminophen - sensitive alternatively spliced variant of cox-1, and it has not been well defined [1113 ]. cox-1 regulates normal physiological processes and is mainly expressed in the gastrointestinal tract, kidneys, platelets, and vascular endothelium. cox-2 is the predominant isoform in the retinal pigment epithelium (rpe) and is upregulated during inflammatory processes, pain, and fever, but it is also expressed under normal conditions in sites such as the brain and kidneys. cox-2 has also been found in choroidal neovascularization (cnv) and in dr [4, 5, 7, 8, 1619 ]. pgs act by upregulating a number of soluble mediators responsible for the expression of vascular endothelial growth factor (vegf), which plays a key role in the cnv and in the dr [2022 ]. in a number of experimental models cox-2 inhibition has been found to inhibit angiogenesis [2326 ], cnv, and dr [17, 18, 27, 28 ].. there are six major classes : salicylates, indole acetic acid derivatives, enolic acid derivatives, fenamates, aryl acetic acid derivatives, and aryl propionic acid derivatives. however, the topical nsaids available for ophthalmic usage are mostly limited to the soluble forms : indole acetic, aryl acetic, and aryl propionic acid derivatives [9, 16 ]. most of the nsaids are weakly acidic drugs, which ionize at the ph of the lacrimal fluid and therefore have limited permeability through the anionic cornea which has an isoelectric point (pi) of 3.2. reducing the ph of the formulation increases the unionized fraction of the drug which enhances permeation. because of their acidic nature, nsaids are inherently irritating ; reducing the ph further increases their irritability and decreases their aqueous solubility. in addition, the anionic nature of nsaids leads to the formation of insoluble complexes with cationic quaternary ammonium preservatives, such as benzalkonium chloride. hence, a nsaid formulation that is comfortable when topically applied is somewhat difficult to formulate. nsaids are adsorbed by the gastrointestinal tract, reaching a peak serum concentration after 13 hours. they are metabolized by the liver and excreted in the urine and bile ; they are highly protein bound in the plasma (> 95%), normally to albumin ; thus their volume of distribution approaches that of plasma. topically administered nsaids follow this distribution, since they are systemically adsorbed by the nasolacrimal outflow system and the mucosal surfaces. nepafenac is a prodrug that is rapidly converted to the more potent amfenac by intraocular hydrolases. since nepafenac is a noncharged molecule, it exhibits greater corneal permeability than the other nsaids. this was demonstrated in an in vitro study that showed sixfold greater corneal penetration by nepafenac than by diclofenac. bromfenac has a similar structure to amfenac, with the exception of a bromine atom at the c4 position. this modification increases the penetration of bromfenac into ocular tissues, increasing its anti - inflammatory activity. ketorolac is reportedly the most potent inhibitor of cox-1, while bromfenac and nepafenac / amfenac are the most potent inhibitors of cox-2 [9, 35, 36 ]. however, ketorolac 0.45% inhibited pge2 more strongly than bromfenac 0.09% and nepafenac 0.1%, reaching significantly greater aqueous concentrations [37, 38 ]. bromfenac has been reported to be a 3- to 18-fold more potent inhibitor of cox-2 than diclofenac, ketorolac, and nepafenac / amfenac, although these data remain to be confirmed in randomized controlled clinical trials [1, 9, 39 ]. it is possible that cox-1 may also play a role in inflammation [1, 16 ] therefore the specific roles of cox-1 and cox-2 in this context require further investigation. after a single eye - drop, peak aqueous drug levels are detectable for diclofenac 0.1% (82 ng / ml ; 2.4 h peak), flurbiprofen 0.03% (60 ng / ml ; 2.0 h peak), nepafenac 0.1% (205.3 ng / ml ; 30 min peak), amfenac (following administration of the prodrug nepafenac 0.1% ; 70.1 ng / ml), ketorolac 0.4% (57.5 ng / ml ; 60 min peak), and bromfenac 0.09% (25.9 ng / ml) [35, 40 ]. more prolonged and more frequent administration of nsaids leads to higher aqueous levels. twelve doses of ketorolac 0.4% over 2 days reportedly result in an aqueous level of 1079 ng / ml, and the same dosing regimen of nepafenac 0.1% results in 353 ng / ml ; both concentrations far exceed that is reportedly required to inhibit cox-1 and cox-2, which is 50 ng / ml. while topical administration of nsaids achieves therapeutic levels in the aqueous humor, thereby reducing the synthesis of pgs in the ciliary body and the iris, such a therapeutic effect is less evident in the retina and the choroid. heier.. measured vitreous drug levels in patients who received ketorolac 0.4% qid, bromfenac 0.09% bid, or nepafenac 0.1% tid for 3 days before vitrectomy. vitreous levels of ketorolac, bromfenac, and amfenac were reportedly 2.8 ng / ml, 0.96 ng / ml, and 2.0 ng / ml, respectively, but only ketorolac resulted in significantly lower vitreous pge2 levels compared to placebo. nsaids inhibit the expression of cox enzymes, thereby reducing the endogenous pgs that act on the iris and ciliary body to induce vasodilation, blood - ocular barrier disruption, leukocyte migration, pain stimulation, iop control, and miosis [2, 3, 16, 43 ]. commercially available pgf2 analogues act by increasing uveoscleral outflow in the ciliary body, while pge2 reportedly increases iop via vasodilation and partial disruption of the blood - ocular barrier. the administration of topical nsaids does not have any effect on iop, as it is not selective with regard to pg class. however, nsaids may have a slight additive effect when administered together with pgf2 analogous [44, 45 ]. a pivotal difference between nsaids and corticosteroids is the effect of the latter on both iop and lipoxygenase, which facilitates a greater anti - inflammatory effect, albeit with an associated increase in the likelihood of iop elevation. there is convincing clinical evidence in the peer - reviewed literature attesting to the capacity of topical nsaids to reduce postoperative inflammation after eye surgery [1, 2, 16, 46 ]. in randomized controlled clinical trials, bromfenac 0.09%, nepafenac 0.1%, diclofenac 0.1%, ketorolac 0.5%, flurbiprofen 0.03%, and indomethacin 1% have been shown to decrease postoperative inflammation following cataract surgery [34, 41, 4758 ]. corticosteroids are also widely used postoperatively to reduce inflammation ; therefore studies comparing the 2 drug classes have been conducted. while significant differences in the reduction of intraocular inflammation after cataract surgery were not observed [55, 56, 59 ], nsaids were more effective at reestablishing the blood - aqueous barrier as indicated by flare, which was measured via either slit - lamp examination or fluorophotometry [16, 46, 52, 59 ]. thus, the collective evidence suggests that topical nsaids may be used in place of topical corticosteroids after cataract surgery or, perhaps preferably, in addition to them ; a number of clinical trials have reported a synergistic effect when nsaids and corticosteroids are administered together [49, 50, 58, 60, 61 ]. despite advances in technique and surgical materials, cystoid macular edema (cme) is the most frequent cause of reduced vision following uneventful modern cataract surgery, with a seemingly rare incidence of 0.12.35% for clinically significant cme [6264 ]. also known as irvine - gass syndrome, it is mainly caused by the accumulation of extracellular fluid within the retina due to leakage from dilated capillaries [1, 16, 63 ]. the pathogenesis of it is not fully understood, but the main trigger is thought to be surgical trauma of the intraocular tissues, involving rupture of the blood - aqueous barrier ; this may cause diffusion of pgs and other inflammatory mediators into the vitreous cavity, inducing a cascade of inflammatory events with subsequent rupture of the blood - retinal barrier, resulting in cme in some patients. therefore it seems reasonable to take strong measures to minimize the inflammatory process, possibly including the administration of both corticosteroids and nsaids together. a recent study by ersoy. that quantitatively assessed aqueous flare after phacoemulsification reported that patients who developed cme had significantly higher flare values than those who did not, suggesting that inflammatory pathogenesis and a breakdown of the blood - ocular barrier may be involved. cme can be diagnosed and classified clinically, on fluorescein angiography and by optical coherence tomography (oct). the range of the reported incidence rates is wide (0.102.35% for clinically important cme, defined as a retinal thickening within 500 microns of the center of the macula causing a significant vision impairment) [62, 64 ], which may be due to the different patient populations, cataract stages, surgical techniques, and, particularly, diagnostic methods utilized by the relevant studies. notably, after small - incision cataract surgery the reported rates of cme range from 9 to 19% based on fluorescein angiography and are as high as 41% as determined by oct [6668 ], although clinically important cme is far less common [1, 69 ]. a number of studies report the effectiveness of topical nsaids in the prophylaxis of cme following cataract surgery [2, 16, 63, 7074 ], although the angiographic reduction of cme is reportedly most evident in the first postoperative month and is no longer statistically significant a year after surgery. however, interpretation of the independent effects of nsaids based on the results of the available studies is difficult, due to the common concomitant administration of corticosteroids. one trial by flach. reported that prophylactic use of ketorolac 0.5% was effective in reducing cme without the use of corticosteroids. prospectively compared the effects of topical diclofenac 0.1% versus fluorometholone 0.1% (a corticosteroid with limited intraocular penetration that therefore could be reasonably approximated to a placebo) in the prophylaxis of cme and reported that 5 weeks after surgery, angiographic cme was present in 5.7% of diclofenac - treated eyes and 54.7% of fluorometholone - treated eyes. a randomized comparison of topical ketorolac 0.4% plus corticosteroid versus corticosteroid alone showed a significantly reduced rate of cme with combination treatment after phacoemulsification. however, the incidence of definite or probable cme (definite cme is intended as the presence of cystoid changes associated with 40 m retinal thickening evident on oct, while probable cme is intended as the presence of changes in retinal contour and increased macular thickness relative to preoperative baseline, but without definite cystoid changes) was low in both groups (2.4% in the corticosteroid group and 0% in the ketorolac / corticosteroid group) and there was no difference in visual outcomes. such results raise the issue of the cost effectiveness of routine administration of cme prophylactic treatment with both corticosteroid and nsaids for patients at low risk of cme. however, cost effectiveness ratio is certainly lower in diabetic and uveitic patients who are at higher risk of cme and are reported to benefit from routine concomitant use of nsaids and corticosteroids. cme following phacoemulsification may be treated early (less than 6 months) or late (6 months or more) following its diagnosis, respectively, defining acute and chronic cme [1, 16 ]. the treatment of chronic cme following cataract surgery has been assessed in a number of studies [16, 7780 ] which have shown an overall beneficial effect of nsaid treatment, although a recent meta - analysis reported that for acute cme, the evidence is not yet conclusive. assessing the efficacy of ketorolac and nepafenac with regard to the prevention of postoperative cme after uneventful phacoemulsification. the authors concluded that prophylactic topical nsaids are not recommended for routine surgery patients without risk factors. in chronic disease, trials by flach. [63, 79, 80 ] suggest that topical ketorolac 0.5% is effective and that treatment for a duration of 3 months provides a longer lasting benefit than treatment for 2 months. however, there are few published trials and they tend to have small sample sizes ; therefore, further controlled studies are required. four topical nsaids (diclofenac 0.1%, ketorolac 0.4%, nepafenac 0.1%, and bromfenac 0.09%) have been evaluated in combination with intravitreal corticosteroid and bevacizumab injections for the treatment of chronic pseudophakic cme. results suggested that while nsaids apparently provide additional benefit to that produced by corticosteroids and anti - vegf, only nepafenac- and bromfenac - treated eyes showed reduced retinal thickness at 12 and 16 weeks, and only nepafenac led to a significant improvement in vision. similarly, in a retrospective and uncontrolled study, nepafenac 0.1% improved retinal thickness and visual acuity in patients with chronic cme. in all reported studies nsaids further studies are needed to enlighten if varying the dosing regimen affects the efficacy of nsaids in cme resolution. in conclusion, although there is no specific approved treatment for pseudophakic cme, overall evidence supports the administration of topical nsaids and also suggests that combining them with topical corticosteroids yields a synergistic effect. however, the advisability of nsaids for cme prophylaxis in patients with low risk factors is debatable, given the low clinically significant incidence and the cost effectiveness ratio. in developed countries, age - related macular degeneration (amd) is the leading cause of visual impairment and blindness in patients over 60 years of age. typical features of neovascular amd include choroidal neovascularization (cnv) beneath the macula, with associated retinal hemorrhages and swelling. involution of the new vessels is accompanied by fibrous metaplasia, permanent loss of photoreceptors, and disciform scarring, which often result in loss of central vision. large - scale clinical trials have demonstrated that monthly intravitreal injection of anti - vegf prevents vision loss and may even improve visual acuity in patients with neovascular amd [87, 88 ]. inflammation plays an important role and some patients exhibit an inadequate response to anti - vegf treatment, along with persistent exudation. in particular, a multitude of recent genetic analyses in human amd patients supports the role of complement factor h in the pathogenesis of it in up to 50% of cases [9093 ]. there are several complement components (c3, c5, the c5b-9 membrane attack complex (mac), and cd46) found in drusen. this indicates that complement components and regulators may contribute to the formation of drusen and upregulate vegf expression [9496 ]. although amd is not a classic inflammatory disease, inflammatory cells have an important role in amd pathogenesis and progression [94, 97 ]. autoimmunity has also been suggested to have a role in drusen formation and amd pathogenesis. it has been suggested that the presence of a number of antiretinal autoantibodies such as anticarboxyethylpyrrole and antiastrocyte antibodies is an early feature of amd pathogenesis [98, 99 ]. recently, morohoshi. demonstrated that 94% of patients with early - stage amd and 83% of patients with late - stage amd had elevated levels of serum retinal autoantibodies, compared with only 9% of normal controls. nsaids may have a protective effect with regard to alzheimer 's disease, reducing its prevalence [101, 102 ], and similarly a prospectively followed group of patients under long - term anti - inflammatory treatment for rheumatoid arthritis showed a very low prevalence of amd. moreover, a larger retrospective study reported reduced rates of cnv among amd patients undergoing aspirin treatment. even the anecdotal use of loxoprofen sodium for toe cellulitis has been reported to improve cnv. however, a more recent australian population - based study reported that regular aspirin use is associated with increased risk of incident neovascular amd. this is consistent with a report emerging from the european eye study that frequent aspirin use is associated with early amd and late wet amd and the odds ratio rises with increasing frequency of consumption. nevertheless, evidence supports the additive role of nsaids in the treatment of cnv, with a protective effect that is probably due to the control of both inflammation, and cox-2 which is a known promoter of angiogenesis and can be found in cnv [8, 19, 24, 108 ]. pharmacological inhibition of cox seems to reduce vegf expression in cultured human rpe cells [8, 109 ]. kim. [17, 18 ] have demonstrated that both topical and intravitreal ketorolac significantly reduce angiographic leakage and retinal levels of pge2 and vegf in an animal model of cnv. therefore, the addition of an anti - inflammatory agent could be a valid option for controlling cnv, as simply inhibiting vegf addresses neither the multifactorial pathogenesis of cnv nor the underlying cause of vegf production. although the evidence coming from human clinical trials is less consistent than that arising from animal models, a favorable effect of additive topical nsaid therapy with regard to anti - vegf for the control of exudative amd has recently been reported in 3 prospective, randomized, and controlled clinical studies (table 2) [4, 5, 7 ]. russo. demonstrated that topical ketorolac acts in conjunction with intravitreal anti - vegf treatment ; central macular thickness (cmt) is significantly lower (37.1 m) after 6 months in patients receiving combination therapy, although there were no differences in either visual acuity or the number of injections between the 2 groups. such results are partially in contrast with the findings of gomi., in which the authors also reported a reduction in the frequency of ranibizumab injections over 6 months when topical bromfenac was used as an adjunctive treatment with ranibizumab. however, in addition to the differences in the pharmacological properties of bromfenac and ketorolac, another point of difference was that gomi. administered just one ranibizumab injection and then treated the patients on an as - needed basis ; therefore the number of injections administered was not consistent. similar results were reported in another recently published trial evaluating the use of topical bromfenac in combination with ranibizumab versus ranibizumab alone. a significantly greater reduction in cmt was found after 12 months in the combination group (28.3% versus 18.9%), without concomitant differences in visual acuity changes between the 2 arms. such findings are in contrast with 2 previous retrospective studies [32, 33 ] that did not detect any improvement in visual acuity or in cmt, with the addition of bromfenac or nepafenac in conjunction with anti - vegf administration. however, these inconsistent results may be due to differences in study design (shorter retrospective design and smaller sample sizes) and the presence of recalcitrant and persistent exudative amd in the examined cohorts, which render direct comparisons problematic. overall the literature supports the concomitant off - label administration of topical nsaids with on - label anti - vegf intravitreal therapy, as nsaids act synergistically to reduce cmt in cnv. it will be important to evaluate the long - term efficacy of nsaids, as amd is a chronic disorder. in particular, careful attention should be paid to the corneal complications associated with long - term use of topical nsaids. dr is the most frequent cause of legal blindness in working - age individuals in developed countries. in addition to dr, diabetic patients can suffer from diabetic macular edema (dme), which is caused by breakdown of the blood - retinal barrier resulting in leakage of plasma and water from small vessels. these leakages result in swelling and thickening of the retina around the macula, the central part of the retina in which fine visual discrimination occurs. in patients with type 2 diabetes growing scientific evidence shows that an immunological cascade has a major role in the pathogenesis of dr. increased levels of inflammation mediators and pgs in dr have been found in the vitreous cavity in both animal and human studies [22, 114, 115 ], and the level of pge2 correlates significantly with vitreous levels of vegf. the role of inflammation in the progression of dr has also been indirectly supported in a recent study by the diabetic retinopathy clinical research network, in which authors concluded that intravitreal triamcinolone appears to be associated with a reduced risk of worsening of proliferative dr. in animal models pgs stimulate vegf expression, and in cultured muller cells agonism and antagonism of the pge2 receptor increase and decreases vegf production, respectively, in a dose - dependent manner. in fact, nsaid treatments have been shown to prevent or delay dr progression in animal models [21, 27, 28, 119 ]. while no benefit was found in advanced dr in the early treatment diabetic retinopathy study examining the effect of 650 mg aspirin, the incidence of dr is reduced in human patients taking salicylates for rheumatoid arthritis, just as previously reported with exudative amd, attesting to the contribution of cox to the development of dr. such findings were confirmed in the dipyridamole aspirin microangiopathy diabetes study (damad) that assessed the effect of 990 mg aspirin in early dr ; a significant protective effect was associated with high doses of aspirin, which slowed the development of retinal microaneurisms. subsequently, either 2 prospective randomized studies confirmed these findings with the administration of sulindac and celecoxib [123, 124 ]. the benefits of topical nsaid therapy for dr control are mainly reported anecdotally or in uncontrolled retrospective case studies. pseudophakic dme showed improvement in retinal thickness and visual acuity after treatment with nepafenac 0.1% for 6 months in a case report. similarly, in a case series of 6 eyes with dme that were treated with nepafenac 0.1%, the average foveal thickness decreased significantly from 417 m to 267 m after a mean of 178 days. authors moreover reported that four eyes gained vision and two eyes maintained vision, with a statistically significant mean visual acuity improvement from 0.78 logmar to 0.67 logmar. such results suggest that nepafenac 0.1% may exhibit activity against diabetic macular edema and warrant further investigation in larger, controlled studies, possibly with and without associated anti - vegf therapy. in this regard a placebo - controlled study to assess the effect of nepafenac 0.1% on macular retinal volume in eyes with noncentral dme is being conducted (clinicaltrials.gov identifier : nct01331005). the intravitreal route is a privileged route for the delivery of drugs to the posterior eye, and it has been proposed as the route of administration for nsaids to treat dme. evidence emerging from published case reports collectively suggests an increase in visual acuity without significant changes in the cmt. soheilian. evaluated the effect of a single dose of intravitreal diclofenac (500 g/0.1 ml) on 5 eyes with clinically significant diabetic macular edema and reported prominent improvements in visual acuity with no significant decrease in cmt. a similar result was reported by do ceu afonso reis. in a study involving 20 patients with dme refractory to retinal photocoagulation, who were treated with intravitreal ketorolac (500 g/0.1 ml) in one eye only. these findings are consistent with a study by maldonado. who treated 25 patients with ketorolac at a dose of 3000 g. on the other hand, elbendary and shahin randomized 32 eyes in a 1 : 1 ratio to treatment with either 500 g/0.1 ml of diclofenac or 4 mg/0.1 ml of triamcinolone and reported a significant reduction in cmt with both treatments, but improvements in visual acuity were only evident in the triamcinolone group. the initial pathological changes in macular edema appear in macular photoreceptors, rpe, bruch 's membrane, and choriocapillaris. while their etiology is not fully understood, it is incontrovertible that inflammation has a critical role in the various manifestations of macular edema and its progression. the fact that inflammation is a common denominator in pseudophakic, exudative amd and diabetic macular edema may explain why anti - inflammatory agents are beneficial as preventive or adjunctive therapies. considering our growing understanding of the underlying role of pgs, complement, and inflammation in eye diseases, the clinical use of topical nsaids will likely continue to expand. the newer and more potent topical formulations emerging are also likely to contribute to this expansion. in summary, topical nsaids could be used alone for pseudophakic cme or as a favorable adjunct together with anti - vegf for exudative amd. cost effectiveness ratio must be considered given the low incidence of pseudophakic cme in low - risk patients ; however, the heavy economic burden of anti - vegf injections that could potentially be reduced if future studies support the use of nsaids should also be considered. | nonsteroidal anti - inflammatory drugs (nsaids) are nowadays widely used in ophthalmology to reduce eye inflammation, pain, and cystoid macular edema associated with cataract surgery. recently, new topical nsaids have been approved for topical ophthalmic use, allowing for greater drug penetration into the vitreous. hence, new therapeutic effects can be achieved, such as reduction of exudation secondary to age - related macular degeneration or diabetic maculopathy. we provide an updated review on the clinical use of nsaids for retinal diseases, with a focus on the potential future applications. |
t4 lysozyme, as a member of the lysozyme family produced by bacteriophage, breaks down the bacterial cell wall by catalyzing the hydrolysis of poly saccharide chains during infection of the bacteria. the enzyme specifically cleaves the glycosidic bonds connecting the repeating subunits of cell walls between n - acetylglucosamine and n - acetylmuramic acid that are substituted with peptide side chains. the three - dimensional structure is clearly organized with two domains connected by a long -helix (figure 1). the active site cleft, where hydrolysis of the glycosidic linkage takes place, is located at the interface between the two domains. it has been widely accepted that t4 lysozyme exhibit hinge - bending conformational motions, referring to rotation of one domain relative to the other domain along an axis running through the interface of the two domains. both ensemble - level and single - molecule measurements have revealed hinge - bending motions in which the opening of the active site cleft is within a nanometer. as we have reported previously, the t4 lysozyme enzymatic reaction involves complex conformational state changes in the enzymatic turnovers. a simplified michaelis menten type of mechanism can be presented as1where e, s, es, es, and ep represent enzyme, substrate, nonspecific enzyme substrate complex, specific or active enzyme substrate complex, and enzyme product complex, respectively. the process of forming the active complex of es involves multiple conformational states. the process of e + s es es essentially involves the enzyme active site opening up to take the substrate, forming a nonspecific es complex, and binding down to form the active complex of es ready to react followed by turnover to ep. the process of reaction and product releasing es ep e + p may not involve significant enzymatic active site conformational changes. while t4 lysozyme hinge - bending motions have been extensively probed by single - molecule fret (fluorescence resonance energy transfer) spectroscopy, the domain motion of t4 lysozyme is rather complex and contains motions besides hinge - bending. it is reasonable to assume that the hinge - bending motion in nature involves multiple coupled nuclear coordinates that can be projected to a nuclear coordinate associated with the -helix. in order to capture the complexity of t4 lysozyme conformational motions, single - molecule spectroscopy is a powerful approach for mechanistic understanding of complex and fluctuating biological processes by resolving time - dependent dynamic process and allowing exploration of hidden heterogeneity beyond nonsynchronized ensemble - averaged measurements. single - molecule fret spectroscopy sensitive to single - molecule conformational fluctuation dynamics has offered possible direct observations of biological conformational dynamics by rendering spatial and temporal information between donor and acceptor fluorophores placed within a certain proximity on individual molecules of interest. this approach has made significant and extensive contributions to the understanding of complex biological dynamics through the perspectives of heterogeneous dynamics of protein molecules, nucleic acids, and their interactions with other molecules. for example, single - molecule fret has been used to study rna folding pathways, hairpin ribozymes intimidate states, dna bubbles kinetics, epidermal growth factor receptor (egfr) dimerization, and conformational dynamics of enzymes. lu and co - workers have focused on conformational dynamics of t4 lysozymes and have observed conformational bunching effects in the process of t4 lysozyme hinge - bending. for complex biological systems, such as protein protein interactions, ion channel receptor activations, protein folding and aggregations, and protein conformational fluctuations under enzymatic reactions, it is more than often that multiple conformational nuclear coordinates simultaneously play critical roles in regulating and gating biological functions. under such complex multiple coordinate conformational dynamic rate processes, one - dimensional fret may be insufficient to characterize the intrinsic complexity of molecular dynamics. hence, it is desirable to have advanced fret techniques capable of probing more than one - dimensional dynamic information. for example, some efforts have been made to develop three - color and four - color fret in which more than one fret pair are used to probe multiple site - to - site distance changes at a time. nevertheless, multicolor fret requires fluorophores with high photostability and clear spectral separation, which are much more difficult to achieve than one fret pair, especially at single - molecule level. in addition, the fact that energy transfer between donor and acceptor obeys orientation dependence () as expected for a dipole dipole interaction has seldom been took into account, instead, the assumption = 2/3 (the fluorephores undergo freely rotation in a time much shorter than fluorescence lifetimes) is widely used for approximate distance estimate. it has been experimentally proved that fluorescence energy transfer between cy3 and cy5 depends on the orientation. it has also been reported that the degree of position accuracy relies on the rotational mobility of single molecules. therefore, the understanding of orientation effect could result in more accurate fret distance approximation and molecular angular information for position determination. for this purpose, several groups have shed light on molecular orientations (or rotations) via imaging and fluorescence anisotropy (or polarization) spectroscopy. for example, out - of - focus and in - focus images have been combined to refer three - dimensional single molecule orientations. dual - color and dual - polarization images of single molecules have been captured by ccd (charge - coupled device) camera. a multiparameter single - molecule fluorescence spectroscopy, photon distribution analysis, along with structural modeling have been developed to quantitatively describe single - molecule fret in which several fret - related parameters such as distance, molecular orientation, and dye quenching (or bleaching) are taken into account. lu and co - workers have demonstrated the use of single - molecule nanosecond anisotropy to study spatially confined rotational diffusion dynamics of individual tethered proteins and nanosecond protein motion dynamics. the possibility of probing multidimensional conformational dynamics of complex biological systems calls for fret - anisotropy correlated measurements on demand. fluorescence anisotropy not only allows for estimating orientation effect on fret or position determination for imaging mentioned above but also allows for acquiring information about the motion of the fluorophore, the rotational dynamics of subdomains, or the entire proteins. the methods of measuring rotational or tilting motions by fluorescence anisotropy in single molecules have been reviewed and discussed. lu and co - workers have successfully probed nanosecond protein motions of calmodulin and t4 lysozyme by single - molecule fluorescence anisotropy. unfortunately, most of fluorescence anisotropy research have been either limited to the study of orientation effect for fret, or limited to the rotational dynamics of molecules ; although the potential ability of fluorescence anisotropy in the identification of multiple conformational states of biological molecules has been mentioned. nevertheless, the potential abilities of correlated single - molecule fret and fluorescence anisotropy for direct observing multidimensional conformational dynamics have not been demonstrated yet, to our best knowledge. in this article, we report our new technical approach integrating single - molecule fret, photo stamping spectroscopy and fluorescence anisotropy to study multicoordinate conformational dynamics of t4 lysozyme under enzymatic reaction conditions. from conformational dynamics perspectives, this approach enables us to simultaneously probe multidimensional or multicoordinate conformational dynamics of proteins, including fret coordinate motions probed by fret pair and rotational motions monitored by fluorescence anisotropy. in this work, we exploit the multidimensional aspect of t4 lysozyme conformational dynamics and demonstrate the potential of our new correlated single - molecule multidimensional photon stamping spectroscopy. multidimensional conformational motions of wild - type t4 lysozyme (pdb - code, 3lzm), including hinge - bending motions along -helix and rotational motions of each domain. cy3 and cy5 are covalently labeled to two cysteines : cys 54 on n - domain and cys 97 on c - domain. cy3-cy5 labeled t4 lysozyme is tethered through an amine - to - sulfhydryl bifunctional cross - linker molecule to thiol - functionalized glass coverslip surface. the approximate 45 nm spacer allows free rotation of single t4 lysozyme without perturbation or confinement from the modified surface. distance changes between the two labeling sites involved in hinge - bending conformational motions can be monitored by tracing the dynamic fluctuations of donor lifetime during the fret process. besides hinge - bending motions along -helix line, the two domains of t4 lysozyme also exhibit other types of conformational motions, for example, rotational motions. wild - type t4 lysozyme plasmid was bought from addgene, which was authorized by prof. two cysteines (residue 54 on n - terminal domain and residue 97 on c - terminal domain) of the wild - type t4 lysozyme are accessible to thiolation reactions. a cy3-cy5 fret pair (ge healthcare) was nonselectively labeled on these two cysteines. the crystal structure of wild - type cy3-cy5 