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heart failure (hf) has a high incidence, is one of the major causes of mortality from cardiovascular diseases in the world and is a major health problem in society. hf is divided into heart failure with reduced ejection fraction (hfref) and heart failure with preserved ejection fraction (hfpef). in the literature, hfref and hfpef patients with systolic dysfunction can also exhibit diastolic dysfunction, particularly in late stage hf. ejection fraction is considered one of the strongest prognosistic factors that influences a poor outcome for hf patients. echocardiography is considered the gold standard for assessing diastolic or systolic dysfunction in patients with hf. however, an expert is required to conduct the examination and not all health facilities provide an echocardiography machine. thus, a simple device that can measure both systolic and diastolic function can help a physician determine the diagnosis. electrocardiogram (ecg) and x - ray examinations are inexpensive tools that are accessible in almost all primary health care settings. a normal or minor change in the ecg is consistent with a low likelihood of left ventricle dysfunction. the ecg is useful because it can serve as an initial investigative tool that physicians can use to determine the presence of systolic and diastolic dysfunction in patients with chronic hf, though it can not replace echocardiography. several scoring systems based on ecg findings have been studied to estimate left ventricular function. however, additional studies have suggested that scoring systems have limitations or are less accurate in estimating left ventricular function in patients with coronary heart disease [8, 9 ]. to our knowledge, no previous study has evaluated a scoring system based on ecg results to predict hfpef or hfref in patients with hf. we used a cross - sectional research design to determine a scoring system based on ecg findings to predict the type of hf (hfpef or hfref). the population consisted of patients with chronic hf that were hospitalized or seen as outpatients in dr. sardjito general hospital between april and july 2015 and in whom both echocardiography and ecg had been conducted. the inclusion criteria were patients with hf diagnosed based on esc (2012) or aha (2013) guidelines, the presence of sinus rhythm, age > 18 years and agreement to participate in the study. the exclusion criteria were patients with congenital heart disease, primary valve disease, massive pericardial effusion, patients with acute coronary syndrome, severe pulmonary disease (cor pulmonale, pneumothorax) or who had pacemakers. the independent variable in this study was the ecg findings in patients with chronic hf. subjects were enrolled in the cardiovascular clinic, the hospital ward, and the echocardiography clinic at dr. subjects who met the inclusion criteria were included in the study and were enrolled consecutively. twelve lead ecgs were obtained by a nurse or a cardiology resident with patients in the supine position at a speed of 25 mm / s. the ecgs were read by three expert physicians (who were blinded to the results of the echocardiogram). the ecg report included an evaluation of heart rhythm and rate, heart axis, presence or absence of chamber enlargement, intraventricular block, and st - t segment changes, and the duration of the qrs complex, qt and qtc intervals. the echocardiogram was supervised by a cardiologist and two expert examiners (who were blinded to the ecg result). echocardiography data included a determination of the ejection fraction (calculated by simpson s method) and the presence or absence of diastolic dysfunction. the criterion for a diagnosis of hfref and hfpef was an ejection fraction 40% and > 40%, respectively. bivariate analysis was used to analyze the relationship between the ecg results and hfref or hfpef. inter - variable bivariate analysis was analyzed with the chi - square test followed by fisher s test. further, variables in the bivariate analysis with a p 100 ms was predominantly found in patients with hfref compared to those with hfpef (81.1% vs. 18.9%), with values of 124 30.4 vs. 97.3 20.7 ms, respectively. murkofsky (1998) showed that a prolonged qrs duration (> 0.10 s) is a very specific, though not a very sensitive, indicator of left ventricular systolic function. a qrs duration of > 0.10 s has a high likelihood of being associated with an ejection fraction 55 mm compared to a posterior wall thickness > 12 mm. this observation explains the strong association between strain pattern and ventricular hypertrophy with eccentric vs. concentric remodeling. the average qtc interval in patients with hfref was also