labeled t4 lysozyme is shown in figure 1. the individual donor acceptor labeled t4 lysozyme can be easily distinguished by four - channel optical images as shown in figure 2, because only donor acceptor labeled molecules can simultaneously exhibit four emission spots (dual color and dual polarization for each color). the substrate for t4 lysozyme used in our experiments is peptidoglycan that is the major component of the bacterial cell wall that t4 lysozyme breaks. peptidoglycan isolated from micrococcus luteus was purchased from sigma - aldrich and was directly used without further purification. the substrate was suspended to a final concentration of 25 g / ml in ph 7.3 pbs buffer during single - molecule experimental measurements. in our single - molecule fret experiments, t4 lysozyme was tethered through a bifunctional nhs - peg6-maleimide cross - linker to a modified glass coverslip surface. this cross - linker is functional between primary amines (nh2) and sulfhydryl (sh) groups in which the n - hydroxysuccinimide ester (nhs) group reacts specifically with primary amino groups of lysine to form stable amide bonds, and the maleimide group reacts with sulfhydryl to form stable thioether bonds. the glass coverslip was first sonicated with acetone for half an hour, followed by rinsing with alcohol solution and distilled water several times. the clean coverslip was treated overnight with a 10% (v / v) mixture of 3-mercaptopropyl - trimethoxysilane and isobutyl - trimethoxysilane (1/1000 ratio) in 15.0 ml of dimethyl sulfoxide (dmso). after rinsing with ethanol and water, the coverslip was put in the ph 7.3 pbs buffer solution for 1 h to remove unreacted solvent. the coverslip was then incubated with 40.0 l of 250 mm bifunctional cross - linker stock solution (nhs - peg6-malemiade, thermo scientific) in 12.0 ml of pbs buffer solution for 2 h at 4 c. the amine - to - sulfhydryl cross - linkers with hydrophilic polyethylene glycol (peg) spacer arms can be attached to the glass coverslip surface. after additional washing, the coverslip was incubated with 0.66 nm t4 lysozyme in the pbs buffer for 2 h at 4 c. after the linkage between amine - reactive group of nhs - peg6-malemiade and primary amine group of t4 lysozyme s lysine, the tethered enzyme sample was assembled on the glass coverslip surface. during our single - molecule measurements, the assembled t4 lysozyme on the coverslip was further incubated with 25.0 g / ml substrate for half an hour at room temperature in pbs buffer solution (ph 7.3) to fulfill the engagement. the effective trolox - oxygen scavenger solution, which contains 0.8% d - glucose, 1.0 mg / ml glucose oxidase, 0.04 mg / ml catalase, and 1.0 mm trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), was added to the above sample chamber to prevent the possible photobleaching or quenching of the cy3-cy5-labeled t4 lysozyme molecules. frster resonance energy transfer (fret) refers to the nonradiative energy transfer from a donor molecule to an acceptor molecule, arising from a dipole energy transfer occurs when the oscillations of an optically induced electronic coherence on the donor are resonant with the electronic energy gap of the acceptor. fret efficiency is sensitive to the interdistance between the donor and acceptor in the range of 3080, although fret is not necessarily accurate in measuring absolute distances, as often being specified as a, single - molecule fret is sensitive to probe the temporal fluctuation dynamics of the distance changes, such as the conformational changes of the biomolecules. the energy transfer efficiency (efret) is given by2where r is the separate distance between donor and acceptor and r0 (the frster radius) is the critical distance at which energy transfer is equal to 50%. r0, as expressed in eq 3, is a function of the orientation factor, the donor acceptor spectral overlap j, the donor quantum yield d, and the refractive index of the medium n. is the orientation factor that varies between 0 and 4 and is defined by the relative orientation of the donor emission and acceptor absorption dipoles. for molecules which rotate very fast, is taken its average value, which equals 2/3 for isotropic rotation. in general, is given by eq 4, in which t is the angle between the donor emission dipole and the acceptor absorption dipole, d is the angle between the donor acceptor connection line and the donor emission dipole, and a is the angle between the donor acceptor connection line and the acceptor absorption dipole.34 any process that influences the distance of donor - to - acceptor affects the fret rate or efficiency, which enables us to probe the conformational fluctuation dynamics of dna, rna, and proteins. for example, fret can be used to sense the distance changes between donor and acceptor that have been labeled on a host molecule or two different molecules, and then the conformational changes of one host molecule or the relative motions of two molecules can be monitored by tracing the fret efficiency. in single - molecule fret measurements, efret from donor to acceptor reflects mutual distance changes, resulting in the capability of probing single molecules conformational dynamics in real time by tracking efret changes. the detection of efret, usually by ratio - metric methods, can be generally classified into intensity - based fret and lifetime - based fret.5where ia is the acceptor fluorescence intensity and i d is the donor fluorescence intensity in fret process. da and d are donor lifetime in the presence (da) and in absence (d) of acceptor, respectively. fret detection on the basis of donor lifetime is more effective and less sensitive to local environment fluctuations, especially in afm tip - enhanced single - molecule spectroscopic and imaging measurements where amplified fluorescence signal by metal tip reflection exists. in the photon stamping spectroscopy, each detected photon is stamped with two parameters : a chronic arrival time and a delay time related to femtosecond laser pulse excitation. in this work, we treat photons distributions detected in each 10 ms (ms) bins as a poisson distribution which gives the mean of delay times in each distribution as the lifetime da. fluorescence anisotropy is capable of determining the rotational correlation time of the fluorescence probe, thus providing insights into the motions of probe, and orientation / rotation or mobility of subdomains or the entire molecule. changes in probe s orientation reflect the rotation or mobility of the target macromolecule where the probe is attached. the fluorescence anisotropy r(t) is defined as the difference between the vertically and horizontally polarized fluorescence emission divided by the total fluorescence emission, given by6where i (t) and i(t) are the fluorescence intensities of the parallelly () and horizontally () polarized emission components under vertically polarized excitation. g is the correct coefficient compensation for the different instrumental detection efficiencies of the various polarized components of the emission, accounting for the ratio of the detection system sensitivities for vertically and horizontally polarized light. in our experiments, bright fluorescence microspheres (0.1 m, 540/560 nm orange spheres, invitrogen molecular probes) were used to measure the g factor by using horizontally polarized excitation. with the horizontally polarized excitation, the excited - state distribution of the molecules is rotated to lie along this observation axis, so that both the horizontally and vertically polarized components are orthogonal to the incident polarization and the intensities of collected signal are equivalent. g factors are averagely estimated to be 1.36 and 2.36 for donor and acceptor, respectively. the unbalanced g factor for donor and acceptor are most likely due to the detection discrepancy of different detectors and the bias of the optics response to different colors. typical anisotropy values are in the range from 0.2 for probes with unrestricted motion to 0.4 for those that are immobile. several factors can depolarize the measured anisotropy to values lower than 0.4 (the maximum theoretical values), such as the numerical aperture of the objective, the angle difference between the absorption and emission dipole, molecular rotation related rotational diffusion, and. in terms of rotational diffusion process, the expected anisotropy is given by the perrin equation7where r0 (assumed to be 0.40 here) is the fundamental anisotropy in the absence of rotational diffusion, rda is donor s anisotropy, and is the rotational correlation time for the diffusion process. we treat donor s anisotropy in the similar way to donor lifetime described in eq 5 : rda is the mean of fluorescence anisotropy in each 10 ms bins measured based on eq 6. fluorescence anisotropy provides information about detailed motions of cy3 or cy5 and conformational dynamics of the t4 lysozyme system being studied here. the time - dependent correlation strength of lifetime as well as anisotropy trajectory is evaluated by autocorrelation function given by8where xi is the donor lifetime (da) or anisotropy (rda), the signal variable in time - dependent lifetime or anisotropy trajectory ; i is the index number of data point ; t is the time lag ; x is the mean value of lifetime or anisotropy in each calculation. by using autocorrelation analysis, the donor lifetime and anisotropy fluctuation decays () or rates (1/) can be identified, providing insights into the t4 lysozyme conformational fluctuation dynamics. combining fret, lifetime, polarization, and anisotropy at single - molecule sensitivity, we show our compact design for probing multidimensional conformational motions. compared to two - color or two - polarization concepts, our new approach combines color discrimination and polarization using a four - channel single - molecule epi - illuminated microscopy, as shown in figure 2a. in short, a femtosecond pulse laser (ti : is combined with an optical parametric oscillator (opo basic, coherent inc.) as well as frequency doubling -barium borate crystal (bbo) to generate linear - polarized pulse laser. the 532 nm linearly polarized pulse laser is aligned and focused into coverslip - solution interface by a confocal objective (plan - apochromat, 1.40 na, 63, carl zeiss) to excite molecules. a piezoelectric scanning stage (nano - h100, mcl) with a positioning resolution of 0.2 nm is further used to scan the coverslip surface and locate individual molecules. the fluorescence signals from single molecule are separated to two pathways (two color) in wavelength ranges below and above 640 nm after passing through a filter (hq545lp, chroma) and two dichroic mirrors (545dcxru, 640dcxr, chroma). signals in each pathway are further divided into four - channel parallel and perpendicular components for each color by using two polarizing beam splitter cubes (pbs 201 420680 nm, pbs 202 6201000 nm, respectively). the photons from four - channel (parallel and perpendicular for both donor and acceptor) are detected by four apds - si avalanche photodiode (spcm - aqr-14, perkinelmer optoelectronics). the time - stamped photon information is recorded through multichannel detector router (hrt-82, becker & hickl gmbh) to a time - correlated single photon counting module (spc-830, becker & hickl gmbh) and a personal computer. figure 2b shows the single - molecule photon counting images of individual cy3-cy5 labeled t4 lysozymes in which dual - color (donor and acceptor) and dual - polarization (and) are captured by four apds. four images are taken simultaneously with an illumination of 532 nm and scanning area of 20 m by 20 m. (a) experimental home - built four - channel single - molecule setup for measuring multidimensional conformational dynamics. basically, it consists of an inverted confocal epi - illumination configured microscopy, a femtosecond pulse laser, four si avalanche photodiode detectors, a time - correlated single photon counting module, and several optics. 532 nm green linearly polarized pulse laser is used to excite cy3-cy5 labeled t4 lysozyme. the emissions (yellow and red) from cy3 and cy5 are discriminated by dichroic mirrors. the polarization of the light emitted is further distinguished into parallel () and vertical () components (relative to the polarization of the laser excitation) by two polarizing beam splitter cubes. (b) single - molecule photon counting images of individual cy3-cy5 labeled t4 lysozymes. dual - color (cy3 and cy5) and dual - polarization (and) images are captured. dm : dichroic mirror ; apd : avalanche photodiode ; pbs : polarizing beam splitter cubes ; lpf : long pass filter ; wp : wave plate ; l1/l2 : lens ; pc : personal computer. figure 3 shows the single - molecule studies of multidimensional conformational motions of t4 lysozyme by recording real - time anisotropy trajectory and donor lifetime trajectory. in our single - molecule multiparameter spectroscopy, time - resolved fret fluctuations detected by donor lifetime and the correlated fluorescence anisotropy are simultaneously recorded. as described before, each detected photon is stamped with two parameters : a chronic arrival time and a delay time related to femtosecond laser pulse excitation. our data analyses are primarily based on those two temporal parameters recorded by single - molecule photon stamping spectroscopy. in this work, we have used donor lifetime (da) to probe fret fluctuations and fluorescence anisotropy (rda) to monitor domain rotational motions of cy3-cy5 labeled t4 lysozyme. figure 3b shows a typical single - molecule lifetime trajectory, { da(t)}. the donor lifetime fluctuates from time to time, implying dynamic change of interdomain distance along fret coordinate. the wide gaussian - like distribution of lifetime (the right panel in figure 3b) indicates the existence of multiple conformational intermediate states characterized with different interdomain distance along -helix, from fret perspective. multiple conformational intermediate states have often been suggested as the general feature of enzymatic mechanisms and protein motions. in single - molecule studies, time - binned fret or lifetime trajectory has been reliably used to identify the presence of multiple intermediate states corresponding to well - defined stable fret values in the case of well - separated, low - noise, two - state or three - state systems. nevertheless, for complex biological systems, due to the influence from local environmental fluctuations, thermal fluctuations, measurement short noise, photophysical effects, and conformational dynamic or static heterogeneity coupled with fluctuating catalytic activity, multiple intermediate states are often buried in a wide - distributed gaussian - like distribution. in this work, the single - molecule lifetime trajectories are limited by 10 ms binning time and the other facts discussed above, resulting in that conformational intermediate states do not present clear separation in the lifetime distribution in figure 3b. furthermore, we have performed advanced quantitative analysis of lifetime, to be discussed later in this paper (figures 4 and 5), to further resolve buried multiple intermediate states. the rotational flexibility of the donor molecule cy3 on t4 lysozyme fluctuates significantly in the course of the enzymatic reaction turnovers, revealed in our single - molecule anisotropy analysis. figure 3a shows a typical single - molecule anisotropy trajectory, { rda(t) } showing that the single - molecule anisotropy fluctuates in a wide range from negative to the maximum value of 0.4. this is most likely associated with the dye molecule fast spinning motions, the slow subdomain motions, and entire protein motions of the t4 lysozyme tethered with the dye molecule. those three types of motions are all involved but contribute differently to the overall anisotropy measured from the experiments. if there are no rotational diffusion restrictions from the subdomains or the whole enzyme, the cy3 probe on t4 lysozyme should exhibit fast wobbling spin rotation and the resulting cy3 lifetime should be in the subnanosecond range consistant with a free cy3 dye in solution. our experimental results of lifetime trajectory and distribution (figure 3a) exclude this scenario because most of the measured lifetimes of cy3 are in the nanosecond range instead of the subnanosecond range. similar behavior of the lack of rotational freedom and nanoseconds lifetime range of tethered dye molecules have also been reported for cy3 covalently linked to dna. the reported cy3 lifetime is around 10 times larger than the fluorescence lifetime of the free dye in solution, which is similar to the result of cy3 on t4 lysozyme in our measurements. the lifetime of cy3 is dependent on physical and chemical properties of surroundings. the longer lifetime of cy3 is directly related to local environment changes deviated from an aqueous environment, such as viscosity. for example, when cy3 is attached to a strand of dna or a protein, an additional increase in local viscosity may occur, leading to a lifetime increase. the interaction between cy3 and dna molecules has also been reported to play a role in longer cy3 lifetime than that measured in aqueous solution. therefore, it is most likely that the increase in local viscosity and the interaction between cy3 and t4 lysozyme result in longer cy3 lifetime which evidence the lack of cy3 free spin and wobbling rotational freedom identified in our anisotropy measurements. the joint distribution of lifetime and anisotropy (figure 3c) further implies that the measured overall anisotropy is not dominated by free dye rotation but rather by restricted rotation, indicating that the cy3 dye probe is significantly regulated by interactions with the t4 lysozyme protein matrix. presumably, there are two possibilities that may contribute to the restricted rotation : the enzyme previous studies have reported that the tethered single t4 lysozyme to the hydrocarbon modified surface is mostly in solution phase, and the interaction between enzyme and surface is weak or insignificant in impacting enzyme rotations, giving rise to the absence of rotational rate fluctuations for most of the time during the measurements. therefore, the restricted rotation of cy3 characterized by the dynamic and fluctuating anisotropy is most likely regulated by the domain rotations of enzyme. figure 3b, c gives a broad anisotropy distribution, implying different rotational flexibility of cy3 regulated by t4 lysozyme domain motions. the different dye rotational flexibility has been found in the study of hiv-1 reverse transcriptase, and they have attributed broad rotational correlation time distribution probably to the clamping of the finger and thumb domains during polymerase activity. in our work, different cy3 rotational flexibility, reflected in the broad anisotropy distribution in figure 3b, c, is likely regulated by t4 lysozyme domain rotations during the interdomain hinge - bending conformational motions. t4 lysozyme exhibits well - known hinge - bending conformational motions in which the distance changes between the opening and closing of the active site cleft are about 46, revealed in crystal structure analyses, md simulation, and our previous single - molecule spectroscopic analysis. conceivably, t4 lysozyme conformational motions involve more than just hinge - bending along its -helix. furthermore, even the hinge - bending motions themselves are actually complex fluctuating conformational motions involving multiple conformations with distinct domain orientations or hinge - bending angles along multiple nuclear coordinates. for example, it has been suggested that t4 lysozyme populates multiple intermediates states with distinct hinge - bending angles trapped in the crystal structures of t4 lysozyme mutants. our single - molecule t4 lysozyme anisotropy fluctuation result (figure 3) suggests that there are a wide range of domain - rotational mobility, indicating different dominant orientations of the domains from time to time, consistent with distinct hinge - bending angles. typically, single - molecule fret spectroscopy only probes the conformational motions associated with the fret donor acceptor distance changes, and most likely, such fret probed conformational motions are actually the projections of much more complex conformational motions of the examined enzyme molecules on the fret sensitive coordinate. nevertheless, besides hinge - bending motions along coordinate probed by single - molecule fret spectroscopy, t4 lysozyme actually exhibits complex and fluctuating rotational motions along multiple orientation coordinates, leading to a comprehension of the multidimensional conformational motions associated with the t4 lysozyme enzymatic reaction turnovers. multidimensional conformational motions of t4 lysozyme probed by single - molecule multiparameter spectroscopy : dynamic anisotropy and lifetime - based fret. all the data are collected from single cy3-cy5 labeled t4 lysozyme under enzymatic reactions with 25.0 g / ml peptidoglycan and are simultaneously recorded by four - channel single photon stamping spectroscopy. (a) single - molecule real - time anisotropy trajectory { rda(t) } and distribution recorded within 100 s. each dot represents the average anisotropy in every 10 ms binning of cy3, calculated on the basis of eq 6. (b) single - molecule real - time donor lifetime trajectory { da(t)}. each dot is the average of photon delay times in each 10 ms binning of cy3 collected from single - molecule photon stamping. (c) 2d joint distribution between anisotropy in (a) and lifetime in (b). we note that the band widths of both lifetime and anisotropy distributions, 1.0 ns and 0.25, are significantly larger than the measurement error bars of 0.30 ns and 0.05, respectively. the broadness of the distributions of the lifetime and anisotropy represent intrinsic physical inhomogeneity beyond the measurement error bars. t4 lysozyme multidimensional hinge - bending conformational motion dynamics presents dynamic and static inhomogeneity, revealed and identified by autocorrelation analysis of the single - molecule lifetime trajectories { da(t) } and anisotropy time trajectories { rda(t)}. autocorrelation function analysis has been extensively applied to identify inhomogeneous fluctuation rates of single - molecule electron transfer, energy transfer fluctuations, and protein conformational changes. we have analyzed the autocorrelation functions, c(t), of lifetime and anisotropy trajectories for 40 cy3-cy5 labeled t4 lysozyme molecules under the enzymatic reaction conditions. figure 4 shows the autocorrelation analysis results of lifetime fluctuation decays (figure 4a) and anisotropy fluctuation decays () (figure 4b). most of the autocorrelation functions of fret donor lifetime trajectories (figure 4a, inset) show nonexponential fluctuation decays, implying the dynamic disorder of fret energy transfer, i.e., the conformational fluctuation rate along fret - probed hinge - bending coordinate changes from time to time under enzymatic condition in one single - molecule measurement. we have analyzed the autocorrelation functions by biexponential fitting and observed a wide range of fluctuation decays from milliseconds to seconds (figure 4a), suggesting a static disorder of conformational fluctuation rate change from molecule to molecule. autocorrelation functions of anisotropy trajectories also give similar results in terms of nonexponential and inhomogeneity of fluctuation correlation function decays (figure 4b). the nonexponential conformational fluctuation dynamics and the wide - range rate constant distributions of the single - molecule anisotropy indicate dynamic disorder and static disorder of conformational rotational fluctuation along multiple orientation coordinates, respectively. dynamic and static disorder of t4 lysozyme multidimensional conformational fluctuations along fret coordinate and orientation coordinates via autocorrelation analysis of lifetime and anisotropy. (a) histogram of fluctuation decays derived from the autocorrelation function of donor lifetime trajectories. (inset) typical nonexponential autocorrelation function calculated from a single molecule fluorescence lifetime trajectory { da(t)}. (b) histogram of fluctuation decays derived from the autocorrelation function of donor anisotropy trajectories. (inset) typical nonexponential autocorrelation function originated from a single molecule fluorescence anisotropy trajectory { rda(t)}. for autocorrelation functions of lifetime or anisotropy, represents the fluctuation decay. correspondingly, 1/ represents the fluctuation rate, that is, conformational fluctuation rate along fret coordinate or multiple orientation coordinates under t4 lysozyme enzymatic reactions. the flexibility of the hinge - bending conformational coordinates regulated by substrate binding to the enzymatic active site most likely contributes to the inhomogeneous and complex fluctuation dynamics of lifetime and anisotropy. the flexibility of conformations associated with the process of forming the nonspecific binding complex (e + s es), the process of enzyme closing down to form the active complex of es es, and the following enzymatic reaction of es ep, involves complex local environment and molecular structures of substrate as well as the enzymatic active site. the conformational motions involve multiple coordinates in nature and are regulated by a fluctuating multiple coordinate energy landscape defined by the dynamically changing and statically inhomogeneous molecular interactions as well as local electric field in the process of open - close hinge - bending enzymatic turnovers. donor lifetime decays exhibit two major distributions, associated with t4 lysozyme open and close states during the hinge - bending motions of the enzymatic active site. mean lifetime trajectory and distribution (figure 3a) do not necessarily give a clear separation of multiple intermediate states but rather present a gaussian - like distribution, due to the limitations from the local environment fluctuations and the time - resolution of our single - molecule spectroscopy. most likely, the convolution of the multiple poisson processes gives rise to the overall wide gaussian - like distribution. to further resolve the intermediate conformational states, we have performed analysis of the temporal decays including all the photons in a single - molecule donor lifetime trajectories. figure 5a shows the representative single - molecule lifetime decay curves of the fret donor. two - component lifetime decays, a faster one (1) and a slower one (2), are derived from biexponential fits. we note that the lifetime decays (1 and 2) in figure 5a reflects different properties of the enzymatic conformational dynamics from the fluctuation decays () in figure 4. as a lifetime - based fret measurement expressed in eq 5, fret donor lifetime decays are derived by fitting donor photon delay times histogram, essentially a time - correlated single photon counting distribution, to identify the fret efficiency reflecting the major conformational states along fret coordinates, whereas the fluctuation decays are calculated by the autocorrelation analysis of mean lifetimes da to characterize the conformational fluctuations and conformational flexibility. we attribute the two - component fret donor lifetime decays corresponding to two different fret efficiency values, to two major conformational states, that is, open and close states along fret coordinates : in each enzymatic reaction cycle, the enzyme active site opens up to interact with the substrate forming a nonspecific enzyme substrate complex (e + s es) and then closes down to form a specific enzyme substrate complex (es es) ready to react and turnover the substrate to product. our result of the two - component donor lifetime decays (figure 5b, c) corresponding to two distinct fret efficiency distributions is consistent with hinge - bending motion that opens and closes the active site cleft along the -helix. from the distributions of two - component lifetime decays of donor fluorescence, we have identified narrowly distributed faster component (1) and widely distributed slower component (2) (figure 5b). the results of two - component lifetime decays imply that t4 lysozyme exhibits open - close hinge - bending conformational motions associated with two - component fret donor lifetime : the faster component (1), when the active - site is closed and the fret efficiency is high, is relatively narrowly distributed, and the slower component (2), when the active - site is open and the fret efficiency is low, is widely distributed. the distinct distribution of each component gives a further indication that close state is rigid and spatially narrowly distributed, and in contrast, the open states involve flexible and broadly distributed conformational fluctuations. on the basis of michaelis menten mechanism in eq 1, in the process of forming the active complex es (e + s es es), the enzyme involves active site opening up to intake the substrate to form the nonspecific enzyme substrate complex es, and binding down to form the active complex es. during this whole open - close hinged - bending rate process, the enzyme essentially involves multiple steps associated with multiple conformational intermediate states. in the process of catalytic reaction and product releasing (es ep e + p), corresponding to the rigid and narrowly distributed close states, the enzyme may not exhibit significant enzymatic active site conformational changes. in terms of t4 lysozyme hinge - bending open - close conformational motions, our result of two - component lifetime decays is consistent with both ensemble - level measurements and single - molecule fluorescence measurements. the different modes of open and close motions also agree with the recent study of conformational dynamics of t4 lysozyme through an electric circuit by means of attaching single molecules to single - walled carbon nanotube field - effective transistors, that is, t4 lysozyme closes up in a single step while the open process requires a minimum of two steps. two - component donor lifetime decays associated with two major open and close conformational states. (a) the representative histograms of photon delay times of donor in a single - molecule measurement. biexponential fitting (red curve) gives the best estimate to the experimental data (gray), and two - component donor lifetime decays (1 and 2) are observed. narrowly distributed faster component (1) and widely distributed slower component (2) are revealed. (c) statistical results of the two - component feature derived from (b). mean and the results of two - component lifetime imply that t4 lysozyme exhibits open - close hinge - bending conformational motions characterized with two - component lifetime decays. the distinct distributions of each component even give a further implication that close state is rigid and spatially narrowly distributed while the open states involve flexible and broadly distributed conformational fluctuations. our work provides a new insight into t4 lysozyme conformational dynamics from a multiple dimensional perspective. along the domain orientation coordinates, significantly different t4 lysozyme conformations can have similar or same donor - to - acceptor (d - a) distance ; therefore, the different t4 lysozyme conformations may not necessarily be identifiable from the fret signal alone associated with this d a sensitive coordinate (figure 6). typically, fret measurement is sensitive to the projected fret - coordinate changes from the multiple t4 lysozyme intermediates states associated with different domain orientation coordinates. the multiple intermediate states involved in the active - site open - close hinge - bending motions and the hinge - bending conformational motions are intrinsically multidimensional, allowing for repositioning and reorienting the subdomains in forming the active enzyme substrate complex conformational state ready for a hydrolysis turnover reaction. while the hinge - bending motions along -helix require spatial proximity of two domains to interact with the substrate within the active site, the domain rotational orientation motions are predicted to be important for the enzyme to function, allowing the substrate to enter and the products to leave the active site. our results of t4 lysozyme conformational dynamics obtained from the single - molecule multiparameter photon - counting spectroscopy highlight the potential significance of probing the multidimensional conformational motions along multiple coordinates for characterizing the enzyme substrate interactions and catalytic efficiency. close hinge - bending motions, involving multiple domain orientation coordinates and fret coordinate. fret - coordinate projections of multiple t4 lysozyme conformations associated with different domain orientation coordinates are presented. along domain orientation coordinates, different enzyme conformations can have the same d a distance, which can be undetectable and hidden in a conventional single - molecule fret spectroscopic measurement. in the past decades, there have been intensive efforts to investigate and understand how the enzymes work and are capable of changing the biological activity pathways and enhancing a biological reaction rate by as much as 10 times. over the years, it has been recognized that the conformational motions are essential for the catalytic functions of enzymes. for example, molecular dynamics (md) simulation and statistical modeling have made significant contributions to characterize conformational motions and reaction fluctuations of enzymes in enzyme - catalyzed reactions. more often than not, subtle conformational changes even play a crucial role in enzyme functions, and these protein conformations are highly dynamic rather than being static, involving in multiple intermediate states and multiple conformational coordinates. the approach, experimental results, and discussion presented in this report are probably just a beginning step in expanding the studies and interpretations of new information about dissecting both the spatially and temporally complex enzymatic conformational dynamics under enzymatic reactions. apparently, there is still a long way toward a detailed and quantitative analysis of function - related conformational motions. for example, fret and anisotropy results suggest the existence of multidimensional conformational motions that are important to enzymatic activities, such as the hinged - bending motions under t4 lysozyme enzymatic reactions. additional efforts are still on demand to give direct spatial and temporal characterizations of the exact angles and positions of multidimensional conformational coordinates. temporal transitions of multiple intermediate states associated with fret coordinate and orientation coordinates or the coupling between them are still unclear. the complementary md simulations will likely be helpful and supportive to address those issues. in this report, we have provided a new insight into t4 lysozyme conformational dynamics from a multiple dimensional perspective. the multidimensional conformational probing from our correlated single - molecule fret and anisotropy measurements implies that t4 lysozyme exhibits much complex conformational motions along multiple orientations and nuclear coordinates beyond hinge - bending coordinate (-helix). significant information about the complex conformational motions are hidden by using only a conventional single - molecule one - dimensional fret analysis that is primarily sensitive to the motions projected from the complex and real conformational motions to the fret distance sensitive coordinate. the results of fret donor lifetime decays and correlated anisotropy suggest that t4 lysozyme open states involve flexible and broadly distributed conformational fluctuations while the close state is more rigid. in addition, the dynamic and static inhomogeneity of multidimensional conformational fluctuations have been revealed by nonexponential features of autocorrelation functions of both lifetime and anisotropy. the developed single - molecule multiparameter photon stamping spectroscopy provides a possible access to probe multidimensional conformational motions of complex enzymatic systems, such as t4 lysozyme, by means of simultaneous acquisition of fret, fluorescence anisotropy, and fret donor fluorescence lifetime. there is still a high call for experimentally technical approaches which are capable of probing the complex enzymatic conformational fluctuations without ensemble - averaging as well as measurement synchronization, and multiple parameter measurements with the sensitivity of analyzing the enzyme rotational motion, translational diffusion, intramolecular domain motions, and intermolecular interactions. our approach reported here holds the promise to characterize not only the enzymatic active site conformational fluctuations and enzyme substrate interactions but also the overall enzyme matrix motions surrounding the active site. evidently, such overall enzyme conformation fluctuations and multiple coordinate in nature, likely play important roles in establishing the catalytic reaction pathways and the overall enzymatic reaction energy landscape. | conformational motions of proteins are highly dynamic and intrinsically complex. to capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one - dimensional fret coordinate or the projected conformational motions on the one - dimensional fret coordinate. t4 lysozyme hinge - bending motions between two domains along -helix have been probed by single - molecule fret. nevertheless, the domain motions of t4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge - bending. it is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged - bending motions have profound impact on overall enzymatic functions. in this report, we have developed a single - molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, fret, and fluorescence lifetime. this spectroscopic approach enables simultaneous observations of both fret - related site - to - site conformational dynamics and molecular rotational (or orientational) motions of individual cy3-cy5 labeled t4 lysozyme molecules. we have further observed wide - distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along fret coordinate by monitoring time - dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of t4 lysozyme open - close hinge - bending enzymatic turnover motions under enzymatic reaction conditions. by analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of t4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental understanding of the enzymatic reaction turnover dynamics associated with overall enzyme as well as the specific active - site conformational fluctuations that are not identifiable and resolvable in the conventional ensemble - averaged experiment. |
pancreatic lipase immunoreactivity (spec fpl) is currently considered to be the most accurate blood test for the diagnosis of feline pancreatitis. in this study, we measured lipase activity in cats using a newer catalytic lipase assay of dry - chemistry system (fdc - v - lip) to determine the reference range and compared the results with those for spec fpl. based on the results of healthy cats, the reference range of fdc - v - lip was determined to be less than 30 u / l. fdc - v - lip did not show a strong correlation with spec fpl in cats with various diseases, which resulted in the low sensitivity and positive predictive value. however, the relatively high (> 90%) specificity and negative predictive value indicated that fdc - v - lip could be a useful patient - side screening test for the exclusion of feline pancreatitis. |
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the results of cancer patient treatment in poland remain unsatisfactory. according to eurocare reports, polish oncology patients have a much lower chance of surviving 5 years after diagnosis than elsewhere in europe [1, 2 ]. this problem, which undoubtedly reflects poland s low healthcare budget, has for several years stirred lively debate in poland. the main causes identified were the low health awareness of polish society at large, the inadequate education of physicians [46 ], lack of funds for delivering healthcare, and the small number of dedicated oncology centers. to improve the situation, on july 1, 2005, the polish government launched the national program for combating neoplastic diseases (npcnd). the program s principal aims were to introduce free public screening for breast, cervical, and colorectal cancers, as well as to procure investment in polish oncology centers in order to provide equipment for radiotherapy and advanced tumor diagnosis. additionally, one part of the program coordinated by wroclaw medical university and implemented from 2006 was to improve the quality of oncological instruction at the undergraduate level. under the npcnd program launched in 2005, short - term financial and organizational supports were planned for the years 20062007 to improve the quality of undergraduate oncology education at all medical universities. after promising results in the first 2 years and recognizing the continued importance of the problem, the npcnd board decided to extend financial support until 2010. the polish academic oncology community has determined [4, 6, 7 ] that the most important tools for improving undergraduate oncology education in poland include the following : development of integrated oncology institutions (radiotherapy + chemotherapy + oncologic surgery + oncologic gynecology) within the structures of medical universitiesbuilding and maintaining a well - trained multidisciplinary staff of academic teachers at each medical universityintroduction of a uniform curriculum complying with european union recommendationsappointment of an oncology education coordinator at each medical universitypreparing an oncology textbook for studentsintroduction of a uniform oncology final examination test at all medical universities. development of integrated oncology institutions (radiotherapy + chemotherapy + oncologic surgery + oncologic gynecology) within the structures of medical universities building and maintaining a well - trained multidisciplinary staff of academic teachers at each medical university introduction of a uniform curriculum complying with european union recommendations appointment of an oncology education coordinator at each medical university preparing an oncology textbook for students introduction of a uniform oncology final examination test at all medical universities. to evaluate the hoped - for improvements in undergraduate training standards, oncology teachers at 11 polish medical universities were asked to fill out a detailed questionnaire. a range of questions were asked, ranging from the appointment of oncology education coordinators, available training facilities and organization of tuition, duration of classes, numbers and types of teaching staff, manner of instruction in palliative medicine, and finally to the main remaining problems associated with oncology teaching. the responses received were then compared with data reflecting the state of affairs in 2004, gathered in an almost identical questionnaire back in 2004 when a report on the state of undergraduate education was being prepared. a summary of responses to the questionnaire are provided in tables 1, 2, and 3.table 1responses to questionnairemedical university (mu)separate course on oncology in the final years of studiesintroductory course on oncology (propedeutics in oncology) in the first years of studieshours of education in palliative medicinenumber of hours per year200420102004201020042010mu bialystok45600152part of oncology course11part of oncology coursemu bydgoszcz457503030separate course30separate coursemu gdansk7970101530separate course30separate coursemu lublin5060004part of oncology course9part of oncology coursemu lodz606018182-day classes part of oncology course3-day classes separate coursemu silesia3045015separate course35separate coursepomeranian mu3045101524separate course24separate coursewroclaw mu406002012separate course12separate coursequestions referring to a separate course in oncology in the final stage of studies, an introductory course in oncology (propedeutics in oncology) in the first years of studies, and education in palliative medicinetable 2responses to questionnairemedical university (mu)is there a university coordinator of oncology educationis there an examination on completion of oncology trainingeducation facilities : yes, provided by mu ; no, provided by an external oncology hospitalyearoutside university200420102004201020042010mu bialystoknoyesnoyesnoyess, rtmu bydgoszcznoyesyesnononos, rt, ctmu gdansknoyesyesyesyesyes mu lublinnoyesnononoyesrtmu lodznoyesyesyesnonos, rt, ctmu silesiayesyesnonoyesyesrtpomeranian muyesyesyesyesyesyesrtwroclaw muyesyesyesyesnonos, rt, ctquestions referring to appointment of a university coordinator of oncology education, final examination to complete oncology training, and training facilities (whether provided by the university or an external oncology hospital) s surgery, rt radiotherapy, ct chemotherapytable 3responses to questionnairemedical university (mu)academic teaching staff in the four basic oncology specialties : radiotherapy, chemotherapy, oncological surgery, and oncological gynecologyproblems considered most significant at the individual universitynumber of oncology teachers in yearlack of20042010mu bialystok67oncological gynecologistno separate palliative medicine course, low number of teachersmu bydgoszcz1111oncological gynecologistno final exam ; inadequate lecture rooms and classroomsmu gdansk1510oncological gynecologist, oncological surgeontoo few lecture roomsmu lublin88oncological gynecologist mu silesia>8>20radiotherapistno exam ; lack of teachers radiotherapistpomeranian mu1520inadequate facilities ; radiotherapy outside of universitywroclaw mu1515inadequate lecture rooms and classrooms, radiotherapy outside of universityquestions referring to numbers of teaching staff in the four basic oncology specialties and problems considered most significant at individual universities responses to questionnaire questions referring to a separate course in oncology in the final stage of studies, an introductory course in oncology (propedeutics in oncology) in the first years of studies, and education in palliative medicine responses to questionnaire questions referring to appointment of a university coordinator of oncology education, final examination to complete oncology training, and training facilities (whether provided by the university or an external oncology hospital) s surgery, rt radiotherapy, ct chemotherapy responses to questionnaire questions referring to numbers of teaching staff in the four basic oncology specialties and problems considered most significant at individual universities the most important achievements of the npcnd program between 2006 and 2010 are listed below.programs of education in oncology were developed at wroclaw medical university s faculty of medicine, faculty of dentistry, faculty of health sciences (nursing, obstetrics, physiotherapy, public health, emergency medicine, dietetics), pharmaceutical faculty, medical laboratory diagnostics faculty, and at the radiology and radiotherapy department. the program plan mainly focused on adjuvant treatment of tumors, including radiotherapy, chemotherapy of solid tumors, oncological surgery, and practical knowledge related to prevention and early diagnosis of tumors. the oncology curriculum was based on the experience and recommendations of the european association for cancer education (eace) and the union for international cancer control (uicc). the program was developed at meetings with representatives of all polish medical universities responsible for oncology education [7, 8 ] (the philosophy of cancer education implemented in the curriculum is described in a separate section below.)at most universities (table 1), the number of classes devoted to oncology education was increased to 70, the postulated program minimum, and an increased number of class hours was devoted to palliative medicine (table 1).an oncology education coordinator was successfully appointed at every medical university (table 2). the main duty of the coordinator is to preside over a team of academic teachers of basic sciences and preclinical and clinical subjects in their efforts to define a detailed program of oncology classes. the coordinator is also responsible for filling gaps in the program and for eliminating contradictions and redundancies.the curriculum and the textbook served as a basis for preparing a set of test questions to ensure that the standard of the final course examination is similar at all medical universities. additionally, all medical universities introduced a set of core lectures and seminars that were considered of key importance for oncology education. there is, however, still no final examination at a number of universities at the end of the oncology course, and students automatically receive a pass credit based only on course attendance (see table 2).an inter - university website for medical students was developed (http://www.e-onkologia.am.wroc.pl/) and contains a series of online lectures in oncology incorporating many of the basics of the uniform oncology education program. the site also includes links to oncology databases, information about the npcnd, the oncology treatment system and oncological specialties, and online tests for students (see next point).online oncology tests (oncotests available at http://www.e-onkologia.am.wroc.pl/onkotest.php) were developed for students to practice their knowledge. these include tests respectively for students of the following : (1) medicine and dentistry, (2) health sciences (nursing, obstetrics, physiotherapy, public health, emergency medicine, dietetics), and (3) pharmacy and medical laboratory diagnostics. the site has been visited over 121,000 times.between 2006 and 2009, several intra- and inter - university conferences and teleconferences were arranged for program coordinators and university academic affairs representatives to discuss progress, difficulties, and challenges.oncology educational institutions were equipped with a variety of teaching aids including computers and software, audiovisual equipment, and anatomical models.a modern comprehensive textbook of oncology for undergraduate students was published in 2007. it presents a multidisciplinary approach to diagnosis and treatment, with special attention given to the practical knowledge indispensable for future physicians (general practitioners).subscriptions to online foreign oncology journals were made to supplement hard - copy polish and foreign publications. programs of education in oncology were developed at wroclaw medical university s faculty of medicine, faculty of dentistry, faculty of health sciences (nursing, obstetrics, physiotherapy, public health, emergency medicine, dietetics), pharmaceutical faculty, medical laboratory diagnostics faculty, and at the radiology and radiotherapy department. the program plan mainly focused on adjuvant treatment of tumors, including radiotherapy, chemotherapy of solid tumors, oncological surgery, and practical knowledge related to prevention and early diagnosis of tumors. the oncology curriculum was based on the experience and recommendations of the european association for cancer education (eace) and the union for international cancer control (uicc). the program was developed at meetings with representatives of all polish medical universities responsible for oncology education [7, 8 ] (the philosophy of cancer education implemented in the curriculum is described in a separate section below.) at most universities (table 1), the number of classes devoted to oncology education was increased to 70, the postulated program minimum, and an increased number of class hours was devoted to palliative medicine (table 1). an oncology education coordinator was successfully appointed at every medical university (table 2). the main duty of the coordinator is to preside over a team of academic teachers of basic sciences and preclinical and clinical subjects in their efforts to define a detailed program of oncology classes. the coordinator is also responsible for filling gaps in the program and for eliminating contradictions and redundancies. the curriculum and the textbook served as a basis for preparing a set of test questions to ensure that the standard of the final course examination is similar at all medical universities. additionally, all medical universities introduced a set of core lectures and seminars that were considered of key importance for oncology education. there is, however, still no final examination at a number of universities at the end of the oncology course, and students automatically receive a pass credit based only on course attendance (see table 2). an inter - university website for medical students was developed (http://www.e-onkologia.am.wroc.pl/) and contains a series of online lectures in oncology incorporating many of the basics of the uniform oncology education program. the site also includes links to oncology databases, information about the npcnd, the oncology treatment system and oncological specialties, and online tests for students (see next point). online oncology tests (oncotests available at http://www.e-onkologia.am.wroc.pl/onkotest.php) were developed for students to practice their knowledge. these include tests respectively for students of the following : (1) medicine and dentistry, (2) health sciences (nursing, obstetrics, physiotherapy, public health, emergency medicine, dietetics), and (3) pharmacy and medical laboratory diagnostics. several intra- and inter - university conferences and teleconferences were arranged for program coordinators and university academic affairs representatives to discuss progress, difficulties, and challenges. oncology educational institutions were equipped with a variety of teaching aids including computers and software, audiovisual equipment, and anatomical models. it presents a multidisciplinary approach to diagnosis and treatment, with special attention given to the practical knowledge indispensable for future physicians (general practitioners). subscriptions to online foreign oncology journals were made to supplement hard - copy polish and foreign publications. regrettably, it proved impossible to increase the number of integrated oncology units at polish universities (table 2). most university teaching (table 2) takes place at oncology hospitals that are not parts of medical universities, and facilities such as classrooms, libraries, and even canteens are scarce or non - existent. also, the number of academic teachers representing the main branches of oncology has not increased significantly (table 3). public awareness of cancer in poland remains low, as is evident from the alarmingly low participation in public screening programs (47 % in breast cancer screening, 23 % in cervical cancer screening efforts are being made, therefore, to ensure that in their first years at a university, students learn about the social and medical aspects of cancer, early symptoms, basics of diagnostics, multidisciplinary treatment, and oncology patient management. special efforts are now being undertaken to emphasize the role of cancer prevention and screening and to describe the role of epidemiology in identifying risk factors. as a result of the program, students in the final stage of their studies now take a course integrating the various concepts and skills taught during other preclinical and clinical classes in etiology, epidemiology, prevention, diagnostics, and treatment of cancer as well as patient post - treatment review and management. lecturers emphasize the role of radiotherapy which is not normally discussed in other classes (in poland, radiotherapy apparatus is usually only available in specialized oncology centers). special importance is also given to the unique nature of solid tumor chemotherapy, oncological surgery and gynecology, and to the need for multidisciplinary therapy, with cooperation between radiotherapists, clinical oncologists, and doctors of other specialties. moreover, lecturers urge primary and secondary prevention and cancer awareness among all doctors and other healthcare professionals and underline the pivotal role that primary care physicians play in diagnosing early stage cancer. students are involved in tumor staging and treatment planning and learn how to inform patients of the disease and of the need for long therapy that often has numerous side - effects. they are also actively involved in diagnosing, treatment, as well as analyzing test results and treatment effects. oncology education also involves management of post - treatment complications that may be discovered by doctors of other specialties during routine examinations. the most important achievements of the npcnd program between 2006 and 2010 are listed below.programs of education in oncology were developed at wroclaw medical university s faculty of medicine, faculty of dentistry, faculty of health sciences (nursing, obstetrics, physiotherapy, public health, emergency medicine, dietetics), pharmaceutical faculty, medical laboratory diagnostics faculty, and at the radiology and radiotherapy department. the program plan mainly focused on adjuvant treatment of tumors, including radiotherapy, chemotherapy of solid tumors, oncological surgery, and practical knowledge related to prevention and early diagnosis of tumors. the oncology curriculum was based on the experience and recommendations of the european association for cancer education (eace) and the union for international cancer control (uicc). the program was developed at meetings with representatives of all polish medical universities responsible for oncology education [7, 8 ] (the philosophy of cancer education implemented in the curriculum is described in a separate section below.)at most universities (table 1), the number of classes devoted to oncology education was increased to 70, the postulated program minimum, and an increased number of class hours was devoted to palliative medicine (table 1).an oncology education coordinator was successfully appointed at every medical university (table 2). the main duty of the coordinator is to preside over a team of academic teachers of basic sciences and preclinical and clinical subjects in their efforts to define a detailed program of oncology classes. the coordinator is also responsible for filling gaps in the program and for eliminating contradictions and redundancies.the curriculum and the textbook served as a basis for preparing a set of test questions to ensure that the standard of the final course examination is similar at all medical universities. additionally, all medical universities introduced a set of core lectures and seminars that were considered of key importance for oncology education. there is, however, still no final examination at a number of universities at the end of the oncology course, and students automatically receive a pass credit based only on course attendance (see table 2).an inter - university website for medical students was developed (http://www.e-onkologia.am.wroc.pl/) and contains a series of online lectures in oncology incorporating many of the basics of the uniform oncology education program. the site also includes links to oncology databases, information about the npcnd, the oncology treatment system and oncological specialties, and online tests for students (see next point).online oncology tests (oncotests available at http://www.e-onkologia.am.wroc.pl/onkotest.php) were developed for students to practice their knowledge. these include tests respectively for students of the following : (1) medicine and dentistry, (2) health sciences (nursing, obstetrics, physiotherapy, public health, emergency medicine, dietetics), and (3) pharmacy and medical laboratory diagnostics. the site has been visited over 121,000 times.between 2006 and 2009, several intra- and inter - university conferences and teleconferences were arranged for program coordinators and university academic affairs representatives to discuss progress, difficulties, and challenges.oncology educational institutions were equipped with a variety of teaching aids including computers and software, audiovisual equipment, and anatomical models.a modern comprehensive textbook of oncology for undergraduate students was published in 2007. it presents a multidisciplinary approach to diagnosis and treatment, with special attention given to the practical knowledge indispensable for future physicians (general practitioners).subscriptions to online foreign oncology journals were made to supplement hard - copy polish and foreign publications. programs of education in oncology were developed at wroclaw medical university s faculty of medicine, faculty of dentistry, faculty of health sciences (nursing, obstetrics, physiotherapy, public health, emergency medicine, dietetics), pharmaceutical faculty, medical laboratory diagnostics faculty, and at the radiology and radiotherapy department. the program plan mainly focused on adjuvant treatment of tumors, including radiotherapy, chemotherapy of solid tumors, oncological surgery, and practical knowledge related to prevention and early diagnosis of tumors. the oncology curriculum was based on the experience and recommendations of the european association for cancer education (eace) and the union for international cancer control (uicc). the program was developed at meetings with representatives of all polish medical universities responsible for oncology education [7, 8 ] (the philosophy of cancer education implemented in the curriculum is described in a separate section below.) at most universities (table 1), the number of classes devoted to oncology education was increased to 70, the postulated program minimum, and an increased number of class hours was devoted to palliative medicine (table 1). an oncology education coordinator was successfully appointed at every medical university (table 2). the main duty of the coordinator is to preside over a team of academic teachers of basic sciences and preclinical and clinical subjects in their efforts to define a detailed program of oncology classes. the coordinator is also responsible for filling gaps in the program and for eliminating contradictions and redundancies. the curriculum and the textbook served as a basis for preparing a set of test questions to ensure that the standard of the final course examination is similar at all medical universities. additionally, all medical universities introduced a set of core lectures and seminars that were considered of key importance for oncology education. there is, however, still no final examination at a number of universities at the end of the oncology course, and students automatically receive a pass credit based only on course attendance (see table 2). an inter - university website for medical students was developed (http://www.e-onkologia.am.wroc.pl/) and contains a series of online lectures in oncology incorporating many of the basics of the uniform oncology education program. the site also includes links to oncology databases, information about the npcnd, the oncology treatment system and oncological specialties, and online tests for students (see next point). online oncology tests (oncotests available at http://www.e-onkologia.am.wroc.pl/onkotest.php) were developed for students to practice their knowledge. these include tests respectively for students of the following : (1) medicine and dentistry, (2) health sciences (nursing, obstetrics, physiotherapy, public health, emergency medicine, dietetics), and (3) pharmacy and medical laboratory diagnostics. several intra- and inter - university conferences and teleconferences were arranged for program coordinators and university academic affairs representatives to discuss progress, difficulties, and challenges. oncology educational institutions were equipped with a variety of teaching aids including computers and software, audiovisual equipment, and anatomical models. it presents a multidisciplinary approach to diagnosis and treatment, with special attention given to the practical knowledge indispensable for future physicians (general practitioners). subscriptions to online foreign oncology journals were made to supplement hard - copy polish and foreign publications. regrettably, it proved impossible to increase the number of integrated oncology units at polish universities (table 2). most university teaching (table 2) takes place at oncology hospitals that are not parts of medical universities, and facilities such as classrooms, libraries, and even canteens are scarce or non - existent. also, the number of academic teachers representing the main branches of oncology has not increased significantly (table 3). public awareness of cancer in poland remains low, as is evident from the alarmingly low participation in public screening programs (47 % in breast cancer screening, 23 % in cervical cancer screening efforts are being made, therefore, to ensure that in their first years at a university, students learn about the social and medical aspects of cancer, early symptoms, basics of diagnostics, multidisciplinary treatment, and oncology patient management. special efforts are now being undertaken to emphasize the role of cancer prevention and screening and to describe the role of epidemiology in identifying risk factors. as a result of the program, students in the final stage of their studies now take a course integrating the various concepts and skills taught during other preclinical and clinical classes in etiology, epidemiology, prevention, diagnostics, and treatment of cancer as well as patient post - treatment review and management. lecturers emphasize the role of radiotherapy which is not normally discussed in other classes (in poland, radiotherapy apparatus is usually only available in specialized oncology centers). special importance is also given to the unique nature of solid tumor chemotherapy, oncological surgery and gynecology, and to the need for multidisciplinary therapy, with cooperation between radiotherapists, clinical oncologists, and doctors of other specialties. moreover, lecturers urge primary and secondary prevention and cancer awareness among all doctors and other healthcare professionals and underline the pivotal role that primary care physicians play in diagnosing early stage cancer. students are involved in tumor staging and treatment planning and learn how to inform patients of the disease and of the need for long therapy that often has numerous side - effects. they are also actively involved in diagnosing, treatment, as well as analyzing test results and treatment effects. oncology education also involves management of post - treatment complications that may be discovered by doctors of other specialties during routine examinations. all doctors encounter patients with cancer, so all medical students should learn about cancer. however, a question raises : what should be learned at the undergraduate level, and what are the most appropriate teaching methods ? the medical universities should be aware that only students with an interest in oncology will receive specialized oncological postgraduate training. several attempts to define core skills and competencies in oncology that medical students should possess can be mentioned. since in 1993, the general medical council published a comprehensive review of medical education ; medical schools in the united kingdom have modified their undergraduate teaching. the australian ideal oncology curriculum for medical students was developed in 1999 by the cancer council australia s oncology education committee. this multidisciplinary group of cancer clinicians and educators representing all medical schools in australia and new zealand extensively consulted their ideal curriculum with the academic staff of all medical schools in australia and new zealand. cancer council australia recommends that the material in the ideal oncology curriculum should appear somewhere in a medical course, not necessarily in a cancer block, to provide a core of knowledge about cancer for the medical graduate. it will assist in enabling the introduction of patient - centered skills simultaneously with a range of technical skills. in 2001,. carried out a questionnaire which covered areas of knowledge and attitudes identified in the australian ideal oncology curriculum as essential for graduating medical students. they observed that although curricula introduced a new course material, it did not produce doctors with better knowledge about cancer. they pointed out that recent students have less exposure to cancer patients than those who trained 10 years ago.. carried out a questionnaire for all 5,143 newly qualified doctors in uk in 2005. they observed low levels of exposure of medical students in the uk to patients with cancer. being aware of those observations, an attempt was made in polish curriculum and study design to hold classes in cancer clinics where combination therapy is used. we observe that the involvement of patients in teaching and learning in oncology is popular with students and improves their knowledge, attitudes, and communication skills. point out that cancer teaching can be fragmented across disciplines with resulting risk of omission or duplication of cancer skills and knowledge. in our program, an oncology education coordinator was appointed at every medical university (table 2) to preside over academic teachers of basic sciences and preclinical and clinical subjects in their efforts to define a detailed program of oncology classes (filling gaps in the program). although none of the authors were able to show that computer - assisted learning improved student learning, in our program, an inter - university website for medical students and online oncology tests were developed (http://www.e-onkologia.am.wroc.pl/). several authors describe oncology courses, among them are the following : portfolio learning, summer schools, oncology module taught by cancer patients, summer oncology fellowship, and attachments in cancer and palliative care. the provided observations suggest that the introduction of courses has better prepared graduating doctors to care for patients with cancer. to increase the quality of education of our students interested in oncology, we established the students scientific societies to provide an opportunity of regular meetings, enhancing motivation for expanding the interest in oncology, promoting self - learning, expanding communications skills, teamwork, and building skills valuable in future professional career as a doctor. the program for improving undergraduate oncology education ran in poland for 5 years (to 2010), and over that period, numerous positive changes in the quality of oncology training were observed. these include the implementation of a uniform course of training at medicine faculties [7, 26 ], an increase in the number and duration of oncology - related classes, and the introduction of a modern textbook for oncology students. special coordinators were appointed to ensure the successful implementation of the program at every university. a constant increase in the number of classes devoted to oncology has also been observed at other faculties. the biggest remaining problem of oncology in an academic setting in poland today is that existing university oncology units are seldom integrated. very few universities have their own facilities and patient rooms for oncology training, and most hands - on teaching still has to be done externally. adult patient oncology at polish universities is now the last and only branch of medicine that lacks a multidisciplinary approach. improving the quality of undergraduate oncology education in poland is a long - term process. it has required the collaboration of all the authorities of each individual medical university and significant financial support to implement the individual elements of the program. via the national program for combating neoplastic diseases, the ministry of health has introduced positive changes in the quality of undergraduate education in oncology by providing financial, legal, and organizational assistance. | cancer patient treatment in poland remains unsatisfactory when compared to that in other countries. in 2005, this alarming situation prompted the polish government to launch the national program for combating neoplastic diseases (npcnd). one part of this project was to improve the quality of oncology instruction at the undergraduate level over the years 2006 and 2007 (subsequently extended until 2010 thanks to promising results and the relatively small financial outlay). the program s main aims were to improve existing oncology therapy and to ameliorate the quality of undergraduate oncology education. to evaluate the changes in the quality of undergraduate education as a result of the npcnd program, medical universities were asked to fill out a questionnaire. responses indicate that the program had a major positive impact on the quality of cancer education mainly as a result of the introduction of a uniform program of training and an increase in the number of classes devoted to oncology. the main unresolved problem is that university hospitals seldom have integrated units catering in - house for surgery, radiotherapy, chemotherapy, etc., and most such hands - on teaching still has to be done externally. |