longer than that of patients with hfpef (499 50.9 vs. 453.2 42.8 ms ; p = 0.000). wilcox (2011) showed that patients with grade ii or iii diastolic dysfunction have longer qtc intervals compared to patients with non - diastolic or grade i diastolic dysfunction (qtc 461 34 vs. 432 32 ms ; p = 0.0003). in our study, most of the patients with hfref had grade iii diastolic dysfunction (restrictive type) (48.3%), while 78% of patients with hfpef had grade i diastolic dysfunction (relaxation type). a limitation of our study is variable duration of hf therapy for each patient, though it is difficult to determine the impact this would have on our results. our study suggests that a scoring system based on ecg findings that include the presence or absence of lah, a wide qrs duration, rbbb, st - t segment changes and a prolonged extended qtc interval can be used to predict the type of hf (hfpef and hfref) in patients with chronic hf. this scoring system has a sensitivity of 76%, a specificity of 96%, a positive predictive value of 95%, a negative predictive value of 80% and an accuracy of 86%. a score of -1 to + 3 suggests the possibility of hfpef, while a score of + 4 to + 6 suggests the possibility of hfref. a limitation of our study is variable duration of hf therapy for each patient, though it is difficult to determine the impact this would have on our results. our study suggests that a scoring system based on ecg findings that include the presence or absence of lah, a wide qrs duration, rbbb, st - t segment changes and a prolonged extended qtc interval can be used to predict the type of hf (hfpef and hfref) in patients with chronic hf. this scoring system has a sensitivity of 76%, a specificity of 96%, a positive predictive value of 95%, a negative predictive value of 80% and an accuracy of 86%. a score of -1 to + 3 suggests the possibility of hfpef, while a score of + 4 to + 6 suggests the possibility of hfref.	backgroundheart failure (hf) is divided into heart failure with reduced ejection fraction (hfref) and heart failure with preserved ejection fraction (hfpef). mortality from hf is inversely related to left ventricular function. additional studies are required to distinguish between these two types of hf. a previous study showed that hfref is less likely when electrocardiogram (ecg) findings are normal. this study aims to create a scoring system based on ecg findings that will predict the type of hf.methodswe performed a cross - sectional study analyzing ecg and echocardiographic data from 110 subjects with chronic hf. hfref was defined as an ejection fraction 40%.resultsfifty people were diagnosed with hfpef and 60 people suffered from hfref. multiple logistic regression analysis revealed certain ecg variables that were independent predictors of hfref, i.e., left atrial hypertrophy (lah), qrs duration > 100 ms, right bundle branch block (rbbb), st - t segment changes and prolongation of the qt interval. based on receiver operating characteristic (roc) curve analysis, we obtained a score for hfpef of -1 to + 3, while hfref had a score of + 4 to + 6 with 76% sensitivity, 96% specificity, a 95% positive predictive value, an 80% negative predictive value and an accuracy of 86%.conclusionsthe scoring system derived from this study, including the presence or absence of lah, qrs duration > 100 ms, rbbb, st - t segment changes and prolongation of the qt interval can be used to predict the type of hf with satisfactory sensitivity and specificity.
first described by gleason in 1903, orbital myositis (om) or orbital pseudotumor is a benign idiopathic inflammatory disease that may affect any structure in the orbit. ocular myositis is now seen as a subgroup within the entity of idiopathic orbital inflammatory syndrome (iois) and it just represent 8% of all idiopathic orbital inflammations. ocular myositis affects mostly females (2:1 females to males ratio) around 32 years old but iois has been reported at any age.while orbital myositis is an inflammation of any orbital structure, ocular myositis describes a rare inflammatory disorder of extraocular eye muscles but the two have a lot of over lapses and so are hard to distinguish. most cases of om have subacute orbital painful diplopia witch is caused by handicapped contraction and distraction of affected eye muscles not by neurogenic associated affections. pain can be exacerbated by eye movement. in most of the patients, beyond orbital discomfort, two major forms of ocular myositis have been described, one with few ocular symptoms and with an additional conjunctival injections called limited oligosymptomatic ocular myositis (loom) and a second form with a severe exophthalmic ocular involvement with additional