the study took place at a 300-bed community hospital that is the primary hospital for an acgme - accredited 3-year internal medicine residency with 36 residents, approved by the institutional review board. as a mandatory part of the residency curriculum, all residents had received formal training in clue as detailed elsewhere (16, 22, 23). the clue involves acquisition of six quick - look views and recognition of six evidence - based signs and can typically be performed within 2 min. the clue is designed to augment the physical examination for accuracy and diagnostic synthesis by providing more accurate detection of left ventricular dysfunction (cardiac dysfunction sign), left atrial enlargement (left atrial enlargement sign), interstitial edema (ultrasonic lung comet - tail sign), pleural effusions (effusion sign), right ventricular enlargement (subcostal right ventricular enlargement sign), and elevation of central venous pressures through plethora of the inferior vena cava (subcostal inferior vena cava sign) (22). as a part of the general residency curriculum, all residents participate in a night hospitalist rotation for 2 weeks, twice during their second and third years of residency, in which they solely admit patients in an independent fashion over a 12-h nocturnal shift. consecutive senior residents were provided with a pocket - sized ultrasound device (vscan with duoprobe, ge healthcare, wauwatosa, wi, usa) during this rotation, in which they were instructed to use the device to perform the clue independently and autonomously, whenever they felt it was necessary to provide good patient care. a convenience sample of 542 patients was obtained over 14 months by meeting with the post - call resident on monday and friday mornings after their admitting shifts. use of the device by the residents was tabulated to better understand the value of providing the device during an admitting shift in a community hospital. it is a retrospective review, and analysis of this usage database forms the basis of this report. residents understood that their use of the device had no effect on their course evaluation and were not separately incentivized nor compelled by any attending to specifically use the psd. no billing charge was submitted for any ultrasound use. of the 36 residents in the program, 25 had already been scheduled for the night hospitalist rotation prior to the study 's design and this schedule was not changed. when residents were sampled, each admitted case was briefly reviewed (age, gender, and chief complaint) and the resident was asked whether he or she performed a clue and his / her reasoning for the same. the primary reason for not performing a clue was categorized as whether there was 1) a lack of perceived necessity, 2) a lack of time, or 3) the presence of patient barriers, such as poor cooperation, pain, and inaccessible or infected imaging sites. in addition, the resident was asked the clue results and whether any view was technically difficult to obtain. many patients had already received extensive pre - admission diagnostic studies in the emergency department, often including laboratory data, chest x - ray, ekg, and ct scans, which were available for review by the resident prior to his / her evaluation of the patient. in a pre - planned analysis, one resident, the 13th to participate, utilized a psd on all patient admissions during his rotation. this resident was questioned after each night 's admitting shift regarding which patients he felt, prior to imaging, would benefit from a clue, whether imaging abnormalities and difficulties existed, and if there was immediate post - imaging clinical utility from the clue. clinical utility was considered as a change of management, defined as a treatment change or referral for further testing based upon the clue results, or significant reassurance directly attributable to the clue, defined as improving certainty for the provisional diagnosis when previously less than moderately certain. to evaluate the accuracy of resident - detected clue abnormalities and diagnoses, examination of the electronic medical record was performed after discharge for information provided by formal echocardiography, other imaging studies, and discharge diagnoses. patient age and the number of patients admitted per resident were reported as meanstandard deviation. fisher 's exact test was used to compare the proportion of abnormalities and clinical utility in patients both with and without perceived indications. calculations were made using prism 5 (graph pad software, la jolla, ca). in a pre - planned analysis, one resident, the 13th to participate, utilized a psd on all patient admissions during his rotation. this resident was questioned after each night 's admitting shift regarding which patients he felt, prior to imaging, would benefit from a clue, whether imaging abnormalities and difficulties existed, and if there was immediate post - imaging clinical utility from the clue. clinical utility was considered as a change of management, defined as a treatment change or referral for further testing based upon the clue results, or significant reassurance directly attributable to the clue, defined as improving certainty for the provisional diagnosis when previously less than moderately certain. to evaluate the accuracy of resident - detected clue abnormalities and diagnoses, examination of the electronic medical record was performed after discharge for information provided by formal echocardiography, other imaging studies, and discharge diagnoses. patient age and the number of patients admitted per resident were reported as meanstandard deviation. fisher 's exact test was used to compare the proportion of abnormalities and clinical utility in patients both with and without perceived indications. calculations were made using prism 5 (graph pad software, la jolla, ca). twenty - four residents admitted 542 patients over 101 admitting shifts (12 h per shift), averaging 15.85.2 patients per resident (range : 526) and 5.4 admissions per shift (range : 18). the clue was performed in 230 patients, 42% of cases, with a wide individual resident use range : 1785%. the most frequent reason why a clue was not performed was that the resident felt the case lacked a clinical need to perform the clue, which accounted for 231 patients, or 74% of the not performed cases. this category included cases in which the clue would not have provided useful information pertaining to the patient 's presenting symptoms as well as cases in which the data that the clue could provide were already known through other laboratory or imaging studies either in past records or from current studies performed in the emergency room evaluation. otherwise, time constraints were felt to have prevented psd use in 44 patients, accounting for 14% of the cases in which a clue was not performed. patient factors, mainly lack of patient cooperation, accounted for 37 patients, or 12% of not performed cases. in the 230 patients in whom the clue was performed, residents identified a total of 269 clue abnormalities (table 1). the most frequently identified clue finding was left atrial enlargement, which was seen in 88 (38%) studies. this was followed by ultrasonic lung comets in 58 (25%) studies, left ventricular dysfunction in 53 (23%) studies, pleural effusions in 36 (16%) studies, and right ventricular enlargement or inferior vena cava plethora in 34 (15%) studies. residents felt 101 patients (44% of the imaged patients) had at least one difficult view, totaling 189 difficult views (16% of total views) that could not be adequately obtained. the subcostal view was the most difficult view accounting for 84 of the failed views (37% of all patients) and was followed by 36 views (16% of patients) for left ventricular dysfunction, 36 views (16% of patients) for left atrial enlargement, 28 views (12% of patients) for pleural effusions, and finally, only 5 views (2% of patients) for ultrasonic lung comets. reported clue abnormalities and difficulties in imaging note : the table shows the distribution of clue abnormalities (n=269) and distribution of technically difficult images (n=189) displayed by each clue finding category (clues), as reported by residents. ivc, inferior vena cava ; lv, left ventricular ; rv, right ventricular. in the detailed analysis, the patients had a mean age of 5118 years (range : 1789 years). the resident felt the clue would be useful in 32 of 71 patients, or 45% of cases. the patient age and the percentage of patients felt to potentially benefit from clue were similar in this subpopulation compared with the overall cohort. of the cases felt to be necessary versus unnecessary, the resident identified clue abnormalities in 50% (16/32) of necessary versus 20.5% (8/39) of unnecessary use (p=0.01). clinical utility was perceived in 28.1% (9/32) of necessary versus 15.4% (6/39) of unnecessary use (p=0.25 ; table 2). in the accuracy analysis of patients in whom clue was felt to be necessary, post - discharge chart review showed concordance in 81% (26/32) cases when compared with formal echocardiography, ct imaging, and discharge diagnoses. the resident 's clue findings were discordant in only two (6%) cases, while four (12%) cases were indeterminate. detailed analysis with detected abnormalities and perceived clinical utility note : the table shows detailed analysis (n=71) to compare reported abnormalities and clinical utility from clue in patients both with and without perceived indications. the patients had a mean age of 5118 years (range : 1789 years). the resident felt the clue would be useful in 32 of 71 patients, or 45% of cases. the patient age and the percentage of patients felt to potentially benefit from clue were similar in this subpopulation compared with the overall cohort. of the cases felt to be necessary versus unnecessary, the resident identified clue abnormalities in 50% (16/32) of necessary versus 20.5% (8/39) of unnecessary use (p=0.01). clinical utility was perceived in 28.1% (9/32) of necessary versus 15.4% (6/39) of unnecessary use (p=0.25 ; table 2). in the accuracy analysis of patients in whom clue was felt to be necessary, post - discharge chart review showed concordance in 81% (26/32) cases when compared with formal echocardiography, ct imaging, and discharge diagnoses. the resident 's clue findings were discordant in only two (6%) cases, while four (12%) cases were indeterminate. detailed analysis with detected abnormalities and perceived clinical utility note : the table shows detailed analysis (n=71) to compare reported abnormalities and clinical utility from clue in patients both with and without perceived indications. the current study demonstrates that ultrasound - trained internal medicine residents would autonomously elect to perform a clue using a psd on a significant proportion (42%) of patient admissions during a hospitalist rotation, even after initial evaluation by the emergency room physician and review of electronic medical records. from the detailed analysis, the data suggest that a resident 's decision on whom to perform a clue is marked by a higher rate of clue abnormalities (50% versus 20%), supporting the clinical decision to perform the clue. the clue provided accurate and pertinent clinical information, based upon a very high concordance with results of subsequent formal echocardiography, other standard imaging studies, and clinical diagnoses at the time of discharge. these data are the first to observe the actual use pattern of a psd for hospital admissions and can be used to understand real - world utility and worth of using such a device to augment initial patient evaluation. prior studies have demonstrated that hospitalists can be trained in point - of - care ultrasound, but little data exist on real - world utilization in hospital admissions. unlike the present study, prior studies may have been biased by the use of motivated physicians (21), often eager to learn and apply the technique, or the enrollment of specific patient populations, such as those already referred for echocardiography (2, 3, 18). actual use is dependent on multiple factors that are rarely studied, including physician familiarity and confidence in the techniques, patient characteristics, and convenience. in a study to investigate the diagnostic influence of a psd when used by six variably trained medical residents on inpatient admissions (14), the residents imaged, despite the study protocol, only 45% of the 446 cases the authors attributed this bias to busy working hours, in - hospital logistics and resident instruction to prioritize standard diagnostics. the present study was able to observe unselected residents acting as hospitalists with over 1 year of specific ultrasound training, in their autonomous and unbiased use of a pocket - sized ultrasound device on typical patients admitted to a community hospital. elective usage, in 42% of 542 cases, suggesting a trained physician would likely apply a psd to augment his or her bedside cardiopulmonary exam at least 14 times per shift and likely hesitate to spend valuable time imaging patients where the diagnostic yield was felt to be low or where no new information would be obtained. the willingness or reluctance of the admitting physician to use psd imaging for soft indications, such as in screening physical examination or confirming what was already known, is likely responsible for the large variation of resident use (1785%) noted in the study and the higher proportion of patients placed in the subjective reason category of not needed. while this study has implications for general medical use of point - of - care ultrasound, there exist limitations. the reluctance to image was categorized using subjective self - reporting by the residents and could be related more to the resident 's comfort with the technique than the true validity of the reason for not imaging. although all residents complete the same training program and document an equivalent level of competency, it is possible that the resident who participated in the detailed analysis was especially skilled or motivated which may have increased his or her specific accuracy or affected his or her pattern of use. however, this resident, who imaged all patients, reported that he would have employed psd in 45% of his cases, which is similar to the mean of the overall group (42%), which attests for the lack of bias in overall resident usage. similarly, the possible bias that all residents, by being aware of which days they would be sampled, could have subsequently increased their frequency of use, is a minimal concern. residents knew that their ability to admit and care for multiple admissions overnight was the overall endpoint used in the course 's evaluation of their performance, which may also have countered unnecessary psd use. we feel such issues of time and efficient data collection represents the true, real - world considerations of an unbiased physician acting as a hospitalist. in summary, when trained residents acting as hospitalists are provided with a pocket - sized ultrasound device for autonomous use in cardiac evaluation, usage is frequent. the current observational study may lay the foundation for future studies to evaluate physician biases and how they may affect studies of patient outcomes in point - of - care ultrasound. all authors had access to the data and a role in the writing of the manuscript. also, there are no funding sources and pertinent conflicts to disclose. | backgroundin actual clinical practice as opposed to published studies, the application of bedside ultrasound requires a perception of need, confidence in one 's skills, and convenience.objectiveas the frequency of ultrasound usage is evidence to its perceived value in patient care, we observed the pattern of autonomous use of a pocket - sized device (psd) by ultrasound - trained residents during a night hospitalist rotation.methodsconsecutive internal medicine residents (n=24), trained in a cardiac limited ultrasound examination (clue) as a mandatory part of their curriculum, were sampled on their psd use after their admitting nights, regarding perceived necessity, deterring factors, detected abnormalities, and imaging difficulties. a detailed analysis was performed with one resident who used a psd on every admission to compare the proportion of abnormal clues and utility in patients with and without a perceived need.resultsresidents admitted 542 patients (mean age : 5517 years, range : 1795 years) during 101 shifts and performed clue on 230 patients (42%, range : 1785%). residents elected not to scan 312 (58%) patients due to 1) lack of perceived necessity (231, 74%), 2) time constraints (44, 14%), and 3) patient barriers (37, 12%). in the detailed analysis (n=71), the resident felt clue was necessary in 32 (45%) patients versus unnecessary in 39 (55%) patients, with abnormality rates of 50% versus 20.5% (p=0.01) and utility rates of 28.1% versus 15.4% (p=0.25), respectively.conclusionwhen unbiased residents acting as hospitalists are provided with a psd to augment initial cardiac examination, usage is frequent and suggests clinical value in hospital medicine. |
the human environment together with the biological processes contributes significantly to free radical production. when these free radicals overwhelm the antioxidant defense mechanisms, oxidative stress sets in with its deleterious effects that lead to cell injury, degenerative diseases, and compromised immune system. antioxidants and immune boosting therapy have become very popular in the treatment of many disease conditions. however, in many rural communities, antioxidant and immune boosting drugs are usually expensive, inadequately accessible, and usually associated with side effects. high reliance on natural products for health care maintenance is therefore very common in many communities and has led to increased search for readily available, cheap, and safe natural products with antioxidant and immune boosting properties. gongronema latifolium benth (asclepiadaceae) is a climber that is widely distributed in tropical africa. it is commonly known as utazi in south eastern nigeria while in ghana, senegal, and sierra leone, it is called akan - asantes, gasub, and ndondo - polole, respectively. the ethanol leaf extract has been shown to normalize renal oxidative stress and lipid peroxidation in diabetic rats. also, the leaf extract has hepatoprotective effect against acetaminophen. the hypoglycemic, hypolipidemic, anti - inflammatory, and antioxidant activities of the leaf extract have all been reported [810 ]. the chemical composition of the leaf extract shows it has abundance of essential amino acids and fatty acids. in south eastern nigeria, the fruit is very popular as a remedy for diabetes and high blood pressure, for maintaining healthy living, and in general body healing, yet no scientific study has been done to evaluate these folkloric uses. this study evaluated the antioxidant, immunomodulatory activities and safety of the ethanol extract and fractions of g. latifolium fruit. the fruits of g. latifolium were collected from nsukka, enugu state, nigeria, in march, 2012 and authenticated by a taxonomist, mr. alfred ozioko of bioresource development and conservation project, nsukka, enugu state, nigeria. the fruits were subsequently cleaned, air - dried under room temperature for 20 days, and pulverized. analytical grades of n - hexane, chloroform, ethyl acetate, methanol, hydrogen peroxide, hydrochloric acid, ethanol, dpph (1,1-diphenyl-2-picrylhydrazyl), epinephrine, trichloroacetic acid, thiobarbituric acid, ovalbumin, and tetanus toxoid were all obtained from sigma chemicals co, usa. all laboratory reagents including distilled water were freshly prepared when required. about 3 kg of the pulverized fruit was cold - macerated in aqueous ethanol (70%) for one week. the extract (116 g) was adsorbed on silica gel and eluted in succession with n - hexane, chloroform, ethyl acetate, and methanol. the eluent was filtered and concentrated in vacuo using rotary evaporator at 40c to obtain the n - hexane, chloroform, ethyl acetate, and methanol fractions. all the extracts and fractions were stored in the refrigerator between 0 and 4c until used. the phytochemical analyses of the fruit extract and fractions were carried out using standard methods [12, 13 ]. swiss male and female albino rats 130 7 g and mice 30 4 g (3 months old) were employed for the study. all the animals were obtained from the animal house of the department of pharmacology and toxicology, nnamdi azikiwe university, agulu campus. the animals were housed in standard laboratory conditions of 12 h light, room temperature, and 4060% relative humidity and fed with rodent feed (guinea feeds nigeria ltd.). albino rats were used for the antioxidant studies while mice were used for immunomodulatory studies. in both studies groups 1 and 2 received ethanol extract while groups 3 and 4 received ethylactate fraction and groups 5 and 6 methanol fraction. the extracts and fractions were administered orally at the doses of 200 and 400 mg / kg. groups 7 and 8 served as the control and received 200 mg / kg of standard (vitamin c or noni capsule) and 10% tween 80, respectively. for the chronic toxicity studies, eighty male rats were randomly divided into four groups (a, b, c, and d) of twenty rats per group. the extract was administered orally at doses 750, 1500, and 3000 mg / kg to groups a, b, and c once daily for 90 days. the control, group d, received 10 ml / kg of normal saline. physical observation of the animals was done on daily basis while their weights were taken every week. all animal experiments were conducted in accordance with nih guide for the care and use of laboratory animals and approved by the institution research ethical committee. acute toxicity study was carried out according to oecd guideline for acute oral toxicity testing. a total of six mice and rats were subjected to limit test at 5000 mg / kg bw. three animals each were administered this dose (p.o.) and observed for 48 hours for mortality or obvious toxic symptoms and thereafter for 14 days. dpph solution (0.6 mmol) was freshly prepared using methanol as solvent and 0.5 ml of this solution was mixed with 0.5 ml of different dilutions (62.5, 125, 250, 500, and 1000 g / ml) of the extract and fractions. the final volume of the resulting solution was adjusted to 5 ml using methanol. the absorbance of the mixtures was measured at 520 nm after incubation in the dark for 30 minutes at room temperature. the absorbance of the test substances and the negative control (0.5 ml of dpph solution and 4.5 ml of methanol) was compared and the value used as parameter for the evaluation of antioxidant property. dpph scavenging activity = 100{(ac as)/ac}. ac = absorbance of negative control. as = absorbance of sample. dpph scavenging activity = 100{(ac as)/ac}. ac = absorbance of negative control. as = absorbance of sample. the animals were anesthetized with light ether anaesthesia and blood samples (2 ml) were collected from the animals through retroorbital plexus into plain tubes for the pretreatment basal serum antioxidant assay. after the blood collection, the animals were treated with the extract and fractions once per day for 21 days. the animals were then given the acute oral dose of ccl4, 1.5 ml / kg bw. blood samples were collected from the animals through retroorbital plexus 8 h after ccl4 administration. the serum was used for the determination of antioxidant enzyme activity (catalase, sod) and lipid peroxidation according to the method described by ogbunugafor.. superoxide dismutase activity was determined by its ability to inhibit the autooxidation of epinephrine and catalase activity, by its ability to react with hydrogen peroxide while lipid peroxidation was quantified through formation of malondialdehyde- (mda) thiobarbituric acid (tba) complex. after 3 hrs posttreatment on the 14th day, the animals were given intravenous injection through the tail vain of 1 ml/100 g of indian ink. blood samples were drawn through retroorbital plexus into heparinised edta tubes at 0 and 15 minutes after injection. 25 l of each of the blood samples was mixed with 3 ml of sodium carbonate and optical density was measured spectrophotometrically at 650 nm. the phagocytic index (k) was calculated as follows : (1)k = inod1inod2t2t1, where od1 and od2 are optical densities at t1 and t2. on the 21st day of daily oral administration of the extract and fractions, the thickness of the right hind foot pad was measured using vernier calliper before subplanter injection of 0.1 ml of 20% sheep red blood cell (srbc). subplanter thickness was remeasured after 24 h and the difference between the pre- and postinjection was taken as the measure of delayed hypersensitivity response. following 14th day of daily treatment, blood samples were collected through retroorbital plexus into heparinised edta tubes and analysed for total (tlc) and differential leucocyte counts (dlc) by fixing blood smear and staining with leishman 's stain. for the ovalbumin (ova) and tetanus toxoid immunization, the animals were intraperitoneally injected with 10 g / ml ovalbumin or tetanus toxoid on the 5th and 14th days. blood samples were collected through retroorbital plexus on the 14th and 21st days for determination of primary and secondary antibody responses, respectively. the antisera were separated and used for the estimation of igg1 and igg2a using elisa as described by duddukuri.. five rats from each group were anaesthetized using light ether anaesthesia on the 31st, 61st, and 91st days of the study. blood samples were collected through retroorbital puncture and heparinised whole blood samples were used for the estimation of haemoglobin (hb), packed cell volume (pcv), red blood cell (rbc), and white blood cell (wbc) counts using the method described by kelly. for the estimation of serum liver enzymes, nonheparinised blood samples were allowed to coagulate and the clear serum separated by centrifuging at 2500 rpm for 10 minutes was then used for the analysis of alanine aminotransaminase (alt), aspartate aminotransaminase (ast), and alkaline phosphatase (alp). after the 90-day treatment studies, the remaining 5 rats in each group were returned to normal diet for 28 days and were used for assessing the reversibility or otherwise the toxic effects of the fruit extract on the haematological and biochemical parameters. histopathological studies were done on liver isolates from different groups of the animals and at various times. differences between control and extract treated groups were considered significant at p 0.5) alteration of the body weights (figure 3), pcv, hb, and rbc counts (figure 4). significant (p 0.05) reduction of pcv, hb concentration, and rbc count is an indication of unlikeliness of the extract to cause anaemia. the significant (p < 0.05) elevation of alt, ast, and alp was noticeable at higher doses. histological examination of the liver showed that the extract at 3000 mg / kg produced vacuolar degeneration at the 91st day which however showed convincing signs of reversibility after 28-day posttreatment. vacuolar changes usually occur due to overaccumulation of its storage products or waste products and can also occur secondarily due to endocrine disorder or increased intake of steroids. this effect could have occurred due to the high concentration of the extract (3000 mg / kg) or due to its high steroid content that may have led to glycogen accumulation. glycogen accumulation has been documented to be a leading cause of vacuolar degeneration in dogs. our findings confirm the value of g. latifolium fruit extract as an antioxidant and immunostimulator in rats. although we are much encouraged by these effects of the extract in our rat model, further study of the mutagenic and teratogenic effects is needed before the use of g. latifolium fruit extract in humans is justified. | gongronema latifolium fruit has wide application in ethnomedicine, especially in maintaining healthy living and general body healing. we therefore investigated the antioxidant, immunomodulatory activities, and safety of its ethanol extract and fractions. the in vitro antioxidant activities of the extract and fractions were determined using 2,2-diphenyl-1-picrylhydrazyl (dpph) test while in vivo activities were determined using carbon tetrachloride (ccl4) induced oxidative stress. cell and humoral mediated immune responses were also evaluated together with toxicity studies. the extract, ethyl acetate, and methanol fractions showed inhibition of dpph radical with ic50s 120, 90, and 60 g / ml, respectively. methanol fraction at 200 mg / kg produced significant (p < 0.05) inhibition of lipid peroxidation (mda conc. 1.2 mol / l) compared to control (2.8 mol / l). both ethyl acetate and methanol fractions at 200 mg / kg produced significant (p < 0.05) phagocytic index of 0.021 and 0.025, respectively, compared with control (0.01). significant (p < 0.05) elevations of white blood cells, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase were noticed on the 91st day at higher doses. generally, this study justified the traditional use of g. latifolium fruit for general body healing and maintenance of healthy living. long term administration is safe on the haematological and biochemical systems especially at lower doses and its toxicity at higher doses is reversible. |
sexual health education (she) has been recognized by international organizations as a human right, a necessity for development, and a promoter of equity. nevertheless, most young people do not receive sufficient education for their sexual lives. worldwide, earlier sexual maturity and marriage at later ages may cause earlier premarital sex. this exposes adolescents to serious threats including sexual coercion, unwanted pregnancy, unsafe abortion, and sexually transmitted infections (stis)/hiv. many studies have demonstrated that equipping adolescents with appropriate sexual information promotes their sexual health. this is achieved by delaying or abstaining sexual debut as well as establishing safe sex. schools are unique settings which have so many advantages and strengths to provide school - based she including having an existing infrastructure, easy access to adolescents and parents, and also opportunities for long - term programs. nonetheless, unaids reported that more than half of the students throughout the world do not receive any school - based education for hiv prevention. there is convincing evidence that adolescents access to information and services is not always effective in delaying sex, reducing pregnancy, or stis. it is likely that the health messages are not tailored properly for the target audiences. a recent review found that effective programs were those which possessed specific characteristics in developing and implementing curriculum, and identifying curriculum content ; in all of these levels, they involved youth themselves. sexual health services and information for adolescents should meet adolescents needs rather than organizers preferences. entitled adults as gatekeepers of sexual education and services for adolescents, because they define the content and design of services. there are some differences between adults and adolescents perspectives regarding sex topics that should be taught. previous research demonstrated deep gaps between students demands and preferences regarding she and what they receive in school. a significant number of students report that their required topics are not covered in school or are not covered in adequate depth. most adolescents obtain important information through informal channels such as friends, siblings and media, and formal sex education could not meet their needs to this type of information. politicians seem to be out of touch with actual adolescents needs. in iran like in most other muslim countries, she for unmarried people is socially unacceptable because of the religious and cultural prohibitions of extramarital sex, in particular for girls. in these countries, denial of sex before marriage among young people and failure to achieve adolescents sexual health is a main barrier to combating hiv / aids. she programs are limited and inadequate, and where it is, teachers skip them over because they are uncomfortable teaching sexual subjects. recent evidence shows that pattern of hiv transmission in iran has been changed from injection form in addicts to unprotected sexual contact among young people. cross - gender romantic friendship and sex outside marriage among young people have been the most obvious changes. despite prohibiting sex outside marriage by recent reports from iran indicated that access to internet and satellite was 43.2% and 60%, respectively. strong social norms such as condemnation of premarital or extramarital sex, expectation to observance of modesty, and sanctity of the family resulted in controversies about appropriateness and necessity of providing sex education to unmarried people. two recent studies have demonstrated that sexual related topics are less than 0.0004 of contents of the textbooks in iranian schools. also, sources of sexual knowledge among high school students are friends, books and magazines, and audio - visual materials, and school training is the last resource. regarding authors experiences with adolescent girls lack of knowledge, as well as health consequences and stigmatization resulting from premarital sex, this study attempts to explore female adolescents perspectives to quality of the she they have received or prefer to receive in schools. this is one of the first studies in iran, which addresses this topic using qualitative method. we are hoping that the findings of this study have some implications for legislators, policymakers, and programmers to legislate, design, and implement she programs for adolescents. qualitative approach was used for its strengths in reflecting voices of adolescents directly and understanding the context of the study. study setting was two large province capitals in iran : mashhad, the most important religious city in which the holy shrine of the eighth imam of shiite muslims is located, and ahvaz, the most important industrial city of iran, in which many residents are immigrants across the country. focus group discussions (fgds) and individual in - depth interviews were data sources for this study. eight high schools (two private and six governmental) in the two aforementioned cities were included in the study. to achieve maximum variation, schools were selected from areas with various socioeconomic statuses through getting help from education offices in both research sites. so, we could expect within - group homogeneity and between - group heterogeneity in terms of age, grade, major, and socioeconomic status. school assistants and teachers helped authors to find students who were eligible, expressive, and interested in participation. data collection was carried out until the answers became repetitive and data saturation was achieved. participants were selected from girls in their late adolescence period, aged 14 - 18, because we supposed they have had enough time to experience sexual issues to be able to talk about. inclusion criteria were having menstrual cycles, living with their family, and being unmarried. because some adolescents might be unwilling to express their own experiences in group, individual in - depth interviews a total of seven fgds (n = 44) and 13 individual interviews were conducted between january and november 2010. one of the fgds was held with adolescents who were members of an adolescent friendly service (afs). because the study topic was culturally sensitive in iran, we obtained official permissions after attempting to convince the authorities. all interviews and fgds were conducted by first author, recorded by two recorders, and then transcribed verbatim. semi - structured in - depth interviews were started with a general question (could you please talk about the current situation of sexual health education for adolescent girls in iranian schools ?). depending on the participants responses during the conversation, the interviewer added or removed some questions to guide interview in order to elicit their experiences. fgds were held in schools in which participants discussed she with the assistance of a facilitator. all fgds and interviews were considered as unit of analysis. in order to get overall insights and to become immersed in the data, data were coded using maxqda, a qualitative data analysis software, and then categorized through process of condensation and reduction. to enhance rigor and trustworthiness of the study, these measures were considered : prolonged engagement with participants in data collection, searching negative cases, member check (confirming samples of coded data by participants), and peer debriefing to enhance the credibility through confirming samples of coded data by experienced qualitative researchers. local committee of ethics in research at mashhad university of medical sciences approved the study. also, they had right to choose not to continue with the study whenever they decided. some of the key experiences and perceptions of adolescents regarding she have been selected to be presented. these experiences have been categorized in six main categories described below. ignoring health education, in particular she, was one of the insufficiencies in iranian educational systems which some of adolescents referred to. they believed that many of their sexual issues and dilemmas could be sorted out through providing she at schools. surely all problems would be sorted out by education, many problems can be solved, but unfortunately there is nothing there is nobody to educate (zahra 17 years). most students were dissatisfied with including inapplicable subjects in curricula and missing their educational needs about their actual life. they believed that instead of educating theoretical and insensible scientific subjects such as some subjects in physics, it is better to include life skills into their curricula. one student in this relation said : some topics in physics have no benefit for the students throughout their life. for instance, we had a chapter in physics regarding mirrors. my question is how much it s important to know about the type of pictures in a curved mirror, for instance ? i think that these topics can be removed from our textbooks and issues related to the life skills could be included instead, you know they believed that all of what they receive as health education at school is limited to few short talks in the entire period of schooling. from students point of view, holding these classes just once regarding issues like puberty and preventing aids does not meet students needs. if somebody wants to come and teach us just on one occasion in a year it does nt work nearly all of the participants wanted she to be offered formally in their curriculum as a formal module such as other courses. i wish we had a specific book and a specific teacher for such things, separately for girls and boys (vida 15 years). most students pointed out to the teachers and parents evading response to their questions about sexual issues and censoring sex topics in their textbooks as great challenges regarding she. adolescents criticized their teachers for their evading response to students questions about sex topics in this way : when they (the teachers) want to talk about sexual issues they rush in discussing the topic, they scare that presumably one of students may ask something in this relation and they can not explain it openly. so, you know, they just pass quickly from the topic and come to an end the manner that teacher responds to sexual questions is an evidence for censoring sex topics at schools : the students asked about its meaning and the teacher said that it s not the right time for you to know about that. we have no understanding about men s sexual issues and in the near future we have to live with a man under the same roof ! (shiva 16 years). another student described censoring sex topics as an important factor in misunderstanding about aids and consequently increasing its prevalence in iran. our biology teacher just said that you should not have sexual relationship to be safe. she did nt elaborate why and, did nt explain how it would be spread. just said no sexual contact adolescents emphasized that this censor is due to the teachers fear of principals to reprimand them for their openness. she should not pay attention to the principal s advice regarding not talking about sexual issues explicitly a further issue was postponing she till the time of marriage. according to adolescents views, she would be valuable when it is timely accessible. they expressed their preference to achieve she in early ages, before initiating sexual debut. they argued that most adolescent girls, who experience sexual coercion, do not have enough knowledge about their opposite sex, importance of virginity, and consequences of unsafe sex, because of tender age. health educators are not sent to the schools to educate the students about sexual issues until the ninetieth minute ; when a boy and girl go for their pre - marriage blood tests, just at that time they receive some sort of sexual counseling and it s too late ! the point is that we need to know just now, not at the time of marriage. when we do nt know how should behave in our first sexual relationship and that how we should show our feelings ? they just leave it to the ninetieth minute ! and it does nt work ! because perhaps we are 30 or 45 years old at that time and probably have a lot of wrongdoings ! adolescents mentioned that the best time for she is in the middle school and even in the elementary school about some subjects such as menstruation. a number of students believed that teachers were concerned about adolescents tendencies to promiscuity, so they resorted to scaring tactics to frighten adolescents from negative consequences of sexual relationships, whereas they probably did not know that new generations of adolescents are very smart and are able to differentiate between reality and erroneously exaggerated issues. they said that the most important factor to trust adults and accepting their advice regarding sexual values is their honesty. regarding the adults efforts to make adolescents frightened of negative consequences of romantic relationships, one of the students said : the teachers try to make us cautious in relation to the opposite sex to that extent that we sometimes get panicked when we see a boy or man at the street (fatima 17 years, fg). one of the students indicated that she knows everything about sexual issues, so would not be convinced by adults reasons who want just to make her frightened of being involved in sexual topics. i ve heard that one of the terrible consequences of having sexual relationships is getting pregnancy. it means that pregnancy is inevitable in case of having sexual intercourse, but i know that there are alternatives for not being pregnant and i ve seen those cases. one of the school counselors who had tried to show masturbation as something dreadful indicated that the students were not convinced and said that they have searched books and found no harm following doing masturbation. participants argued that one of the most obvious insufficiencies is inconsistency of she with their needs. the majority of participants considered the real needs of adolescents to obtain information about boys, romantic relationship, sexual instinct and the way of controlling it, sexual relationship and related issues, while educators often limit themselves to discuss commonplace, ordinary, and repetitive subjects. one of the students said : there are many students in middle school who ve got boyfriends and have various risky behaviors and i m saying if the school made us alert about sexual issues instead of educating about menstrual periods ! (laugh), it was so much better. many students in the high school do not like to remember their time at middle school at all, because it was so terrible ! (parisa 15 years, fg). adolescents also mentioned that superficiality and inadequate explanation of subjects does not convince them and can not answer to the several questions of their curious minds. they criticized their teachers for not explaining sex topics in detail and enough depth : one student referred to not using of logic and rationale to justify the poor consequences of sexual risky behaviors by the teachers in their sexual education. one of the students in this relation indicated : schools just say this is bad, but do not explain why they do nt draw our attention to this point that we might get a serious illness, or get pregnant or may be faced by a physical or psychological problem another adolescent stressed the effectiveness of logic argumentation on justifying adolescents to accept adults advice : when we were at the middle school, they (principals) reproved us not to wear short and tight uniform. we said we like it ! and so did not pay attention to these issues at all. but now we ve realized that when boys meet us with such condition, they may be sexually irritated. so now we try to keep our hijab (islamic cover), it was better if they gave clear explanations for the issue they reported superficial and inadequate knowledge of health providers that they found the health providers to be unqualified in sexual health, particularly adolescents she. i assume that our information is more than the health providers who come to educate us. they believed that there is no any trustworthy and honest person at schools to help students when they face any sexual problems. if one student who s got any problem regarding her sexual relationships wants to get help from one teacher, the teacher s behavior would be changed thereafter due to knowing the matter based on adolescents opinions, not only their textbooks are insufficient in providing she, but also they have no access to any appropriate book for learning about sexuality. most books are written in a jargon language or targeted adults so that adolescents are unable to understand them. it has been explained in the books, but we can not understand anything from the book. for instance, regarding fertilization, there is something in our textbook on biology, but we understood nothing from that chapter, as it is so short and ambiguous. ignoring health education, in particular she, was one of the insufficiencies in iranian educational systems which some of adolescents referred to. they believed that many of their sexual issues and dilemmas could be sorted out through providing she at schools. surely all problems would be sorted out by education, many problems can be solved, but unfortunately there is nothing there is nobody to educate (zahra 17 years). most students were dissatisfied with including inapplicable subjects in curricula and missing their educational needs about their actual life. they believed that instead of educating theoretical and insensible scientific subjects such as some subjects in physics, it is better to include life skills into their curricula for instance, we had a chapter in physics regarding mirrors. my question is how much it s important to know about the type of pictures in a curved mirror, for instance ? i think that these topics can be removed from our textbooks and issues related to the life skills could be included instead, you know they believed that all of what they receive as health education at school is limited to few short talks in the entire period of schooling. from students point of view, holding these classes just once regarding issues like puberty and preventing aids does not meet students needs. if somebody wants to come and teach us just on one occasion in a year it does nt work nearly all of the participants wanted she to be offered formally in their curriculum as a formal module such as other courses. i wish we had a specific book and a specific teacher for such things, separately for girls and boys (vida 15 years). most students pointed out to the teachers and parents evading response to their questions about sexual issues and censoring sex topics in their textbooks as great challenges regarding she. adolescents criticized their teachers for their evading response to students questions about sex topics in this way : when they (the teachers) want to talk about sexual issues they rush in discussing the topic, they scare that presumably one of students may ask something in this relation and they can not explain it openly. so, you know, they just pass quickly from the topic and come to an end the manner that teacher responds to sexual questions is an evidence for censoring sex topics at schools : the students asked about its meaning and the teacher said that it s not the right time for you to know about that. we have no understanding about men s sexual issues and in the near future we have to live with a man under the same roof ! (shiva 16 years). another student described censoring sex topics as an important factor in misunderstanding about aids and consequently increasing its prevalence in iran. our biology teacher just said that you should not have sexual relationship to be safe. she did nt elaborate why and, did nt explain how it would be spread. just said no sexual contact adolescents emphasized that this censor is due to the teachers fear of principals to reprimand them for their openness. she should not pay attention to the principal s advice regarding not talking about sexual issues explicitly a further issue was postponing she till the time of marriage. according to adolescents views, she would be valuable when it is timely accessible. they expressed their preference to achieve she in early ages, before initiating sexual debut. they argued that most adolescent girls, who experience sexual coercion, do not have enough knowledge about their opposite sex, importance of virginity, and consequences of unsafe sex, because of tender age. health educators are not sent to the schools to educate the students about sexual issues until the ninetieth minute ; when a boy and girl go for their pre - marriage blood tests, just at that time they receive some sort of sexual counseling and it s too late ! the point is that we need to know just now, not at the time of marriage. when we do nt know how should behave in our first sexual relationship and that how we should show our feelings ? they just leave it to the ninetieth minute ! and it does nt work ! because perhaps we are 30 or 45 years old at that time and probably have a lot of wrongdoings ! adolescents mentioned that the best time for she is in the middle school and even in the elementary school about some subjects such as menstruation. a number of students believed that teachers were concerned about adolescents tendencies to promiscuity, so they resorted to scaring tactics to frighten adolescents from negative consequences of sexual relationships, whereas they probably did not know that new generations of adolescents are very smart and are able to differentiate between reality and erroneously exaggerated issues. they said that the most important factor to trust adults and accepting their advice regarding sexual values is their honesty. regarding the adults efforts to make adolescents frightened of negative consequences of romantic relationships, one of the students said : the teachers try to make us cautious in relation to the opposite sex to that extent that we sometimes get panicked when we see a boy or man at the street. one of the students indicated that she knows everything about sexual issues, so would not be convinced by adults reasons who want just to make her frightened of being involved in sexual topics. i ve heard that one of the terrible consequences of having sexual relationships is getting pregnancy. it means that pregnancy is inevitable in case of having sexual intercourse, but i know that there are alternatives for not being pregnant and i ve seen those cases. one of the school counselors who had tried to show masturbation as something dreadful indicated that the students were not convinced and said that they have searched books and found no harm following doing masturbation. participants argued that one of the most obvious insufficiencies is inconsistency of she with their needs. the majority of participants considered the real needs of adolescents to obtain information about boys, romantic relationship, sexual instinct and the way of controlling it, sexual relationship and related issues, while educators often limit themselves to discuss commonplace, ordinary, and repetitive subjects. one of the students said : there are many students in middle school who ve got boyfriends and have various risky behaviors and i m saying if the school made us alert about sexual issues instead of educating about menstrual periods ! (laugh), it was so much better. many students in the high school do not like to remember their time at middle school at all, because it was so terrible ! (parisa 15 years, fg). adolescents also mentioned that superficiality and inadequate explanation of subjects does not convince them and can not answer to the several questions of their curious minds. they criticized their teachers for not explaining sex topics in detail and enough depth : one student referred to not using of logic and rationale to justify the poor consequences of sexual risky behaviors by the teachers in their sexual education. one of the students in this relation indicated : schools just say this is bad, but do not explain why they do nt draw our attention to this point that we might get a serious illness, or get pregnant or may be faced by a physical or psychological problem another adolescent stressed the effectiveness of logic argumentation on justifying adolescents to accept adults advice : when we were at the middle school, they (principals) reproved us not to wear short and tight uniform. we said we like it ! and so did not pay attention to these issues at all. but now we ve realized that when boys meet us with such condition, they may be sexually irritated. so now we try to keep our hijab (islamic cover), it was better if they gave clear explanations for the issue they reported superficial and inadequate knowledge of health providers that they found the health providers to be unqualified in sexual health, particularly adolescents she. i assume that our information is more than the health providers who come to educate us. they believed that there is no any trustworthy and honest person at schools to help students when they face any sexual problems. if one student who s got any problem regarding her sexual relationships wants to get help from one teacher, the teacher s behavior would be changed thereafter due to knowing the matter not only their textbooks are insufficient in providing she, but also they have no access to any appropriate book for learning about sexuality. most books are written in a jargon language or targeted adults so that adolescents are unable to understand them. it has been explained in the books, but we can not understand anything from the book. for instance, regarding fertilization, there is something in our textbook on biology, but we understood nothing from that chapter, as it is so short and ambiguous. adolescents presented a sort of unhappiness and dissatisfaction with she they received because of inadequacy, discontinuity, lack of educational materials, emphasizing on negative aspects of sexuality, superficiality, and not having enough depth. they perceived the lack of sexual health - related knowledge and skills of their teachers, school counselors, and health care providers to address their she needs, particularly in relation to psychological aspects of sexual health. most of these critiques have been also reported (more or less) by adolescents in the other studies across the world. one of the most important reasons for the silence about sexuality is the concerns of adults that she can encourage adolescents to be sexually active. they believe that having early and accurate sexual information does not increase the likelihood of wanting to experience sex, whereas having limited knowledge increases students curiosity and likelihood of desire to have sex. giving no priority to she by authorities and schools in iran similar to some other regions adolescents criticized adults negative attitude to sexuality and trying to use scaring tactics to make adolescents frightened from health consequences subject to sexual contacts. as islam recognizes human sexuality as an endowment and emphasizes on parents role in sexuality education to their children, it should encourage parents to discuss sexual health topics to children and avoid employing scaring tactics. according to islamic thought, achieving the human transcendence and maturation depends on concordant development of all dimensions of him / her including sexual dimension ; hence, sex education is a necessity for his / her life. indeed, main rationale for sexuality education is not just preventing from sexually transmitted diseases such as aids and hepatitis ; rather, sexuality is a part of individual and social human identity, which can be influenced by the sexual education. previous studies have also reported adolescents dissatisfaction with focusing she on negative consequences of unhealthy sexual decisions. shoveller. reported that adolescents criticized she which is directed toward protecting against pregnancy and stis. they described this as pathologizing sex. in other words, sex has been seen as a reason for diseases and issues related to emotional aspects of sexual relationship have been overlooked. views of adolescents about appropriate time of education in our study were consistent with the results of previous studies. adolescents urgent need to she at middle schools was found in this study ; even some students believed that beginning of she in some topics such as menstruation at last years of primary schools would be more useful. they mentioned that postponing she to the time of marriage for marrying candidates - as it is currently implemented in iran - is too late and pointless. some studies have shown that iranian parents agree with she for adolescents, but other research has found that iranian parents and teachers believe that the appropriate time for educating most sexual health topics is the marriage time. this incompatibility between adults and adolescents views indicates decreased age of sexual orientation in the new generation that is unacceptable for adults yet. there are common concerns that exposing adolescents to sex education results in starting sexual activity earlier. in fact, such evidences suggest this opinion that a kind of she is avoiding to talk about it. thus, controversy among adults about appropriateness of she for adolescents is unanswered yet, but adolescents themselves were ever complaining about evading and reticence in their parents and teachers. it seems that not educating sexual health issues to the adolescents does not warrant that they will not be exposed to such information. in most studies, adolescents have reported that they receive important information about sex topics from their friends, siblings, and media, which has never been provided formally at schools. bleakly. demonstrated that learning about sex from peers and media is related to the increased likelihood of having sex, whereas learning from adults (parents and religious leaders) is associated with delaying sex. in this study, adolescents considered their teachers to be unqualified to educate sex topics. in communities such as iran in which sexuality education has not been adequately provided both in students curricula and training programs for teachers, inadequate knowledge and skills of teachers to teach sex topics seems normal. but it is notable that adolescents distrusting to their teachers and their dissatisfaction with teachers judgmental reactions has been also reported in research studies which have been conducted in the settings in which sexuality education has been formally included in the school curricula. they have recognized their teachers neither as the best educators for teaching she nor as possessing qualifications. on the other hand, most teachers prefer not to deal with sensitive subjects because they fear that society may reproach them. in addition, teachers need some skills to be able to teach sex topics successfully. unesco emphasized the key role of well - trained and motivated teachers in the provision of effective she. supporting teachers in this regard by establishing supportive rules helps them to deliver qualified she. this matter indicates that any sex education program should consider trained and skilled educators as an important part of program ; otherwise, the program will not be effective and successful. adolescents overwhelmingly revealed their urgent need to information about virginity, romantic relationships, knowing opposite sex, and handling sexual desire. asian cultures share stigmatization of sexuality and condemnation of pre- and extramarital sex, especially for daughters whose family s honor depends on their chastity, and muslims particularly have more fanatical beliefs to virginity standard. critical importance of virginity makes adolescents to rate this topic as the most important subject to include in she content. the main strength of this study was adopting a qualitative approach in order to attaining first - hand experiences of adolescents and hearing their voices directly. furthermore, conducting the study in two cities that seem to be different in terms of socio - cultural context (religious vs. industrial) enabled us to compare findings from these two different contexts. these findings confirm emerging of a global youth culture as united nations population fund introduces. it means that youth are under the influence of a global culture among them, as they share values and ideas through mass media and communication technology rather than being limited to their socio - cultural context. another importance of this study was that only a few studies have addressed adolescents perspectives on their sexual information needs in iran. another limitation was that because study settings were restricted to two provinces of iran, generalization of findings to iran is not necessarily correct. conducting another study that includes both male and female adolescents will be more comprehensive and presents interesting comparison between boys and girls perspectives toward she. their views and suggestions provide insights for developing and tailoring she for adolescents, therefore any program for sexual health promotion in adolescents ought to address adolescents needs, demands, and aspirations. we found some similarities between expectations of iranian adolescents and those of adolescents from other cultures about an she program. | background : despite so many unmet sexual health education (she) needs of adolescents, socio - cultural challenges have caused this issue to be ignored in different scoieties. this study investigated iranian female adolescents experiences and perceptions with respect to she that they received at schools, and what they really needed, expected, and preferred.materials and methods : in this qualitative study, seven focus group discussions (44 adolescents) and 13 individual in - depth interviews were conducted among female adolescents aged 14 - 18 in mashhad and ahvaz, iran, to explore adolescents experiences and perceptions towards she in iranian schools. data were analyzed using qualitative content analysis.results:analyzing adolescents perspectives and experiences revealed their great dissatisfaction with she in schools. emerged categories included : lack of obligation and priority for she, sexual reticence and evading, making adolescents frightened of sexual issues, inconsistency of she with adolescents needs, unqualified educators, and lack of appropriate educational materials.conclusion:this study found some similarities between expectations of iranian adolescents and those of adolescents from other cultures about an she program. adolescents showed great abilities to appraise health services delivered for them, and so any program for sexual health promotion in adolescents ought to address adolescents needs, demands, and aspirations. their contribution can provide insights for tailoring she programs for adolescents. |