ptosis, chemosis, and proptosis, called severe exophthalmic ocular myositis (seom). also signs and symptoms of other subgroups of iois such as dacryoadentitis, periscleritis and perineuritis many inflammatory, vascular, neoplastic or infectious disease can affect the extra - ocular muscles and mimic orbital myositis. muscle biopsies of eoms have reporting mixed infiltrates of plasma cells, lymphocytes, macrophages and polymorphonuclear cells while chronic forms of idiopathic ocular myositis are associated with fibrosis of the affected muscles. specific myositis either by bacterial or viral infections can be associated to orbital myositis while others can be a local manifestation of a systemic immunomediated disease. while specific forms of ocular myositis will be resolved with specific systemic treatment, idiopathic ocular mysositis is treated with corticosteroids or with radiotherapy. we present the case of a 43 years old caucasian male, ex - smoker (15pa), stopped 6 years ago, was admitted in our hospital in september 2012 for a history of recurrent palsy of left abductor nerve with left gaze diplopia. these symptoms began in march 2012 were treated first by his general practitioner with non - steroidal anti - inflammatory drug and b complex vitamins for 10 days with no results. the patient had a history of arterial hypertension, ischemic cardiomiopathy, dyslipidemia, rhinitis, maxilar sinusistis and he was treated with calcium blocker channels, indapamidum and statin. physical examination revealed a overweight patient (mc=26 kg / m2), horizontal gaze diplopia, left nerve vi paresis, mild left retroorbital pain, no dizziness, no nystagmus, no motor deficite, no difficult in swalloing liquids or solids, normal osteotendinous reflexes, bilateral plantar flexion, normal language. blood pressure was 120/60mmhg, pulse was 74 bpm, respiratory rate was 18 breaths / minute, oxygen saturation was 99% while breathing ambient air ; the rest of the examination were in normal limits. at this point several diagnoses were taken into consideration, such as thyroid disease, ocular myasthenia gravis, infection, ocular - faringial syndrome and ocular myositis. results of laboratory tests revealed a moderate hypercholesterolemia (237mg / dl) and hypertryglideridemia (147mg / dl) ; the rest of the results were in normal limits. the patient was tested for different pathologies in parallel as following : tsh=0,4mclu / ml, ft4=10,6pmol / l, t3=1,01ng / ml, igm antibodys for lymme were 2.3au / ml, igg for borellia were less than 5 au / ml, anti musk antibodys were less than 5 nmol / l, anti - acetylcholine receptor (achr) antibody less than 0,1 nmol / l,, hba1c=5%, anca, anticardiolipin, anti - dsdna, hiv were all negative. the chest x - ray showed no active pulmonary lesions, so it did nt raised the suspicion of sarcoidosis or malignancy. the otorhinolaryngology exam revealed signs of maxillar chronic sinusitis, chronic amigdalistis, deviated nasal septum and chronic rhinitis. the ophthalmology consult observed just edema in the left orbit, with no other modifications. the orbital mri also did nt offer new information : mild external edema on the left eye, with normal tendon aspect, no thickening or enhancement of the muscle belly and also normal aspect of the bony orbit (fig.1). cerebral angiography : normal aspect at injection in both internal and external carotid artery (fig.2). also functional testing through visual evoked potentials using a patter - reversal stimuli was normal (fig.3). orbital mri showed for both t2 (a) and t1 (b) mild external edema on the left eye, with normal tendon aspect, no thickening or enhancement of the muscle belly and also normal aspect of the bony orbit (c). normal left eye visual evoked potential according with the standard of the lab. given these results a diagnose of ocular myositis was made without having the biopsy confirmation. corticosteroid treatment was reinitiated with high doses as 80mg / day of methylprednisolonum for 1 month with a slow decrease in dosage. all the fallow - up evaluation found the patient struggling between asymptomatic periods and diplopia with left nerve vi paresis periods, according to the corticosteroid dosage. it is not precisely known why extraocular muscles (eoms) are preferentially affected by inflammation in iois. eoms have a unique myofibrillar protein isoform composition reflecting their structural and functional properties. compared with skeletal muscle, eoms have smaller motor unit sizes, higher motor neuron discharge rates, higher blood flow with higher mitochondrial volume fractions which can suggest that the energy requests and susceptibility to mitochondrial dysfunction are higher as