however, the exact mechanisms of vasoconstriction of pulmonary vessels during hypoxia (hpv) have not been identified yet. many investigators have reported the role of reactive oxygen species (ros) in hpv. however, there are conflicting results on whether ros decreases or increases during hpv. furthermore, the contribution of ros in the acute and sustained phases of hpv is still arguable. superoxide anion (o2) is one of the oxygen free radicals which is supposedly involved in hpv. therefore, it should be eliminated from the cells by a carrier or an anion channel. so far, no special carrier is known for o2. however, the rate of o2 release from the isolated pulmonary rat artery increases during short - term hypoxia in the presence of bicarbonate buffer. furthermore, inhibition of cl - hco3 exchanger (anion exchanger, ae) prevents the release of o2 in rat s pulmonary artery and isolated rabbit lung. two isoforms of ae, na - dependent, and na - independent were detected in the pulmonary vessels. na - dependent ae leads hco3 anions to enter the cells in exchange for chloride anions. the activity of this transporter increases during hypoxia and leads to intracellular alkalinization in the pulmonary smooth muscle cells. therefore, hco3 may enter into the smooth muscle cells in exchange for o2 instead of chloride anion during hypoxic condition. some studies have reported the role of extracellular hco3 on the regulation of pulmonary vascular tone. hypercapnia with normal ph (corrected by bicarbonate) is indicated to increase pulmonary artery wall tension in rats. also, pulmonary artery pressure increases during hypoxia and hypercapnia with ph adjusted by hco3 in the isolated perfused rabbit lung. a few studies have addressed the role of ae on the regulation of pulmonary vascular resistance. dids, acetazolamide (carbonic anhydrase inhibitor), and sodium - free buffer decrease pulmonary vascular tone during hypercapnia with normal ph, which suggests the role of na - dependent ae on the pulmonary vasculature. moreover, high dose of dids prolongs acute phase and prevents sustained phase of hpv in the isolated rabbit lung, which proposes the different role for ae in acute and sustained phases of hpv. however, it is not fully clear by which mechanism ae may regulate pulmonary vascular tone, and whether changes in intracellular ph or entrance of hco3 have an important role in hpv there is a general agreement that nitric oxide (no) is involved in the regulation of pulmonary vascular tone. both in vivo and ex vivo studies have revealed that no production decreased in hpv. therefore, at least a part of vasoconstriction during hypoxic gas ventilation is in concomitant with the reduction of no production, which could influence superoxide release too. many studies indicated interactions between o2 and no in the lung during normoxic and hypoxic conditions. inhibition of each may increase the availability of the other and induce pulmonary vasoconstriction by o2 or pulmonary vasodilatation by no. therefore, it may be concluded that dids increases no production by preventing o2 release through the smooth muscle cells and thereby decreasing hpv. however, no study has indicated the interaction between ros and anion exchanger and a possible role of no during ventilation of the lungs with hypoxic gas. with the above background, this study was performed to clarify the interaction of o2 and ae in hpv. therefore, we added dids as an ae inhibitor and antioxidant trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, to the perfusate before hypoxic gas ventilation in the isolated rabbit lung and measured changes in pulmonary artery pressure during the time course of experiments. furthermore, in order to reveal the effects of trolox or / and dids on no production, we measured no metabolites in the perfusate at above conditions. experiments were approved by the center of comparative and experimental medicine and the ethical committee of animal care at shiraz university of medical sciences, shiraz, iran. animals were housed in standard cages under controlled laboratory conditions of temperature, humidity, and 12:12-hour light - dark cycle. they had free access to water and standard food a few days before starting the experiments. briefly, 20 male new zealand rabbits with body weight (bw) of 2.030.07 kg were anaesthetized by i.v. administration of ketamine (30 - 50 mg / kg bw) and xylazine (6 - 10 mg / kg bw) and heparinized (1500 u / kg bw). the trachea was cannulated and animals were ventilated with room air (tidal volume : 18.480.58 ml, respiratory rate : 30/min). chest wall was opened and about 40 ml heparinized blood was collected by cardiac puncture. the blood was then centrifuged at 4 c at 4000 g for 7 minutes and about 20 ml of plasma was stored in ice. then, pulmonary artery was cannulated and the lung perfused with 4 c air bubble - free krebs - henseleit solution (perfusate) through the pulmonary artery cannula connected to a peristaltic pump with a flow rate of 20 ml / min. finally, isolated ventilated perfused lung was placed in a temperature - equilibrated housing chamber and freely suspended from a force transducer and recirculation was performed. meanwhile, the flow rate was slowly increased from 20 to 140 ml / min. the left atrial pressure and positive end - expiratory pressure (peep) were set at 1.5 - 2.5 cm h2o and 2 cm h2o, respectively. after 20 minutes of steady state period, the perfusate was changed, plasma was added to the perfusate, and the lung was stabilized for additional 20 minutes. two different gas mixtures were employed in this study ; (i) normoxic normocapnic gas : 21% o2, 5.5% co2 balanced with n2 and (ii) hypoxic normocapnic gas : 3% o2, 5.5% co2 balanced with n2. the perfusate used in this study contained in mm : 120 nacl, 1.1 k2hpo4, 1.3 mg po4, 4.3 kcl, 2.4 cacl2, and 13.32 glucose. ph was adjusted to the normal range of 7.35 - 7.40 with nahco3 in all experimental groups. after 40 minutes of the steady - state period, lungs were ventilated in a humidified chamber with hypoxic gas for 10 minutes to evaluate lung viability and its responses to short alveolar hypoxia as a routine test procedure. subsequently, the lungs were randomly divided into four groups, namely control (hox, n=5), dids (200 m, sigma, n=5) treated, trolox (20 m, sigma, n=5) treated, and trolox+dids treated group (n=5). trolox and/or dids were solved and added to the perfusate 10 minutes before the onset of the hypoxic gas ventilation. pulmonary artery pressure, left atrial pressure, airway pressure, and lung weight were continuously registered using a data acquisition system (power lab, ad instrument, australia) connected to a pressure and force transducers. pvr was calculated from pulmonary artery pressure, left atrial pressure, and perfusate flow rate by using ohm equation. pvr was also calculated to evaluate changes in pvr irrespective of its basal values in the experimental groups. perfusate samples were taken during steady - state and at 30 minutes in order to measure po2, pco2, hco3, ph, and no metabolites of each group. perfusate samples were taken from pulmonary venous outflow and were stored at -80c until measurement. after incubating the samples and standards by griess reagent and vanadium chloride in a 96-well plate, the absorbance of samples was detected at 540 nm wavelengths using a microplate reader (biotek, usa) and the concentrations of no metabolites were calculated. the analysis of variance (anova) with lsd post hoc test was used for the comparison of means between the experimental groups. paired t - test was used for comparison of the values in each group at 5 and 30 minutes. experiments were approved by the center of comparative and experimental medicine and the ethical committee of animal care at shiraz university of medical sciences, shiraz, iran. animals were housed in standard cages under controlled laboratory conditions of temperature, humidity, and 12:12-hour light - dark cycle. they had free access to water and standard food a few days before starting the experiments. briefly, 20 male new zealand rabbits with body weight (bw) of 2.030.07 kg were anaesthetized by i.v. administration of ketamine (30 - 50 mg / kg bw) and xylazine (6 - 10 mg / kg bw) and heparinized (1500 u / kg bw). the trachea was cannulated and animals were ventilated with room air (tidal volume : 18.480.58 ml, respiratory rate : 30/min). chest wall was opened and about 40 ml heparinized blood was collected by cardiac puncture. the blood was then centrifuged at 4 c at 4000 g for 7 minutes and about 20 ml of plasma was stored in ice. then, pulmonary artery was cannulated and the lung perfused with 4 c air bubble - free krebs - henseleit solution (perfusate) through the pulmonary artery cannula connected to a peristaltic pump with a flow rate of 20 ml / min. finally, isolated ventilated perfused lung was placed in a temperature - equilibrated housing chamber and freely suspended from a force transducer and recirculation was performed. meanwhile, the flow rate was slowly increased from 20 to 140 ml / min. the left atrial pressure and positive end - expiratory pressure (peep) were set at 1.5 - 2.5 cm h2o and 2 cm h2o, respectively. after 20 minutes of steady state period, the perfusate was changed, plasma was added to the perfusate, and the lung was stabilized for additional 20 minutes. two different gas mixtures were employed in this study ; (i) normoxic normocapnic gas : 21% o2, 5.5% co2 balanced with n2 and (ii) hypoxic normocapnic gas : 3% o2, 5.5% co2 balanced with n2. the perfusate used in this study contained in mm : 120 nacl, 1.1 k2hpo4, 1.3 mg po4, 4.3 kcl, 2.4 cacl2, and 13.32 glucose. ph was adjusted to the normal range of 7.35 - 7.40 with nahco3 in all experimental groups. after 40 minutes of the steady - state period, lungs were ventilated in a humidified chamber with hypoxic gas for 10 minutes to evaluate lung viability and its responses to short alveolar hypoxia as a routine test procedure. subsequently, the lungs were randomly divided into four groups, namely control (hox, n=5), dids (200 m, sigma, n=5) treated, trolox (20 m, sigma, n=5) treated, and trolox+dids treated group (n=5). trolox and/or dids were solved and added to the perfusate 10 minutes before the onset of the hypoxic gas ventilation. pulmonary artery pressure, left atrial pressure, airway pressure, and lung weight were continuously registered using a data acquisition system (power lab, ad instrument, australia) connected to a pressure and force transducers. pvr was calculated from pulmonary artery pressure, left atrial pressure, and perfusate flow rate by using ohm equation. pvr was also calculated to evaluate changes in pvr irrespective of its basal values in the experimental groups. perfusate samples were taken during steady - state and at 30 minutes in order to measure po2, pco2, hco3, ph, and no metabolites of each group. perfusate samples were taken from pulmonary venous outflow and were stored at -80c until measurement. after incubating the samples and standards by griess reagent and vanadium chloride in a 96-well plate, the absorbance of samples was detected at 540 nm wavelengths using a microplate reader (biotek, usa) and the concentrations of no metabolites were calculated. data are presented as meanse. the analysis of variance (anova) with lsd post hoc test was used for the comparison of means between the experimental groups. paired t - test was used for comparison of the values in each group at 5 and 30 minutes. there was no significant difference between po2, pco2, and ph in all hypoxic groups at 30 minutes of the experiments. however, concentration of hco3 in the trolox group was lower compared to the control (p=0.006) and dids (p=0.05) groups. also, hco3 in the trolox+dids group was less than the control (p=0.001) and dids (p=0.011) groups (table 1). gas parameters in the perfusate at 30 minutes of hypoxic gas ventilation in the experimental groups data are presented as meanse (n=5 in each group) ; p<0.01 in trolox vs. control group ; p<0.01 in trolox vs. dids group ; p<0.05 in trolox+dids vs. control group ; p<0.05 in trolox+dids vs. dids group there was no difference between pvr in the control and dids groups at 5 minutes of the experiments (p=0.667). the administration of trolox decreased pvr compared to the control (p=0.016) and dids groups (p=0.007). pvr in the trolox+dids group was also lower than the control (p=0.001) and dids (p<0.001) groups. furthermore, pvr in the trolox+dids group was insignificantly less as compared with the trolox group (p=0.130) (figure 1). pulmonary vascular resistance (pvr) in trolox - treated and trolox+dids - treated groups were lower than other groups at 5 minutes. p<0.01 in trolox vs. control group ; p<0.01 in trolox+dids vs. control group ; p<0.01 in trolox vs. dids group ; p<0.001 in trolox+dids vs. dids group. there was no difference between pvr in the control and dids groups at 30 minutes of the experiments (p=0.457). however, pvr decreased in the trolox group compared to the control (p=0.005) and dids (p=0.020) groups. similar reduction in pvr occurred in the trolox+dids group compared to the control (p=0.005) and dids (p=0.022) groups. there was no variation between pvr in the trolox and trolox+dids groups (p=0.836) (figure 2). pulmonary vascular resistance (pvr) in trolox - treated and trolox+dids - treated groups were lower than other groups at 30 minutes. data are presented as meanse (n=5 in each group). p<0.01 at each group vs. the control group ; p<0.05 at each group vs. the dids group. the concentration of no metabolites in the perfusate of the trolox+dids group was significantly higher than the control group at 30 minutes (p=0.007). however, there was no significant difference between no metabolites in the dids (p=0.169) and trolox (p=0.101) groups compared to the control group (figure 3). concentrations of no metabolites of the perfusate in trolox+dids - treated was more than other groups at 30 minutes. data are presented as meanse (n=5 in each group). p<0.01 in trolox+dids vs. control group. there was no alteration between pvr in the control group at 30 and 5 minutes (p=0.286). furthermore, pvr in the dids group at 30 minutes decreased insignificantly compared to 5 minutes (p=0.184). however, pvr decreased significantly at 30 minutes compared to 5 minutes (p=0.038). also, the reduction of pvr in the trolox+dids group at 30 minutes was significant as compared with 5 minutes (p=0.013) (figure 4). pulmonary vascular resistance (pvr) in trolox - treated and trolox+dids - treated groups at 30 minutes were lower than their values at 5 minutes. although hpv has been appreciated for about seven decades, but its mechanism is still controversial. so far, various mechanisms are attributed to this physiologic phenomenon, including endothelial derived substances and special characteristic of pulmonary smooth muscle cells. it has been reported that ros may increase or decrease during the exposure of pulmonary vessels to hypoxic gas, thus leading to hpv. furthermore, any changes in ros result in no change in opposite direction. since there is no special carrier for o2 release from the pulmonary smooth muscle cells, it may exit through other transporters such as ae. the aim of this study was to investigate the interaction of o2 and ae in hpv. in addition, our findings indicate the effect of ae and ros inhibition on no production, as a factor for modulation of hpv. pco2, hco3 and ph in the perfusate did not alter throughout the experiments in the hypoxic groups, which confirm stable conditions over the time course of experiments. a similar reduction of po2 in all hypoxic groups verifies identical conditions of experiments between groups. we tested pulmonary vascular responses to hypoxic gas ventilation immediately after the steady - state period in order to examine the viability of the lungs and found no variation between the results in all groups (data not shown). therefore, any alteration in hypoxic response of pulmonary vessels after the administration of drugs could be independent of the initial responses of the lungs. furthermore, we calculated pvr in order to rule out the effect of the left atrial pressure and perfusate flow rate on pulmonary artery pressure. ventilation of the lungs with hypoxic gas induced hpv being higher at 5 minutes and then started to reduce insignificantly until 30 minutes of the experiments. this data is comparable with data from previous studies showing a biphasic response of hpv in the isolated lung. the administration of trolox decreased hpv at 5 minutes and entirely prevented it at 30 minutes. trolox is a water - soluble analogue of vitamin e. it diffuses into both lipid and aqueous compartments and has a high potency for ros scavenging. the effects of trolox in our experiments confirm the role of ros during acute as well as the sustained phase of hpv, which is in line with previous studies. however, the role of different ros derivatives (onoo, o2, h2o2, and oh) on hpv can not be differentiated by trolox because of its wide spectrum antioxidant effects. the administration of dids had no further effect on hpv compared to the control group. the data is challenged by other studies postulating that the inhibition of ae or carbonic anhydrase decreases pulmonary vascular tone during hypercapnia. furthermore, 400 m of dids extends the acute phase of hpv in the isolated rabbit lung whereas dids 200 m has no effect on pulmonary artery pressure compared to the control hypoxic group during 60 minutes of the experiments. since dids is an inhibitor of ae and chloride channels, it can be assumed that the high doses of did have more effect on hpv via inhibition of both anion transporters and channels. the expression of na - dependent ae increases the pulmonary artery smooth muscle cell during hypoxia. the activity of this exchanger leads to the influx of hco3 into the cells in exchange for chloride anion. inhibition of ae prevents hco3 to enter the cells and lead to intracellular acidosis. however, there are conflicting results with regard to the effect of hpv on intracellular ph. some researchers have reported intracellular alkalosis and a few studies have indicated intracellular acidosis during hpv. it remains unclear whether changes in ph is a fundamental trigger for the induction of hpv. the combination of trolox and dids resulted in lower hpv compared with the effect of trolox at 5 minutes even though it was not statistically significant. however, reduction of hpv in the trolox+dids group compared with the control and dids groups was more than the trolox group compared to the control and dids groups. o2 anion may exit from the pulmonary smooth muscle cells through ae in the isolated rat pulmonary artery and ischemia / reperfusion model in the isolated rabbit lung. o2 release in exchange for hco3 may lead to the enhancement of intracellular ph in the pulmonary smooth muscle cells. overall, one could suggest that dids reduces hpv a fraction more than trolox per se, perhaps by the prevention of intracellular alkalosis. it seems that the presence of ros is fundamental for the induction of hpv and the activity of ae is necessary for alkalinization during hpv. however, additional studies with different doses of ros inhibitors and ae inhibitors could specify the contribution of each in hpv. no metabolites in the perfusate were significantly higher in the trolox+dids group compared to the control group. inhibition of ros increases no production and the inhibition of ae prevents ros release from the pulmonary artery. each inhibitor insignificantly increased no metabolites of the perfusate in the trolox or dids group. no is a vasodilator involved in the regulation of pulmonary vascular tone. more production in no may lead to more reduction in hpv in the trolox+dids group. we did not measure extracellular and intracellular ph, and o2 due to certain limitations in our preparations. these measurements would help us to confirm our hypothesis with regard to the possible interactions between ros and ae. in the present study, the interaction between ros and ae in hpv has been assessed. a possible interaction is found by a mechanism partly linked to no production and availability. | background : the mechanism of hypoxic pulmonary vasoconstriction (hpv) is still debatable. it has been proposed that reactive oxygen species (ros) might be involved in hpv. however, there is no special transporter for superoxide anion in the cell membrane and it may release from the cells via anion exchanger. therefore, the aim of this study was to investigate the interaction of ros and anion exchanger in acute hpv.methods:the present study was performed in the isolated rabbit lung. after preparation, the lungs were divided into four hypoxic groups of control, trolox (antioxidant)-treated, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (dids, anion exchanger inhibitor)-treated, and trolox+dids - treated. pulmonary artery pressure, left atrial pressure, and lung weight were continuously registered and pvr was then calculated. po2, pco2, hco3-, ph, and no metabolites of the perfusate were measured during steady - state and at the end of experiments (30 minutes). all data were compared with anova and t - test and significance was considered when p<0.05.results : ventilation of the lungs with hypoxic gas induced hpv in the control group. dids did not have a further effect on hpv compared with the control group. the combination of trolox and dids decreased hpv rather than trolox per se at 5 minutes. furthermore, hpv was abolished in both the trolox and trolox+dids groups at 30 minutes. concentrations of no metabolites in the trolox+dids group were more than other groups.conclusion:the present study indicates a possible interaction between ros and anion exchanger in acute hpv. it also suggests the modulatory effect of no at above condition. |
osteoblastoma is a rare benign tumor that accounts for less than 1% of all bone tumors and most commonly involves the spine and sacrum of young individuals. less than 10% of osteoblastoma are localized to skull and nearly half of these cases affecting the mandible, especially the posterior segments. the first well - documented case of osteoblastoma of the jaw bones is attributed to borello and sedano in 1967. the osteoblastoma nomenclature has had a wide synonymy since its discovery by jaffe and mayer in 1932, when it was named osteoblastic osteoid tissue forming tumor. other names have been proposed, such as giant osteogenic fibroma and giant osteoid osteoma. in 1956, the lesion was definitely separated from osteoid osteoma and recognized as an entity by jaffe and lichtenstein, in separate reports, under the name of benign osteoblastoma. this is the name that has been adopted by the world health organization classification of bone tumors and the armed forces institute of pathology. this central bone tumor usually occurs in young adults, with a mean age of 20 years ; with a male to female ratio of 2:1. the lesion is characterized clinically by pain, which is traditionally said to be unresponsive to pain and swelling at the tumor site, the duration being just a few weeks to a year or a more. radiographically the lesion may appear as radiolucent that can be either ill or well defined containing variable amounts of mineralization. although, conventional radiography play an important role in diagnosis ; however, the final diagnosis can only be confirmed after histopathological examination. this report describes a case of aggressive osteoblastoma in posterior segment of mandible, in a 26-year old female. a 26-year old female reported with the chief complaint of a slowly growing painful swelling with respect to right mandibular posterior region of 3 years duration. pain associated with the swelling was mild and intermittent in nature. extra - oral examination revealed a 3 cm 3 cm smooth - surfaced swelling involving right body of the mandible, extending from the parasymphyseal region anteriorly to the right mandibular second molar posteriorly causing obvious facial asymmetry. the lesion contained calcified mass approximating the distal root of the second molar and few radio - opaque flecks scattered within the radiolucency. the lesion was well - delineated, causing expansion and thinning of the lower border of the mandible [figure 1 ]. panoramic view revealed an expansile unilocular radiolucent lesion in relation to right mandibular second molar with variable amount of mineralization the differential diagnosis included cementoblastoma, osteoid osteoma, ossifying fibroma, and focal cemento - osseous dysplasia. the curettings and the second molar were submitted for the histopathological examination [figure 2 ]. photomicrograph of the gross specimen showing curetted lesion along with the right mandibular second molar light microscopic examination of sections stained with h - e revealed irregular wove bone like tissue within areas of hypercellular and loose fibrovascular connective tissue stroma. large amounts of osteoid and focal areas of cementoid deposition were also seen. woven bone showed large osteocytes within, along with a prominent plump osteoblastic rimming [figures 3a and b ]. maturation of the trabeculae could be seen at the margin of the lesion, with appreciation of demarcation from the adjacent normal bone. photomicrograph of histopathologic section reveals irregular wove bone like tissue with osteoblastic rimming within fibrovascular connective tissue stroma. (h - e stain, 100) photomicrograph demonstrates presence of large and irregular areas of woven bone showing large osteocytes within, along with the presence of plump and even bizarre - appearing osteoblast - like cells, bordering the osteoid substance. osteoblastoma is a rare bone forming tumor that very rarely involves the maxilla and mandible, particularly the posterior mandible. this diagnostic challenge may cause relevant problems with the differential diagnosis in view of the tumor 's rarity, ambiguous clinico - radiologic presentation, and histopathologic features, which sometimes resemble osteosarcoma. conventional osteoblastomas are biologically benign with limited growth potential and typically do not exceed 4 cm in diameter. however, there is a small subgroup of borderline osteoblastomas that possesses a locally - aggressive growth pattern, usually exceeding 4 cm. these tumors can not easily be classified as conventional osteoblastomas or osteosarcomas and have, thus, been separated from the classic lesion and designated as osteoblastoma - like osteosarcoma and malignant osteoblastomas or aggressive osteoblastomas. the aggressive osteoblastoma occurs in the older age group than benign osteoblastoma. on the clinical side it is able to extend into adjacent tissues and to recur in 1021%, but does not metastasize. the histologic findings in the aggressive osteoblastoma are those that suggest the possibility of osteosarcoma rather than an obviously benign lesion. some authors advocate that lesions described as aggressive osteoblastoma are, in fact, well - differentiated osteosarcomas resembling osteoblastomas. the diagnostic evaluation is based on the histologic features and the clinical behavior of the lesion. histologically and clinically, differential diagnosis for osteoblastoma ranges from a variety of benign and malignant tumors, ranging from cementoblastoma, osteoid osteoma, fibrous dysplasia, ossifying fibroma, focal cemento - osseous dysplasia, to borderline forms of the above malignancies, up to low - grade osteosarcoma. benign cementoblastoma is to be considered in the differential diagnosis, since its radiographic features are similar to osteoblastoma. the feature to differentiate it from osteoblastoma, however, is the merging of the lesion and the radicular surface of the tooth. in the case described here there was no contact of the tumoral mass with the tooth, in spite of their proximity. this microscopic similarity indicates that a lesion should not be diagnosed as benign cementoblastoma unless it adheres to the tooth. another diagnostic pitfall in connection with the benign osteoblastoma is the possibility of its confusion with osteoid osteoma. on the clinical side, the benign osteoblastoma does not tend to produce pain, so characteristic of osteoid osteoma. also, osteoblastoma is a larger lesion, which by definition exceeds 1 cm in its greatest diameter and is not generally associated with outstanding bony sclerosis typical of osteoid osteoma. at microscopic examination, the bony trabeculae of osteoblastoma are slightly wider than those of osteoid osteoma and there is less irregularity in their arrangement ; the number of osteoblasts is much greater in osteoblastoma but osteoid osteoma lacks giant cells and is not as well vascularized as osteoblastoma. histopathologically, ossifying fibroma and fibrous dysplasia of bone may share many similarities with osteoblastoma but usually are less mineralized lesions, revealing fine calcifications rather than large clusters of mineralized material. in addition, fibrous dysplasia is less circumscribed radiologically than osteoblastoma and may be multifocal, a feature that is exceedingly rare for osteoblastoma. focal cemento - osseous dysplasia is a fibro - osseous lesion that may share similar radiographic features with osteoblastoma. although microscopically, both may feature immature bone ; focal cemento - osseous dysplasia lacks a large number of plump and actively proliferating osteoblasts. histologic examination is paramount in confirming a definitive diagnosis and excluding osteosarcoma, especially the osteoblastic variant. the confusion arises mainly due to the presence of plump or even bizarre - appearing osteoblast - like cells in osteoblastoma, often bordering osteoid substance, mimicking the atypical cells that characterize osteosarcoma. in such instances, the diagnosis is greatly facilitated by the accurate interpretation of the histologic features in view of the radiologic findings that do not disclose the aggressive characteristics of osteosarcoma. believe that the chief microscopic features that separate osteosarcoma from osteoblastoma is the lack of tumor maturation at the margins of osteosarcoma, with permeation of tumor into adjacent tissues, in contrast to maturation at the margins and lack of permeation into the adjacent bone. further, in view of the purported benign nature of this tumor, conservative surgical excision is the treatment of choice ; because recurrence is a rare event (13.6%) and usually attributable to incomplete excision. moreover, apparently no special histological features exist that provide clues to the biological behavior of this neoplasm. based on these various reports and findings, it is clear that the neoplasm has no constant behavior, and varies from case to case. the importance of conceiving the individual bone tumors as clinicopathologic entities should be pointed out again. in the delineation of differential entities, the clinical facts and radiologic findings are very important in the diagnostic evaluation of the lesion, and must be considered along with the histologic findings. at the same time, adequate representative sections of the entire lesion must be submitted to ensure adequate histologic diagnosis so that appropriate treatment can be instituted to the specific pathology. | the clinical facts and radiologic findings are very important in the diagnostic evaluation of jaw swellings, and must be considered along with histologic findings. osteoblastoma, an uncommon primary lesion of the bone that occasionally arises in the jaws, is one such lesion causing a localized jaw swelling. clinically, osteoblastoma can be symptomatic or even remain symptom - free, and may be diagnosed only on routine radiographic examination. histologically and clinically, differential diagnosis for osteoblastoma ranges from a variety of benign and malignant tumors that poses a diagnostic dilemma. stressing the importance of the correct diagnosis of such lesions, this report discusses a case of aggressive osteoblastoma of the mandible posing as a diagnostic dilemma. |
over 500 000 gene sequences have been discovered encoding glycoside hydrolases that are grouped into more than 130 families according to the carbohydrate active enzyme classification (cazy : www.cazy.org). endo--1,2-mannosidases are eukaryotic proteins involved in n - linked glycan maturation, folding, and quality control and are of clinical significance as they provide a means for viruses and cancer to evade the effect of exo - glucosidase inhibitors.endo--1,2-mannanases are produced by bacteroides spp., bacterial residents of the gut microbiota. they facilitate the degradation of dietary yeast mannan consumed in bread and fermented foods, facilitating the breakdown of these complex carbohydrates, with beneficial effects on the gastrointestinal tract and, possibly, mitigating the symptoms of crohn s disease. given the importance of family gh99 enzymes in n - linked glycan maturation and carbohydrate processing by the microbiota, the development of inhibitors has been of particular importance to allow assessment and manipulation of their roles in these complex processes. in this work, we investigate several mechanism - inspired inhibitor design concepts for family gh99 endo--1,2-mannanases from the gut microbiota constituents bacteroides thetaiotaomicron and bacteroides xylanisolvens ; btgh99 and bxgh99, respectively. our results cast light on the importance of structural mimicry of shape and charge of species along the reaction coordinate for achieving potent inhibition of this enzyme family. glycosidases that cleave their substrates with retention of anomeric configuration typically operate through a two - step mechanism that proceeds via a covalent glycosyl - enzyme intermediate. such enzymes utilize enzymatic amino acid residues that in the first step act as general acid and nucleophile to assist in departure of the anomeric substituent while simultaneously substituting the anomeric group ; in the second step the first carboxylate acts as a general base to deprotonate a nucleophilic water molecule that hydrolyzes the covalent glycosyl enzyme intermediate (figure 1a). important exceptions include a range of -hexosaminidases that perform catalysis through mechanisms involving neighboring group participation by the 2-acetamido group of the substrate (figure 1b). these enzymes also operate through a two - step mechanism : in the first step an amino acid residue acts as a general acid to assist in departure of the leaving group while the 2-acetamido group performs a nucleophilic attack on the anomeric center, forming a bicyclic oxazoline / oxazolinium ion intermediate. in the second step the same amino acid residue acts as a general base, assisting nucleophilic attack by a water molecule that opens the oxazolinium ion ring, reforming the 2-acetamido group and completing the hydrolysis reaction. (a) mechanism for a canonical retaining -glycosidase that proceeds through a covalent glycosyl - enzyme intermediate. (b) mechanism for a retaining -hexosaminidase involving neighboring group participation by the 2-acetamido group, via an oxazolinium ion intermediate. (c) proposed mechanism for family gh99 -mannosidases involving neighboring group participation by the 2-oh group, via a 1,2-anhydro sugar (epoxide). x - ray structures are available for gh99 enzymes in complex with a variety of ligands based on sugar - shaped heterocycles. however, in x - ray structures of b. xylanisolvens bxgh99 with various substrate - mimicking ligands, it was not possible to identify an appropriately positioned enzymatic nucleophile within the typical <3 distance from the reactive anomeric center, leading to the proposal of a nonclassical mechanism. in particular, in structures of glcifg (2) and manifg (3) with bxgh99, there were no close contacts with a likely candidate enzymatic nucleophile, at odds with the usual observation of a carboxylate situated typically 2.62.7 away in classical retaining glycosidases. moreover, in complexes of glcdmj (1) with bxgh99 a conserved carboxyl residue (e333 ; numbering refers to bxgh99) was located 2.7 from the 2-oh group ; similar observations extend to the binding of a substrate (-man-1,3--manmu) to the carboxamide mutant bxgh99 e333q. collectively these data supported the proposal of a two - step reaction involving in the first step the formation of a bicyclic 1,2-anhydro sugar intermediate, through e333 acting as a general base residue to deprotonate the 2-oh and facilitating a nucleophilic substitution at c1 coincident with departure of the leaving group, assisted by e336 acting as general acid (figure 1c). in the second step of this proposed mechanism, e333 acts as a general acid, assisting ring opening of the epoxide, while e336 acts as a general base, promoting nucleophilic attack by a water molecule. while such a mechanism lacks precedent in enzymes, there is strong evidence that the base - catalyzed solvolysis of 4-nitrophenyl -d - mannoside and -mannosyl fluoride proceed through similar neighboring group participation mechanisms. the most stable conformation of 1,2-anhydro--d - mannose is a h5 half - chair ; applying the principle of least nuclear motion, a c1 e h5 conformational itinerary has been proposed for the first step of the family gh99 reaction coordinate. intensive efforts have been invested in the rational development of glycosidase inhibitors, and many fundamental principles have been articulated inspired by our deep mechanistic understanding of this class of enzyme. based on pioneering insights from pauling and wolfenden, it is recognized that a common principle underpinning catalysis is the selective affinity of an enzyme for a reaction transition state, relative to the ground state. accordingly, inhibitor design by transition state mimicry, which can take advantage of the high transition state affinity of a glycosidase, has proven a useful guiding strategy. while it is widely appreciated that perfect transition state mimics are chemically unstable and thus unattainable, a general design principle is to develop analogues incorporating features that mimic the shape and charge of the transition state. three features have been highlighted for consideration in the development of effective glycosidase inhibitors : configuration, conformation and charge. configuration is the simplest to address and not surprisingly it is usually found that glycosidases are normally best targeted by inhibitors with stereochemistry matching that of the substrate. in the case of bxgh99, an enzyme that has the ability to cleave both -glc-1,3--man - or and -man-1,3--man - or configured substrates (with a preference for the latter), optimal inhibition is achieved by inhibitors matching the preferred substrate configuration. glycosidases typically operate through transition states with substantial oxocarbenium ion character, and partial double bond development between o5 and c1, leading to a flattened conformation at the transition state. consequently, mimicry of the flattened conformation expected at the transition state has proven a second effective strategy, with inhibitors bearing sp - hybridized atoms at the anomeric or endocyclic oxygen positions, such as glyconolactones and -lactams, identified as fairly broad spectrum glycosidase inhibitors. finally, partial charge development at c1 and the endocyclic oxygen at the transition state can be mimicked by the protonated forms of nitrogen - containing heterocycles, exemplified by deoxymannojirimycin (dmj) with a nitrogen in place of the endocyclic oxygen, and isofagomine with a nitrogen in place of c1. motivated by the unusual mechanism proposed for family gh99 enzymes and the complexity of their biochemical roles, our understanding of which should benefit from the development of potent inhibitors, we undertook a study of several inhibitor designs inspired by the concepts of charge and shape mimicry. glcdmj (1), an inhibitor reported by spiro and co - workers, was the first effective inhibitor of mammalian endo--1,2-mannosidase, and was subsequently shown to bind to and inhibit bxgh99 and btgh99 (table 1). more potent inhibition was achieved by glcifg (2), which also proved to be a more effective inhibitor than glcdmj of mammalian endo--1,2-mannosidase in cell - based studies, demonstrating that varying the position of charge can provide improvements in potency. enzymes as preferential endo--1,2-mannanases, we were able to configurationally match the substrate and develop the inhibitor manifg (3), the most potent inhibitor yet reported for any gh99 enzyme. however, manifg and glcifg lack the 2-oh group of the substrate and thus can not benefit from specific interactions with the putative acid / base e333. separately, spiro and co - workers reported that two other neutral compounds were almost as effective as glcdmj in the inhibition of mammalian endo--1,2-mannosidase, namely, glcddman (4) and glcglucal (6). we were intrigued by these observations and sought to investigate whether the equivalent configurationally matched species, manddman (5) and manglucal (7), and the related cyclohexene derivative (8) were inhibitors of bacterial gh99 enzymes and to understand how they bind to the enzyme. manddman (5) and manglucal (7) were prepared from -1,3-mannobiose (10) (scheme 1). acetylation, followed by bromination afforded mannobiosyl bromide (12), which was converted to the protected glucal (13) using zn in meoh. alternatively, reduction of 13 using h2/pd c, followed by zempln transesterification afforded manddman (5). the preparation of glcchex (8) will be described elsewhere. dissociation constants for the binding of compounds 5 and 7 to btgh99 and bxgh99 were determined by nmr spectroscopy (figure s1). the 2d nmr sofast - hmqc spectra of n - labeled enzymes determined in the presence or absence of a saturating amount of the ligands revealed several h n peaks that displayed significant chemical shift perturbations. for instance, new signals for an arginine residue (assigned as r295 in bxgh99 and r291 in btgh99 on the basis of analysis of inter - residue noes from the 3d - hsqc - noesy spectra ; see annotation to figure 4a) appeared during the titration experiments, which were in slow exchange with the initial ones in the chemical shift time scale (figure 2). therefore, since the relative intensities of these signals are proportional to the populations of the bound and unbound forms (see experimental section and supporting information (si)), the dissociation constants (kd) were readily calculated. no evidence for binding of glcchex could be obtained by either nmr or itc. excerpt of the side chain hn -arginine region in the 2d sofast - hmqc nmr spectra of n - labeled btgh99 in the absence (blue) or presence of an excess (top) of manglucal (7 ; red) or (bottom) manddman (5 ; green). the arrow highlights the chemical shift perturbation observed for the hn- signal corresponding to r295. in order to analyze the mode of binding of the conformationally restricted compounds, 3-d structures of complexes of bxgh99 with 7 and 8 were determined by x - ray crystallography at near atomic (approximately 1.0) resolutions (table 2, figure 3a, b). manglucal 7 (kd 111 m) binds to bxgh99 in the 2 and 1 subsites, with the 1 glucal ring intact in a h5 conformation. for reasons most likely related to its poor affinity for the enzyme, we were unable to obtain a complex of glcchex with wildtype enzyme, but were serendipitously successful in obtaining a complex with the catalytically inactive bxgh99 e333q mutant. in this complex glcchex 8 also bound in the 2/1 subsite with the cyclohexene ring in a h5 conformation (figure 3b). relative to manglucal, glcchex suffers both by replacement of the endocyclic oxygen with methylene and by the presence of a nonreducing - end glucosyl moiety, the latter of which is known to reduce binding to the bacteroides spp. owing to the unmeasurable binding of glcchex, the synthesis of the mannose analogue was not pursued. the lack of oxygen atoms within the glcchex ring means it can not form hydrogen bonds with active site residues y252, e333 or e336. ms experiments indicated that the compound is not affected by the enzyme or its variants so the reason for binding to only the inactive variant is unclear. (a) bxgh99 with manglucal (7), (b) bxgh99-e333q with glcchex (8), and (c) bxgh99 with manddman (5). depicted electron density maps are refmac maximum - likelihood/a weighted 2f0 fc syntheses contoured at 1.5 (0.57, 0.59, and 0.62 e, respectively). glycals are often effective inhibitors of classical retaining glycosidases. inhibition is typically found to be time - dependent, owing to a chemical reaction in which the conjugate acid of the nucleophile protonates the enol ether of the glycal, and forms a 2-deoxy - glycosyl enzyme. this mode of reactivity has been exploited to allow the identification of the catalytic nucleophile by peptide sequencing, and the 2-deoxyglycosyl - enzymes are sufficiently stable to be studied by x - ray crystallography. to date, only two classes of retaining glycosidases have been identified upon which glycals bind as competitive inhibitors without exhibiting this mode of reactivity. these are n - acetylhexosaminidases that use neighboring group participation and retaining sialidases, and in both cases these enzymes lack a typical carboxylate nucleophile. retaining n - acetylhexosaminidases use a 2-acetamido group, which is able to catalyze the slow hydration of the enol ether. retaining sialidases lack a carboxylate nucleophile capable of protonating the glycal, and instead use a less acidic tyrosine residue as the catalytic nucleophile. in the case of sialidases, the corresponding glycal 2,3-didehydro-2-deoxy - n - acetylneuraminic acid has been elaborated into the extraordinarily potent inhibitor zanamivir, a clinically used antiinfluenza drug. despite its potency, quantitative examination of transition state mimicry by zanamivir reveals it to be a poor transition state mimic. the observation that manglucal binds to bxgh99 without a chemical reaction despite the retaining mechanism of the enzyme provides further evidence in favor of the unique neighboring group participation mechanism proposed for this family. in order to harness the tighter binding of the bacterial enzymes with a mannoside in the 2 subsite manddman (5) nmr titration revealed manddman to bind to bxgh99 and btgh99 with kd values of 221 and 53 m, respectively (table 1). by comparison, glcddman (4) is an inhibitor for rat endomannosidase with an ic50 value of 3.8 m for inhibition of cleavage of c - labeled glcman9glcnac, only slightly worse than that of glcdmj (1) (ic50 = 1.7 m). the structure of bxgh99 in complex with manddman was determined at 1.03 resolution. compound 5 binds in the 2/1 subsites of the enzyme in an undistorted c1 conformation. this conformation matches that of the ground state of the substrate, and while this is consistent with the modest dissociation constant, it is noteworthy that the tight - binding inhibitor manifg 3 also binds in a c1 chair. as manifg (3), manddman (5), and manglucal (7) all lack an o2 group, in considering the contribution of shape and charge to inhibition, it would appear that the cationic nature of manifg contributes most significantly to inhibition, in spite of its conformational resemblance to the ground state. we therefore decided that it would be appropriate to investigate a charged inhibitor based on manifg that was able to make the correct o2 interactions, in particular with e333. inspired by the work of bols and co - workers on the development of noeuromycin, a 2-hydroxy analogue of isofagomine that binds 24000 times more tightly than isofagomine to various glycosidases, we therefore synthesized the noeuromycin derivative, mannoe. this inhibitor was synthesized by the regioselective mannosylation of the nitrile diol 16(35) by trichloroacetimidate 15scheme 2. the sole acetate group of the glycoside 17 was cleaved by treatment with hcl / meoh to afford alcohol 18, and the nitrile group was reduced using bh3me2s, followed by protection as the boc derivative 19. hydrogenolysis of the benzyl ethers of 19 using h2/pd c, and then cleavage of the boc group with hcl, afforded mannoe.hcl (9) [as a mixture of -hydroxypiperidine and pyranose isomers (not drawn) ; see the si ]. isothermal titration calorimetry revealed mannoe (9) to bind to bxgh99 with kd = 13 nm, and to btgh99 with a kd = 30 nm, 17- and 5-fold more tightly than manifg (3) to the respective enzymes, commensurate with improvements seen for binding of ifg versus noe for other enzymes, and demonstrating that better matching of the substrate by reinstatement of the 2-oh group absent in the latter compound provides more effective inhibition (table 1, figure s4). 3-d structures were solved of a binary complex of bxgh99mannoe, and a ternary complex of bxgh99mannoe-1,2-mannobiose at resolutions of 1.14 and 1.05, respectively (figure 4). the poses of mannoe in both complexes were essentially identical and the more informative ternary complex, with mannoe in the 2/1 subsites, and -1,2-mannobiose in the + 1/+2 subsites will therefore be discussed. the noe heterocycle binds in a c1 conformation, similar to that seen for manifg with the same enzyme. a close contact with e333 oo2 of 2.58 is evident, similar to that seen in the complex of bxgh99 with glcdmj (2.54, pdb 4ad3). (a) x - ray structure of ternary complex of bxgh99 with mannoe (above) and -1,2-mannobiose (below). the + 1 subsite mannose residue electron density is best modeled by two mannose conformations with 0.6/0.4 occupancy, rotated by about 30 with respect to + 2 mannose. assembled using ccp4 mg. in order to understand the intrinsic conformational preferences of the d - glucal, 1,2-dideoxymannose (ddman), chex, and noeuromycin (noe) inhibitor warheads, so as to help rationalize the conformations observed on - enzyme, we calculated their conformational free energy landscapes (fels). fels were computed by ab initio metadynamics (see experimental section), and the cremer pople puckering coordinates and were used as collective variables, yielding a mercator representation for each inhibitor fel (figure 5). the same procedure has been previously used to analyze the conformational preferences of related gh inhibitors (mannoimidazole, glucoimidazole, and ifg). conformational free - energy landscapes (fels, mercator projection) of isolated d - glucal (a), chex (b), ddman (c), and noe (d), contoured at 1 kcal mol. fels have been annotated (yellow star) with the inhibitor conformations of manglucal 7 (for a), glcchex 8 (for b), manddman 5 (for c), and mannoe 9 (for d) that have been observed on - enzyme in this work. the fels of both d - glucal and chex (figure 5a, b) exhibit a main energy minimum centered at h5. h5e5, with an arm toward b3,o, indicating substantial conformational freedom around h5. there is also a local minimum in the southern hemisphere, centered at h4. however, this is 3 kcal mol higher in energy and thus less populated at room temperature. interconversion between the two minima is hindered by an 8 kcal mol energy barrier. therefore, both d - glucal and chex display only one main accessible conformation at room temperature, h5, but can readily adopt the nearby e conformation predicted for the transition states leading to and from the proposed 1,2-anhydrosugar intermediate. these data suggest that d - glucal and chex provide good shape mimicry of the transition state or intermediate for the gh99 catalyzed reaction. that both d - glucal and chex adopt a h5 conformation when complexed to bxgh99 (table 2 and yellow star in figure 5a, b) indicates that the conformational preference of the isolated molecules are not significantly perturbed on - enzyme. this is also the case for ddman, for which the fel (figure 5c) is strongly biased toward the c1 chair (the local minima on the equator are 8 kcal / mol higher in energy), as observed in the x - ray structure on - enzyme. however, the fel of ddman does not exhibit any stable minimum around h5, thus it can not be considered a gh99 transition state shape mimic, and should instead be considered a mimic of the substrate conformation in the michaelis complex. noe differs from the other inhibitors considered here as it is a basic molecule and was therefore considered as both the neutral (figure 5d) and protonated species (figure s5). while the topographies of the fels for the two protonation states are broadly similar, they differ greatly in relative energies, most importantly for the global and local minima, such that the most stable state in noe, a c1 chair, becomes the ring - flipped c4 conformer in protonated noe. in that case, the two most stable species, the c4 and s5 states, are characterized by the presence of transannular hydrogen bonds between nh2 and o6 or o3, respectively. what then is the most appropriate fel to consider in relation to the enzyme - bound state, which is expected to be protonated, but which in a c1 conformation lacks a transannular hydrogen bond ? it is known that in the absence of solvation (in the gas phase) flexible molecules in low charge states tend to compensate charge effects by forming stabilizing intramolecular interactions that do not take place in other environments (e.g., in solution). for example, low - charge state proteins in the gas phase tend to adopt more compact structures owing to increased intramolecular interactions. on balance, we consider the fel of neutral noe to be a more relevant representation of the enzyme - bound conformations, because interactions of the inhibitor with active site residues prevent the formation of intramolecular hydrogen bonds that dominate the conformations of charged species. accordingly, the fel displays a wide main minimum situated close to c1 (figure 5d), and is thus similar to that of ddman. the noe fel is also reminiscent of the one previously computed for the closely related neutral ifg inhibitor (which differs from noe by the absence of the 2-hydroxyl group). interestingly, the transition state region between the north pole and the equator in noe (h3/e) is shifted by 60 in in ddman. this is likely due to a vicinal intramolecular hydrogen bond formed between the 2-oh and the 3-oh, which stabilizes the h3/e conformations in noe ; this interaction is not present in ddman or ifg as they both lack a 2-oh. overall, noe most closely resembles the conformation of the substrate in the michaelis complex and in its protonated state on - enzyme provides mimicry of an oxocarbenium - ion - like transition state ; it is therefore best considered a charge-mimicking inhibitor with no significant shape mimicry of the transition state. the proposed gh family 99 neighboring group participation mechanism, via a 1,2-anhydro sugar, allows prediction of a c1 e h5 conformational itinerary for the first step of the reaction coordinate. the fels for glucal and chex suggest that when these inhibitor warheads are extended to manglucal and glcchex, their flattened conformations provide mimicry of the e transition state and h5 intermediate conformations. x - ray structures of these compounds in complex with bxgh99 revealed them to bind in a h5 conformation, most closely matching the proposed intermediate conformation. the modest dissociation constant of manglucal, and lack of detectable binding for glcchex, suggests that shape mimicry of the intermediate or transition state provides only weak affinity for the enzyme. on the other hand the fel for the neutral sugar ddman reveals a preference for a c1 conformation, and the corresponding complex of manddman with bxgh99 revealed this compound to bind in the same conformation, albeit with an affinity relative to manglucal 8- or 18-fold worse for bx or btgh99 enzymes, respectively. as manglucal, glcchex and manddman all lack a 2-oh group, these ratios provide an estimate for the contribution of shape - mimicry of the ts or intermediate to enzyme binding. based on the above analysis, and combined with the previous discovery that the best inhibitor for gh99 endo--mannanases is manifg, which provides charge mimicry, but poor transition state shape mimicry, we were inspired to reinstate the 2-hydroxyl group missing in this compound. mannoe was synthesized and shown to bind to bx and btgh99 with kd values of 30 and 13 nm, the most tightly binding ligand for these enzymes yet reported, and a 20-fold enhancement of affinity relative to manifg. the x - ray structure of mannoe in complex with bxgh99 reveals a c1 ground - state conformation mimicking the michaelis complex, and we conclude that this inhibitor acts primarily to mimic the charge of the transition state. data set hkl index was matched to a previous solution using aimless software and freer set was generated from bxgh99manddman data, and then used for every other data set. initial polypeptide chain model was obtained from the same previous solution, refined against bxgh99manddman data and used as starting model for other structure solutions. refmac5 with prosmart was used for restrained refinement and coot for real - space refinement. during fcalc difference map was examined at 3. validation was performed using coot and edstats. mannoe binding to bt and bxgh99 was measured using a microcal autoitc200 instrument (malvern instruments, formerly ge healthcare) at 25 c in 25 mm hepes ph 7.0, 100 mm nacl buffer. protein concentration was kept at 5 m and ligand concentration at 50 m. as mannoe exists as a 5:2 ratio of d - gluco and d - manno noe isomers ; n sofast - hmqc spectra were recorded at 298 k for 1 h using n - labeled btgh99 and bxgh99 on a bruker avance iii 800 mhz spectrometer with cryoprobe. upon binding of manglucal or manddman, chemical shift perturbations a signal corresponding to n-h of r295 (numbering based on bxgh99), and which is close to the enzyme active site, was chosen as binding reporter. in the case of bxgh99, this signal shifts from 7.33, 87.5 ppm (h, n) in the free state to 7.41, 87.2 ppm (h, n) in the bound state. bound and free protein populations at different protein / ligand ratios were calculated from peak intensities. nmr measurements were made in 50 mm potassium phosphate ph 7.0, 50 mm kcl with 5% d2o added. protein concentration was determined by measuring 280 nm uv absorbance after denaturing the solution in 6 m guanidinium chloride (average protein concentration : 58 m). ligand concentrations were cross verified by integrating the h peaks against the internal standard tsp - d4 (sigma - aldrich). the dissociation constants (kd values) were estimated using in - house matlab 2015b scripts, using the following equation : where l is the free ligand concentration and ([pl]/pt) is the ligand - bound protein fraction. errors were propagated using a monte carlo algorithm to estimate the uncertainty in the kd values. a distribution of kd values (n = 10 000) were obtained from data sets randomly varying within the error bars, and the standard deviation of was used for the kd error estimation. to obtain the conformational free energy landscapes of ddman, glucal, chex, and noe, quantum mechanical calculations were performed using density functional theory based molecular dynamics (md), according to the car parrinello (cp) method. each molecule was enclosed in an isolated cubic box of 12.0 12.0 12.0. a fictitious electron mass of 700 au was used for the cp lagrangian and a time step of 0.12 fs was used in all cpmd simulations. the kohn sham orbitals were expanded in a plane wave (pw) basis set with a kinetic energy cutoff of 70 ry. the perdew, burke, and ernzerhoff generalized gradient - corrected approximation (pbe) was selected in view of its good performance in previous work on isolated sugars, glycosidases, and glycosyltransferases. the metadynamics algorithm, provided by the plumed 2 plugin, was used to explore the conformational free energy landscape of the systems, taking as collective variables and of the puckering coordinates of cremer and pople, in the spirit of the pioneering work by dowd, french, and reilly. initially, the height of these gaussian terms was set at 0.6 kcal mol and a new gaussian - like potential was added every 250 md steps. once the whole free energy space was explored, the height of the gaussian terms was reduced to half of its initial value (0.3 kcalmol) and a new gaussian - like potential was added every 500 md steps. the simulations were stopped when energy differences among wells remain constant, which was further confirmed by a time - independent free energy estimator. for all molecules, the phase space was fully explored in less than 60 ps and the simulations were further extended up to 140 ps for chex and glucal, 160 ps for ddman, and 240 ps for noe. the errors in the principal minima, taken as a standard deviation (sd) from the last 60 ps, are below 0.6 kcal mol (figure s6). | inhibitor design incorporating features of the reaction coordinate and transition - state structure has emerged as a powerful approach for the development of enzyme inhibitors. such inhibitors find use as mechanistic probes, chemical biology tools, and therapeutics. endo--1,2-mannosidases and endo--1,2-mannanases, members of glycoside hydrolase family 99 (gh99), are interesting targets for inhibitor development as they play key roles in n - glycan maturation and microbiotal yeast mannan degradation, respectively. these enzymes are proposed to act via a 1,2-anhydrosugar epoxide mechanism that proceeds through an unusual conformational itinerary. here, we explore how shape and charge contribute to binding of diverse inhibitors of these enzymes. we report the synthesis of neutral dideoxy, glucal and cyclohexenyl disaccharide inhibitors, their binding to gh99 endo--1,2-mannanases, and their structural analysis by x - ray crystallography. quantum mechanical calculations of the free energy landscapes reveal how the neutral inhibitors provide shape but not charge mimicry of the proposed intermediate and transition state structures. building upon the knowledge of shape and charge contributions to inhibition of family gh99 enzymes, we design and synthesize -man-1,3-noeuromycin, which is revealed to be the most potent inhibitor (kd 13 nm for bacteroides xylanisolvens gh99 enzyme) of these enzymes yet reported. this work reveals how shape and charge mimicry of transition state features can enable the rational design of potent inhibitors. |
a previously healthy teenage girl, who had immigrated to oregon from mexico as an infant, sought care in january 2000 for progressively severe headaches of several months duration. autopsy showed no evidence of trauma, and results of a toxicology screening were negative. examination of the brain showed obstructive hydrocephalus, bilateral uncal herniation, flattening of the cerebral gyri, and an intact cysticercus compressing the inferior 4th ventricle (figure 1). a) coronal section of brain showing dilation of ventricles, flattening of the cerebral gyri, and uncal herniation. we searched the oregon hospital discharge database to identify oregon hospitals that had discharged patients with the icd-9-cm code for neurocysticercosis (123.1) from january 1995 through december 2000 and requested the medical records of these patients. we searched the oregon death certificate database for additional neurocysticercosis deaths during the same period. a case of neurocysticercosis was defined as any person with a hospital discharge code of 123.1 or death certificate diagnosis of neurocysticercosis during the study period, and a record of imaging studies or pathology reports consistent with neurocysticercosis. an incident case was defined as one for which no reference to any previous diagnosis of neurocysticercosis was found in the medical record. incidence rates were calculated by using u.s. census bureau yearly population estimates for oregon as the denominator (2,3). data were analyzed with epiinfo, version 6.04d (centers for disease control and prevention, atlanta, ga). we found 89 hospital discharges coded for neurocysticercosis during the study period among 18 hospitals in 10 counties. review of these records confirmed the diagnosis of neurocysticercosis for 59 persons (17 were admitted more than once). thus, including the death described above, we found 61 persons who met the case definition for neurocysticercosis ; 43 (70%) of these were incident cases. the mean annual incidence rate from 1995 to 2000 was 0.2 per 100,000 general population and 3.1 per 100,000 hispanic population. number of neurocysticercosis hospital discharges and new diagnoses by year ; oregon, january 1995december 2000. among the 61 confirmed patients, 40 (66%) were male, and 52 (85%) were hispanic. the median age at the time of first hospitalization was 24 years (range 2 to 79 years) ; 13 (21%) were < 18 years old. agriculture or other manual labor was the most common job type listed (20/37 ; 54%) ; 21% patients were unemployed or disabled ; 40% of patients had no health insurance. 41 (72%) were born in mexico, 10 (18%) in the united states, 3 (5%) in guatemala, and 1 (2%) each in korea, saudi arabia, and an unspecified african country. of the 10 neurocysticercosis patients born in the united states, four of these five cases occurred among children (ages 3, 5, 5, and 12 years) ; their source of exposure to taenia eggs was not documented. the source of exposure for the adult patient was presumed to be a household contact visiting from mexico. travel histories of four of the remaining u.s.- born patients included a caribbean cruise by a retired pig farmer, extensive travel in madagascar and tanzania by a student, annual visits to puerto rico by an 11-year - old, and 1 week spent in mexico city by a toddler. no information about travel was documented for one patient, a 3-year - old child. treatment included craniotomy with cyst removal (16%),ventriculoperitoneal shunting (12%), albendazole (18%), praziquantel (19%), anticonvulsive therapy (63%), and corticosteroids (49%). two of these deaths also occurred unexpectedly before diagnosis ; both resulted from acute hydrocephalus caused by an obstructing cyst on the 4th ventricle. the causes of death in these patients were aspiration pneumonia after craniotomy and cyst removal, intracerebral hemorrhage, and hydrocephalus due to recurrent cerebrospinal fluid shunt failure. the median age at time of death was 33 years (range 1773 years). this study demonstrates that neurocysticercosis causes substantial illness and death among hispanic populations in oregon. many of those affected are young immigrants from mexico without medical insurance, who are either unemployed or are working in agriculture or other manual labor. our study did not address where disease transmission occurred for persons born outside of the united states ; some of these cases could also have been acquired locally. several previous reports have documented the increasing recognition of neurocysticercosis in the united states, but the emphasis has been primarily on disease occurring in southwestern states bordering mexico (4) and small outbreaks elsewhere (57). previously, the only available information about the occurrence of neurocysticercosis in the pacific northwest was a study of seizure patients at 11 university - affiliated, urban emergency departments throughout the united states. in that study, cases were concentrated in southwestern states ; four cases were found in oregon during a 2-year period (8). the study underestimated the incidence of this disease in the northwest, and perhaps other areas of the united states, by looking for cases only in selected urban emergency departments. the average annual incidence of neurocysticercosis among oregon s hispanic population found in our study is higher than that previously reported in los angeles county (1.6/100,000) (9) and in mexico (0.8/100,000) (10). census bureau population estimates due to undercounting of hispanic migrants could have resulted in a falsely elevated incidence rate. in addition, the higher observed incidence in oregon may have been a result of greater case ascertainment because hospital discharge data were used rather than physician and laboratory reports. nevertheless, because of our study design, we probably underestimated the true number of incident cases. persons in outpatient clinics and emergency departments in whom neurocysticercosis had been diagnosed (who did not become hospitalized) were not counted in our study. we also did not include in the analysis of cases 15% of hospital discharge diagnoses that we could not confirm by chart review ; if these had been confirmed as neurocysticercosis, then the true number of new cases may have been even higher. although the hispanic population in oregon grew by an estimated 67% during the study period (2,3), the number of hospitalizations and new diagnoses did not increase. whether this finding represents a true decrease in incidence or a shift in diagnosis and management of these cases to the outpatient setting, is unclear. neurocysticercosis has not previously been a reportable condition in oregon, and no public health followup of the patients with locally acquired cases was performed to determine the source of these persons exposures to taenia eggs. previous serologic studies (11,12) would suggest that these patients acquired disease from household contact with a t. solium carrier. early identification and public health followup of neurocysticercosis patients may lead to the recognition and treatment of tapeworm carriers, thereby preventing additional cases. however, since excretion of taenia eggs is intermittent, direct parasitologic examination of stools is not a sensitive test (11). a serologic test for detection of taenaisis has been developed (13), but it is not yet commercially available. public health providers need simple, inexpensive, and readily available techniques for rapidly identifying taenia carriers in order to conduct optimal followup of cases and prevent additional cases. the world health organization has estimated that more than 2 million persons are infected with adult tapeworms (14). as a result of increasing immigration and foreign travel, t. solium will likely continue to emerge as an important pathogen in the united states. we recommend that public health surveillance activities more accurately define the incidence and risk factors for illness in oregon, allow identification and treatment of tapeworm carriers, and provide epidemiologic and clinical data to physicians caring for patients in at - risk populations. | the unexpected death of a teenager from neurocysticercosis prompted an investigation of this disease in oregon. we found 89 hospitalizations, 43 newly diagnosed cases, and 6 deaths from 1995 to 2000. at least five cases occurred in persons who had not traveled or lived outside the united states. enhanced surveillance for neurocysticercosis is warranted. |
pact suicides are those wherein two or more individuals mutually agree and execute to terminate their lives together. the usual profile of persons involved in pact suicides includes those females, older, married and of high social class. if one relies on the media reports, whenever two or more deaths occur together, one of whom is a child ; it is usually considered as a homicide - suicide without the consent of the child. for establishing that the suicides were an outcome of suicide pact, objective evidence in the form of a suicide note is required to establish that the deaths were an outcome of suicide pact. in indian context, pact suicides generally involve issueless couples, disappointed lovers, unmarried sisters, frustrated individuals or a woman ending her own life and that of her children due to family circumstances or financial difficulties. this usually occurs in the religious, political or military reasons. in our literature search, we did not come across a pact suicide involving four persons (quadruple pact suicide). in this report, we describe a family (of four persons, three of whom were adolescents), which entered into a pact suicide due to the social reasons. a man and his three children were brought to the emergency department by their neighbors. he and two of the children had allegedly consumed celphos (aluminum phosphide) tablets, which is a commonly used insecticide in this part of india. the father was found to have respiratory distress and had to be immediately intubated and ventilated. detail information was obtained from the neighbors who had brought them and the third child. he was managing a nuclear family with his wife and three children of ages 14 (girl), 12 (boy) and 11 (boy) years. two days prior to the alleged suicidal attempt, the man came to know about the extra - marital relationship of his wife. according to the informants, the fact that she was having and extra - marital relationship was true and the extra - marital relationship was continuing for a long time prior to patient coming to know about the same. due to extreme distress, man discussed the issue of humiliation with his children, who all agreed that they were also having the same distressing emotions and did not know how to face the society. following which the man suggested to his children that, it would be best to end life to escape the humiliation from the society. as per the information provided by the child who had escaped with only minor head injury, all of them agreed to end their life and signed a suicide pact, for the same. the following morning, the man procured eight tablets of aluminum phosphide from a local vendor. after ensuring that the house was securely closed, the man handed over two tablets of aluminum phosphide to each of his three children. the two elder children consumed the tablets, but as the turn of the youngest son 's came, he tried to run toward a window to escape. to prevent him from escaping the man threw a blunt object, which hit the youngest son on his head, but he managed to escape and raised alarm for help. within minutes, several villagers rushed toward their home and when they managed to break open, they reportedly found the man and the two children vomiting profusely. they were initially shifted to a local primary care health center and gastric lavage was done. however, the physical status of the man and the two elder children worsened, which led to patients being shifted to our emergency. besides intubating and providing ventilatory support the man could not be saved and he succumbed to the aluminum phosphide toxicity. the youngest child, who described the whole event, did not have any evidence of intracranial bleeding on the computerized tomography of the brain. the description of pact suicide involving an adult and a child usually describe the involvement of a mother and her child, in contrast in the present case the pact suicide involved father and children. in the majority of the pact suicides, the two persons who enter into a pact to end their life do so to avoid separation from each other because of love and closeness among themselves. in contrast, in the case described by us, the people who entered the pact did the same so as to avoid social humiliation. further, this case suggests that many a time 's children do enter into a pact to end their life impulsively without understanding the consequences. this was highlighted by the behavior of the youngest child in our report, who initially agreed to end his life along with other family members, but later tried to escape when he realized the consequences. this fact reflects that adolescents are very vulnerable and many of them may be suggestible to execute the act of self - harm. another unique situation, which was seen in this case, was that three of the persons who entered the pact survived and only one lost his life. occasional reports involving pact suicide have given similar descriptions of one of the survivors who is usually a co - operator, rather than the instigator. in the present report too, the pact suicide was preceded by a psychosocial stressor, in otherwise well - adjusted individual as has been observed in many of the previous reports from india. in contrast to the commonly described cases of mass suicide, which involves many people for religious, political or military reasons, the current case highlights quadruple pact suicide following a social stressor. from a legal standpoint, the case suggests that the children were influenced to sign on to the suicide note and commit suicide. the mere act of attempting to commit suicide is punishable as per the indian penal code (section 309). however, the survivors being minors may absolve the children of responsibility and hence a trial. otherwise too, | pact suicides involving families have been reported in the scientific literature, but reports have been few from india. we report the case of a family, in which the father and three children had entered into a suicidal pact and executed it due to social reasons. a 41-year - old man, with no past psychiatric or substance use history, had reportedly come to know that his wife had been involved in an extra - marital affair. as expressed by him in a suicide note, he could not bear the humiliation due to this and also did not want his children to face disrespect from the society. he along with his daughter and 2 sons, aged 14, 12 and 11 years respectively, thus entered into a suicide pact to end their lives and wrote a suicide note. man and two of his children consumed aluminum phosphide. however, the youngest son did not consume the poison and raised alarm, following which they were rushed for medical care. the father died, but the three children recovered completely. the case highlights the rare phenomenon of suicide pacts involving an adult and children. |