compared with skeletal muscle. due to the high blood flow and vascularisation, inflammatory blood cells can access more easily this specialized body compartment [2, 10 ]. thyroid orbitopathy is the most common cause of orbital disease with a insidious onset is rather than acute leading to lid lag and lid retraction in down - gaze which is characteristic for this disorder. usually patients will have bilateral eye involvement with signs of inflammation like edema leading to secondary fibrosis and in a few cases to optic neuropathy. because 50%80% of myasthenia gravis initial manifestation can be an isolated eye muscle weakness with blepharoptosis or ophthalmoparesis this diagnostic has to be taken in consideration in all patients presenting with diplopia. usually asymmetric ptosis is frequent associated with myasthenia gravis and up to 60% of the patients have acetylcholine receptor antibodies present. other pathologies can be taken in consideration in this patient, some can be excluded by carful conducted history interview like tolosa - hunt syndrome, while others need specific tests to confirm (oculopharyngeal muscular dystrophy, myotonic dystrophy type 1, congenital cranial dysinnervation disorders and congenital fibrosis of extraocular muscles and mitochondrial myopathies). although not an often pathology, when dealing with a patient with recurrent palsy of eoms, ocular myositis must be always be taken in consideration.	we report the case of a 43 year old man who presented recurrent left abducens palsy. his medical history included arterial hypertension, ischemic cardiomiopathy, dyslipidemia, rhinitis, maxillary sinusitis. physical examination revealed a overweight patient, horizontal gaze diplopia, left nerve vi paresis, mild left retro - orbital pain. the orbital mri also did nt offer new information : mild external edema on the left eye, with normal tendon aspect, no thickening or enhancement of the muscle belly and also normal aspect of the bony orbit. recurrent palsy of eoms can be caused in rare cases by ocular myositis.
the nuclear envelope (ne) is a biostructure, which separates the nuclear and cytoplasmic parts of eukaryotic cells. the ne is a dynamic structure composed of the outer nuclear membrane (onm) and the inner nuclear membrane (inm). the ne is embedded with nuclear pore proteins, through which molecules selectively pass to move between the nucleus and the cytoplasm. the nuclear membranes are bilipid structures supported by a network of proteins. within the nucleus, the inm is underlined by the nuclear lamina, a dynamic filamentous protein network. the nuclear lamina is predominantly composed of lamins, which are dynamically anchored to the inm via posttranslational modifications. the molecular composition of the nuclear lamina has been previously discussed in several reviews [1, 2 ]. in brief, lamins are divided into a- and b - types (lmna and lmnb, resp.) and play a central role in the integrity of the nuclear lamina. both proteins bind to the chromatin at highly defined regions, creating a regulatory role for lamina - chromatic interaction. while lmnb is constitutively expressed, the expression of lmna is developmentally regulated, and expression levels differ between cell types. it has, therefore, been suggested that lmna also plays a regulatory role [1, 2 ]. silencing of lmnb causes dramatic changes in the lmna meshwork, while lmna downregulation has no effect on the structural organization of lmnb filaments. this suggests a crucial and indispensable role for lmnb in maintaining the structural integrity of the nuclear lamina. lamins are brought into the inm via a farnesyl group, which is attached to the c - terminus of the molecule by a posttranslational modification. the maturation step of the b - type lamins ends with a farnesylated molecule, while the mature lmna protein is generated after a reversible anchorage to the inm via the farnesylated group. a de novo mutation in the last intron - exon boundary in lmna causes removal of the 3 of exon 11 and a deletion of the c - terminus, including the farnesylation signal. as a result, farnesylation is irreversible, and the progerin protein predominantly attaches to the imn. progerin causes premature cell senescence, which is associated with genome - wide transcriptional changes and telomere shortening [6, 7 ]. progeria patients exhibit premature aging, starting in young children, only a few years old. progerin is also expressed during normal aging [6, 9 ] and during closure of the ductus arteriosus in neonates. in the closing ductus arteriosus the function of progerin in those cells is still obscure, and the mechanism regulating progerin expression in normal physiological situations