to report a case of corneal perforation associated with oral administration of erlotinib and its spontaneous healing after temporary discontinuation of drug treatment. a 65-year - old man with metastatic lung cancer was treated with erlotinib (150 mg / day), a specific tyrosine kinase inhibitor of the epidermal growth factor receptor. he was referred to our corneal service for the treatment of bilateral corneal disorders, diagnosed with mild aqueous - deficient dry eye, and treated by insertion of punctal plugs. his corneal epithelial disorders initially improved, but subsequently worsened, as manifested by the development of bilateral corneal ulceration with corneal perforation in the right eye. the oral administration of erlotinib was interrupted in preparation for tectonic keratoplasty, but 2 days later the corneal perforation of the right eye and the bilateral epithelial defects had healed spontaneously. treatment with erlotinib was resumed at half the initial dose, and the cornea of both eyes has remained apparently healthy. the development of corneal epithelial disorders in patients receiving this drug may be reversed by reducing its dose. a 65-year - old japanese man was diagnosed in april 2010 with stage iv lung adenocarcinoma, manifesting multiple metastases in the lung, adrenal gland and brain. he received 31 gy of basicranial radiation for the brain metastases. given that he was found to harbor an epidermal growth factor receptor (egfr) mutation, he was also treated with the egfr - specific tyrosine kinase inhibitor (tki) gefitinib (250 mg / day). as a result of the development of adverse effects including fatigue, dermatitis, and liver dysfunction, however, the dose of gefitinib was initially reduced by half and treatment with the drug was subsequently discontinued after a total of 2 months. computed tomography and magnetic resonance imaging revealed that the size of the original lesion and the number of brain metastases had increased. treatment with the combination of cisplatin (125 mg) and pemetrexed (0.75 mg) was initiated. after 8 weeks, this regimen was switched to vinorelbine (32 mg) and docetaxel (80 mg) because of the development of adverse effects. this latter treatment was also not tolerated well, and so administration of the egfr - tki erlotinib (150 mg / day) was initiated., he developed a foreign body sensation in both eyes and visited his local ophthalmologist, who referred him to the corneal service at yamaguchi university hospital for the treatment of corneal epithelial disorders with corneal thinning. corneal ulceration on his right eye was also observed, but no signs of infection or inflammation were apparent. schirmer 's test detected bilateral mild hypolacrimation (5 mm), and his corneal and conjunctival epithelial disorders were thus attributed to aqueous - deficient dry eye. three weeks after plug insertion, his corneal and conjunctival epithelial disorders had apparently improved. however, 2 months after his first visit to our clinic, he was referred to us again for the treatment of bilateral corneal ulcers, which were confirmed by slitlamp microscopy (fig. slight stromal edema, ulceration with an opaque epithelium, and a shallow anterior chamber were also observed in the right eye. the next day, the anterior chamber of the right eye was flat, and so we treated this eye with a bandage soft contact lens. the right anterior chamber remained flat, however, and the patient was diagnosed with noninfectious corneal perforation of unknown cause and was prepared for tectonic keratoplasty. the oral administration of erlotinib was interrupted in preparation for general anesthesia, and the patient was treated with gatifloxacin eyedrops only. two days later, the anterior chamber of his right eye had spontaneously reformed and the epithelial defects of both eyes had healed (fig. the keratoplasty was therefore canceled and we monitored the patient carefully, but his corneal condition remained stable. treatment with erlotinib at half the original dose was reinitiated and both eyes were maintained by the administration of artificial tears, with the punctal plugs remaining in place. we present the case of corneal perforation that underwent spontaneous healing after a temporary suspension of the oral administration of erlotinib for the treatment of metastatic lung cancer. the clinical course of this case suggests that erlotinib might be secreted into tear fluid and thereby affect the condition of the corneal epithelium. the clinical course of the present patient was characterized by the development of corneal epithelial disorders after the onset of erlotinib treatment, the temporary improvement of these disorders after the insertion of punctal plugs for treatment of dry eye, the subsequent development of corneal ulceration, the spontaneous healing of corneal perforation, and corneal epithelial disorders after cessation of erlotinib administration as well as the maintenance of a healthy cornea after the resumption of erlotinib at a reduced dose. this course suggested that erlotinib may have been secreted into the tear fluid of the patient and thereby adversely affected the corneal epithelium, and that punctal plug insertion may have increased the concentration of erlotinib in the tear fluid and eventually worsened its effects on the ocular surface. the treatment of dry eye by pooling of tear fluid may thus have exacerbated the clinical course in this case. administration of artificial tears, which would remove secreted drugs from the ocular surface, may thus be preferable to punctal plug insertion for the amelioration of dry eye in patients treated with egfr - tkis. the dose of the latter drugs may need to be reduced in patients who develop corneal epithelial disorders. the development of corneal perforation in the present patient was likely attributable to erlotinib exposure, but the etiology of the condition is unclear. various proteinases released from bacteria, infiltrated neutrophils [2, 3 ], or resident keratocytes degrade collagen in the corneal stroma of patients with infectious corneal ulcer. however, we did not observe any clinical signs suggestive of corneal infection or inflammation in the present patient. it is thus possible that erlotinib or other factors in tear fluid stimulated the release of proteinases from resident keratocytes and thereby gave rise to degradation of stromal collagen secondary to the epithelial defects. previous studies have described corneal disorders in patients treated with erlotinib, including a case of corneal perforation that required treatment with tectonic keratoplasty, a case of corneal melting that was successfully treated with autologous serum eyedrops, and a case of persistent epithelial defects. laboratory investigations [8, 9 ] have also suggested that inhibition of egfr function by drugs such as erlotinib may adversely affect the corneal epithelium. the recent introduction of egfr - tkis for the treatment of various types of cancer might be expected to result in an increase in the number of patients with corneal disorders induced by these drugs. ts-1, a combination of tegafur, gimeracil, and oteracil potassium, is widely administered for the treatment of various types of cancer in japan. the widespread application of this drug has resulted in the development of lacrimal duct dysfunction or corneal epithelial disorders in many patients. these various observations and the present case therefore suggest that individuals treated with certain anticancer drugs should be monitored for the development of ocular surface disorders. | purposeto report a case of corneal perforation associated with oral administration of erlotinib and its spontaneous healing after temporary discontinuation of drug treatment.case reporta 65-year - old man with metastatic lung cancer was treated with erlotinib (150 mg / day), a specific tyrosine kinase inhibitor of the epidermal growth factor receptor. he was referred to our corneal service for the treatment of bilateral corneal disorders, diagnosed with mild aqueous - deficient dry eye, and treated by insertion of punctal plugs. his corneal epithelial disorders initially improved, but subsequently worsened, as manifested by the development of bilateral corneal ulceration with corneal perforation in the right eye. the oral administration of erlotinib was interrupted in preparation for tectonic keratoplasty, but 2 days later the corneal perforation of the right eye and the bilateral epithelial defects had healed spontaneously. treatment with erlotinib was resumed at half the initial dose, and the cornea of both eyes has remained apparently healthy.discussionerlotinib may be secreted into tear fluid and thereby adversely affect the corneal epithelium. the development of corneal epithelial disorders in patients receiving this drug may be reversed by reducing its dose. |
a 23-year - old healthy female was admitted to our hospital with a new - onset tender bulge in her left groin, associated with subjective fevers and chills. four days prior to presentation, she noted a sudden protrusion in her left groin with associated pain. given worsening pain and intermittent nausea, she presented to our emergency room. on admission, physical examination revealed a soft, non - distended abdomen that was tender to palpation in the left lower quadrant. a soft, mobile, exquisitely tender, and non - reducible mass laboratory data were notable for a leukocyte count of 6.7, a hematocrit of 36.9 and a lactate of 1.4 in the setting of normal electrolytes. an outside ultrasound showed an avascular complex cystic lesion measuring 2.1 cm in the left groin. a subsequent computed tomography scan of the abdomen and pelvis revealed a tubular cystic structure along the left inguinal canal, coursing along the expected path of the round ligament, without surrounding fat stranding or herniation of bowel loops. given her symptoms and the unclear etiology of the hernia, she was admitted to the hospital and taken to the operating room for exploration (fig. 1). in the operating room, a 6-cm dark purple cystic structure was visualized within the sac of an indirect inguinal hernia, travelling along the round ligament down toward the mons pubis (fig. 2). the cystic structure was dissected away from the round ligament and the cyst was opened and noted to contain clear liquid. ultimately, the patient underwent left indirect inguinal hernia repair, with gore bio a absorbable mesh, and excision of a left inguinal hydrocele of the round ligament. final pathology revealed fibromuscular tissue lined by mesothelium consistent with a hernia sac and a hydrocele with concordant endometriosis. the patient 's post - operative course was uneventful and she was discharged home on post - operative day 1. figure 2:purple cystic structure within the sac of an indirect inguinal hernia, travelling along the round ligament down towards the mons pubis. left indirect inguinal hernia. purple cystic structure within the sac of an indirect inguinal hernia, travelling along the round ligament down towards the mons pubis. endometriosis is defined as the presence of ectopic endometrial glands and stroma at extrauterine sites. it is a relatively common, benign, estrogen - dependent disorder that affects 10% of women of reproductive age. it is characterized by chronic pain, severe dysmenorrhea and infertility, although the disease may be asymptomatic or discovered incidentally. endometriosis can affect almost any organ system, although most ectopic implants are located in the pelvis. extrapelvic endometriosis is a rare disease entity that may cause a variety of unusual symptoms, including pain and physiologic dysfunction, dependent on the location and size of lesions as well as the organ system affected. it has been described at multiple sites including the gastrointestinal tract, urinary tract, thoracic cavity, central nervous system, umbilicus, skin (including abdominal wall incisions and episiotomy scars) and inguinal canal. inguinal endometriosis is a condition characterized by the presence of endometrial stroma and glands in the extraperitoneal portion of the round ligament and in the surrounding connective and lymphatic tissues. first described in 1986, it is an extremely rare affliction with < 60 cases reported in the literature. typically, patients are in their reproductive years, with a peak incidence between 3040 years, and present with tender inguinal swelling. inguinal endometriosis is difficult to recognize and thus often confused with more common conditions such as hernias, lymphadenopathy, granulomas, soft tissue tumors, cysts and hydroceles. catamenial symptoms, such as variation in size and tenderness of the mass, are present in 50% of cases and should raise the index of suspicion for inguinal endometriosis. in many cases, the patient is brought to the operating room with a preoperative diagnosis of incarcerated hernia and the endometriosis is discovered incidentally or on histologic examination. in over 90% of cases, inguinal endometriosis occurs on the right side. the endometriosis most often affects the extraperitoneal portion of the round ligament and is rarely found in association with occult hernias. however, in a minority of cases, inguinal endometriosis can coincide with inguinal hernias and other groin pathology, increasing the diagnostic difficulty. in one series, 37% of patients suffered from inguinal endometriosis associated with a groin hernia. the presence of isolated endometriosis contained within a left - sided inguinal hernia sac has, to our knowledge, never been reported. in all but two cases reported in the literature, or 91% of cases, the treatment of choice for both inguinal hernias and inguinal endometriosis is surgical excision. in order to be locally curative, surgical excision must include the removal of the extraperitoneal portion of the round ligament. given the high concordance of pelvic endometriosis, concurrent diagnostic laparoscopy or post - operative referral to a gynecologist is indicated to evaluate for other lesions and the extent of endometriosis. in some cases, there may be a role for hormonal suppression with oral contraceptives or gnrh agonists. our patient underwent complete excision of a left inguinal hydrocele of the round ligament and repair of left indirect inguinal hernia with mesh. she was started on hormonal therapy with an oral contraceptive and subsequently referred to a gynecologist for subsequent evaluation of her newly documented endometriosis. all authors were members of the clinical team who saw the patient and treated the patient at the beth israel deaconess medical center. the case report was written in large part by k.a. with contributions from all other authors. | endometriosis is a common gynecologic disorder wherein ectopic endometrial glands and stroma are found at extrauterine sites. extrapelvic endometriosis is a well - documented, yet rare, disease entity that can affect almost any organ system. inguinal endometriosis is an extremely rare disease entity characterized by tender inguinal swelling. here we report a case of a sudden - onset and acutely painful left inguinal hernia with concordant endometriosis. a review of the literature is presented. the presence of isolated endometriosis contained within a left - sided inguinal hernia sac has, to our knowledge, never been reported. often diagnosed incidentally or on histologic examination, general surgeons should consider inguinal endometriosis in the differential diagnosis of inguinal masses, even in the absence of catamenial symptoms. surgical excision, with gynecologic follow - up, is locally curative and the treatment of choice for inguinal endometriosis. |
cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. to this end, three applications of epithelial cells cultured with 3t3 feeder layer support are described. first, treatment of the premalignant human epidermal keratinocyte line scc-12f2 with the tumor promoter 12-o - tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. this agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nm, but much less above 100 nm. expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nm. second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. the two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. great differences were evident even among those cells derived from stratified squamous epithelia (epidermal, esophageal, vaginal, forestomach) despite their expression of aryl hydrocarbon hydroxylase activities to similar degrees. finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the camp - elevating agent forskolin. maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. although expressing keratinocyte character (transglutaminase activity and envelope forming ability), the cells thus retain some hormonal character that may be modulated by camp - dependent kinase activity. pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.imagesfigure 1.figure 4. |
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lactic acid bacteria (lab) isolated from dairy products have received increased attention as a potential food preservative due to their antagonistic activity against many food born pathogen such as listeria monocytogenes. lab are widely distributed in the nature, they are typically involved in a large number of the spontaneous food fermentation, and they have been extensively studied. some members of lab produce bacteriocins and bacteriocins - like substances which may inhibit growth of spoilage and pathogenic microorganisms. bacteriocins from lab are bioactive peptides or proteins with antimicrobial activity toward gram positive bacteria, including closely related strains and/or spoilage and pathogenic bacteria. bacteriocins are ribosomaly synthesized and extracellulary released bioactive peptides or peptide complexes which have bactericidal or bacteriostatic effect. use of either the bacteriocins or the bacteriocin - producing lab like starter cultures for food preservation has received a special attention. moreover, bacteriocins are innocuous due to proteolytic degradation in the gastrointestinal tract [7, 8 ]. s. thermophilus is a lactic acid bacterium of major importance in food industry like the manufacture of yoghourt. some of s. thermophilus strains produce a bacteriocin named thermophilin which is active against several lab and food spoilage bacteria such as clostridium sporogenes. in view of its technological and biochemical properties some of other lab like enterococcus, lactococcus, and pediococcus are also widely used as natural preservatives, due to the potential production of metabolites with antimicrobial activity such as organic acids, hydrogen peroxide, antimicrobial enzymes and bacteriocins. the aim of the present study is to assess antimicrobial activity of some lactic acid bacteria strains isolated from traditional fermented dairy products prepared from raw milk, raib, which is obtained after spontaneous curdling of raw milk within 24 to 36 hours at ambient temperature. in addition, preliminary investigations on a bacteriocin produced by s. thermophilus strain isolated in this work will be presented. the bacterial strains used in this study were isolated from fermented traditional milk, raib, manufactured without starter cultures. samples were collected all over chlef regions and obtained with collaboration of bioressources research laboratory. lab were isolated from raib, by homogenizing 10 g samples of cheese in 90 ml saline solution and then plating suitable serial dilutions onto different media : bhi, mrs, and m-17 (biokar diagnostics, beauvais, france). the plates were incubated aerobically at 30c for 48 hours, and then several colonies were picked at random for identification. cell morphology and gram - staining reaction were examined by light microscopy, and the catalase activity was carried out. phenotypic identification was based upon physiological and biochemical characteristics ; sugar fermentation profile, in the api-20 strep ch and api-50 ch fermentation, was carried out according to the manufacturer 's instructions (biomerieux, marcy letoile, france). for detection of antagonistic activity, the agar spot test was a modification of that described by tom.. overnight cultures, on mrs medium, of the strains to be tested for production of antimicrobial compound were centrifuged (10 minutes at 15000 g, 4c). cell - free supernatants were filtered across cellulose acetate filter (0.2 m) to remove residual cells. an overnight culture (37c) of the target strain was diluted in sterile mueller - hinton medium, and 2 ml of ca 10 cfu ml were spread on solid mueller - hinton medium. after 5 minutes of contact, the excess was removed and the petri dishes were dried for 10 minutes. samples (10 l) of filtered cell - free supernatants were spotted on the agar plate. the target strains used in this study are bacillus cereus cip 6624, bacillus subtilis atcc 6633, escherichia coli cip 35218, enterococcus faecalis cip 29212, listeria innocua atcc 51742, salmonella typhimurium cip 5858, staphylococcus aureus cip 29213, and staphylococcus epidermitidis atcc 14990. the biochemical nature of the antibacterial agent was studied on both chloroform extract and cell - free supernatant ; all the samples were incubated for 1 hour at 37c before the antilisterial essay. the ph of cell - free supernatants was adjusted to 6.5 with naoh (1 n) and then treated with catalase (sigma ; 500 iu ml). the cell - free supernatant was also submitted to heat treatment (6095c) and to several ph (48). the chloroform extract was treated with -amylase (sigma ; 1 mg ml 100 mm phosphate buffer, ph 6.9), -chymotrypsin (sigma, 1 mg ml, 0.05 m tris hcl buffer (ph 8.0)0.01 m cacl2), pronase e (sigma ; 1 mg ml in 100 mm tris hcl buffer, ph3), proteinase k (sigma ; 1 mg ml in 100 mm tris hcl buffer, ph 7.5), and trypsin (sigma, 1 mg ml 50 mm tris hcl buffer ph 8.0). prior to being assayed for bacteriocin activity, preparations containing pronase e were adjusted to ph 6.0. neutralized cell - free supernatant neutralized cell - free supernatant treated with catalase, heat - treated supernatant, and chloroform extract were spotted against l. innocua. untreated bacteriocin plus buffer, bacteriocin plus buffer treated 5 minutes at 100c, buffer alone and enzymes solutions served as controls [13, 14 ]. growth experiments were performed in erlenmeyer flask of 500 ml containing 250 ml of mrs broth (ph 6.5) at 37c without shaking. an overnight pre - culture of s. thermophilus was used for the inoculation of the mrs broth at initial cell density of ca 10 cfu ml. at different time intervals, samples were removed from the culture and used for optical density measurement (660 nm), viable and cultivable count (cfu ml), extracellular ph measurements, and bacteriocin production. the bacteriocin concentration arbitrary unit ml (au ml) was calculated as the inverse of the strongest dilution which induces the inhibition of l. innocua. the experiments were repeated three times, and results are expressed as mean standard error to the mean. the extraction was realized from cell - free culture supernatant of s. thermophilus obtained after centrifugation of overnight culture (20 minutes at 15000 g at 4c). the culture supernatant (100 ml) was stirred vigorously for 20 minutes with chloroform (v / v) and transfer in separation funnel, the interface layer between the aqueous and organic phases, which contain bacteriocin, was harvested, and the residual chloroform was eliminated by speed vacuum (50 hours, unique, martinsried, germany). then bacteriocin activity was measured in the interface layer, aqueous and organic phases. the plasmid extraction was performed from a cell pellet of an overnight culture of s. thermophilus (250 ml) using miniprep spin kit together with the corresponding buffers purchased from qiagen (hilden, germany). s. thermophilus plasmidic dna analysis was performed by electrophoresis (1 hour, 100 v) using a 0.7% agarose gel dissolved in tris 45 mm ; borate 45 mm ; edta 1 mm ; ph 8. the electrophoresis gels were analyzed under uv using molecular imager gel doc system (bio - rad, hercules, usa). the conditions for bacteriocin isolation were realized, through analytical rp - hplc, on the chloroform extract. the liquid chromatographic system consisted of a waters 600 e automated gradient controller pump module, a waters wisp 717 automatic sampling device, and a waters 996 photodiode array detector. all of the chromatographic processes were performed on an uptisphere c18 column (150 mm 4.6 mm, up5odb615qs, interchim, montluon, france). the mobile phase was water / trifluoroacetic acid (1000 : 1, v / v) as eluent a and acetonitrile / trifluoroacetic acid (1000 : 1, v / v) as eluent b. the flow rate was 1 ml / min. the gradient applied was 050% (v / v) b over 100 minutes then 50%100% (v / v) b over 5 minutes and 15 minutes at 100% (v / v) b. online uv absorbance scans were performed between 200 and 300 nm at a rate of one spectrum per second with a resolution of 1.2 nm. twenty lab strains, isolated from algerian dairy milk (table 1), were screened for their antagonistic activity against listeria innocua, enterococcus faecalis, bacillus cereus, bacillus subtilis, staphylococcus aureus, staphylococcus epidermitidis, escherichia coli, and salmonella typhimurium. the results of table 2 show that six isolates were active against one or more tested strains. however, s. thermophilus t2 strain showed a wide inhibitory spectrum against all the gram positive target bacteria used in this study except against staphylococcus aureus (table 2). in addition, s. thermophilus t2 did not show any inhibitory activity against gram negative bacteria used in this study : escherichia coli and salmonella typhimurium. our results showed that the free - cell supernatant remained active, against sensitive target strains, even when the ph was adjusted to ph 7. however, when the cell - free supernatant and the chloroform extract were exposed to the proteolytic enzymes (table 3) no inhibitory activity was observed against listeria innocua by contrast to the control tests which showed an inhibitory activity against the target strain (table 3). in addition, when the cell - free supernatant and the chloroform extract were exposed to the action of -amylase and catalase similar inhibitory activity was measured when compared with the control test against l. innocua. these results suggest that the biochemical nature of the molecule produced by s. thermophilus is peptidic. thus, the inhibitory activity of the chloroform extract was still measured after a heat treatment of 30 minutes at 90c. our results showed also that in a range of ph 48 similar antibacterial activities of the chloroform extract were obtained against l. innocua. the extraction of the bacteriocin produced by s. thermophilus t2 strain from culture supernatant was realized with chloroform, a water - immiscible solvent. the method used concentrates the bacteriocin at the interface between chloroform and the aqueous culture of the producing bacterium. we demonstrated that no bacteriocin activity was detected in the solvent phase (data not shown). in addition, the precipitate at the interface between the chloroform and culture supernatant fluid contained most of the bacteriocin activity in the mixture. the precipitate at the interface was harvested, and the residual chloroform was eliminated by speed vacuum. after hplc reversed - phase chromatography, bacteriocin activity was associated with two peaks eluting at 17 minutes and 110 minutes (figure 3). these results showed that the antibacterial activity of s. thermophilus t2 could be associated with two molecules which present different hydrophobicity. growth and bacteriocin production of s. thermophilus was studied in mrs broth at 37c at ph 6.5. under these conditions bacteriocin activity was detected at 4 hours of incubation at the beginning of the exponential phase, at a cell concentration of ca 10 cfu ml (12 au ml). the results of figure 1 showed that bacteriocin production increases with the increase of cell concentration to reach a maximum of 90 au ml with a bacteriocin production rate of 9.3 (au ml) h. this concentration was reached between 12 and 14 hours of incubation at 37c. during the stationary phase both bacteriocin concentration and the cell concentration remained at a steady state (figure 1). antibacterial activity decreased after 24 hours of incubation after having reached maximum levels after 14 hours of incubation (data not shown). the genes encoding for bacteriocin are either chromosomic or plasmidic [18, 19 ]. the aim for this preliminary investigation is to assess the presence of plasmid in s. thermophilus cells. the analysis of the plasmidic dna extraction showed that s. thermophilus seems to have a single plasmid as shown in figure 2. in addition, drai fragmentation pattern of the plasmid resulted in three restriction fragments with approximately 2.2 kb, 1.5 kb, and 0.5 kb. this preliminary result is of importance since in many lactic acid bacteria bacteriocins and carbohydrate fermentation exopolysaccharide production and antiphage mechanisms are carried by the same plasmid as reported previously by martinez - bueno. and turgeon and moineau. moreover, the use of more than one lab bacteriocin as a combination of biopreservative may have major applications in improving food safety. in the present study, the inhibitory effect of the cell - free filtrates of each of the 20 isolates was evaluated. the biochemical nature of the antibacterial molecule produced by s. thermophilus t2 was studied in both the cell - free supernatant and the chloroform extract. our results showed that the molecule, produced by s. thermophilus, is peptidic since the antibacterial activity of the molecule was lost after digestion with proteolytic enzymes. however, the neutralization (ph 7) and addition of catalase or -amylase to the cell - free supernatant did not result in the loss of the antilisterial activity. our results showed also that the bacteriocin produced by s. thermophilus is heat stable (up to 30 minutes at 95c) ; these results are similar to what has been reported for thoenicin. in addition, the bacteriocin was stable over a wide ph range, this indicates that such bacteriocin may be useful in acidic as well as nonacidic food ; similar ph stability results have been reported for propionicin plg1. growth and bacteriocin production profiles showed that the maximal bacteriocin production was measured by the end of the late - log phase. the level of production remained at a steady state during the stationary phase ; similar results were obtained by ivanova. however, bacteriocin production decreases after 24 hours of incubation after having reached maximum levels after 14 hours. preliminary characterization of the bacteriocin produced by s. thermophilus t2 was realized in the present study. it was found that the bacteriocin inhibits closely related gram positive strains like listeria innocua and enterococcus faecalis. active substance from culture supernatant of s. thermophilus t2 was obtained according to the procedure described by burianek and yousef. chloroform was added to the cell - free supernatant in a separator funnel, the bacteriocin was concentrated at the interface between chloroform and the aqueous phase. recovery of bacteriocin by the chloroform extraction was 10-fold higher when compared with ammonium sulphate precipitation (data not shown). this study allowed to underline the presence of at least one plasmid, of 4.2 kb as reported for many strains of s. thermophilus. in conclusion, the study of autochthonous lab will help to select the best candidates for improving the microbiological safety of traditional food products such as raib and may increase their shelf life. such a collection could be used for construction of specific starter cultures for fermented food products. | in the present study, the antibacterial effect of 20 lactic acid bacteria isolates from a traditional cheese was investigated. 6 isolates showed antibacterial effect against gram positive bacteria. streptococcus thermophilus t2 strain showed the wide inhibitory spectrum against the gram positive bacteria. growth and bacteriocin production profiles showed that the maximal bacteriocin production, by s. thermophilus t2 cells, was measured by the end of the late - log phase (90 au ml1) with a bacteriocine production rate of 9.3 (au ml1) h1. in addition, our findings showed that the bacteriocin, produced by s. thermophilus t2, was stable over a wide ph range (48) ; this indicates that such bacteriocin may be useful in acidic as well as nonacidic food. this preliminarily work shows the potential application of autochthonous lactic acid bacteria to improve safety of traditional fermented food. |
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