remains to be studied. next to genetic conditions, a deformed nuclear structure is also found in senescent, aging, and apoptotic cells as well as during cell division [1113 ]. moreover, overexpression of lmna in cell culture also causes severe changes in the nuclear shape. in those cells, folding of the nuclear lamina the molecular mechanisms driving the changes in the nuclear lamina in senescent and aging cells are not fully understood. the changes in the nuclear lamina during cell division have, however, been studied extensively and are described in several reviews [1, 4, 16 ]. in this review, we will focus on the structural changes of the nuclear lamina that are associated with cell senescence and aging. in apoptotic cells, changes in the structure of the nuclear lamina in human mesenchymal stem cells (hmscs) precede dna fragmentation, the hallmark of cell apoptosis. this suggests that structural deformation of the nuclear lamina precedes irreversible degradation of lamins during caspase-8 induced apoptosis. changes in the shape of the nuclear lamina also precede well - known bioimaging markers for cell senescence [11, 13, 17 ]. this suggests that monitoring changes in the nuclear lamina organization could mark early changes in cellular function. architectural changes should, therefore, be investigated in living cells expressing lamins fused to fluorescent proteins. as high overexpression of lamins leads to structural deformation of the nuclear lamina, only cells with low expression level should be considered for analysis of the lamina structure., analysis can be also performed on three - dimensional reconstructions from immunofluorescence. those are, however, less robust compared with those obtained from living cells. the pixels associated with the nuclear lamina are then identified using automated image processing procedures. several studies reported the quantification of the shape of the cell nucleus in two dimensions [19, 20 ]. those analyses only account for the edge of the nucleus and do not provide an internal architectural description. recently, we developed an automatic method to detect pixels of the nuclear lamina from three - dimensional images. quantitative analysis of the lamina architecture was performed subsequently, resulting in a robust and reliable procedure to study cell populations. the nuclear shape can be described by curvature measures. in conditions where cells exhibit a deformed nuclear shape, as in senescent cells or disease situations, folding of the nuclear lamina is associated with high curvature values and often shows high fluorescence intensity values of lmna, indicating protein accumulation. in young and healthy cells, lmna is evenly distributed along the nuclear rim, and it accumulates mainly in the short edges of the nuclear surface. in aging or senescent cells, the main changes in the structure of the nuclear lamina are associated with an uneven distribution of intensity of lmna and folding along the structure (examples are depicted in figure 1(b)). regions with high intensity values are often surrounded by high curvature values (figure 1(b)), suggesting a biomechanical association where protein accumulation induces a mechanical pressure that causes bending of the structure. several approaches can be used to quantify the three - dimensional structure of the nuclear lamina. a simple method is quantifying the length - to - width ratio of nuclear objects. structural changes along the lamina would then not be detected, because only the overall length and width of the cell are measured. a more accurate description can be provided by curvature measures of the folding of the structure. the distribution of fluorescence intensity at the nuclear lamina can provide a quantitative description of the structure as well. the intensity distribution can be summarized by the mean value and the skewness of the intensity distribution. absolute curvature and intensity values can not be directly compared between cells ; the measured values should be normalized first. a cell - based accurate and robust description of the nuclear lamina can then be obtained by combining these features. with a linear classification method it is possible to determine whether cell populations can be classified based on the nuclear lamina structure. a comparison between different cell populations is possible, because the measures themselves are normalized. the result of this robust analysis demonstrated that young and aged cell populations can be discriminated based on mean intensity and curvature values of the nuclear lamina (figure 3). a confined accumulation of proteins is often a result of a decrease in protein mobility. using frap analyses, lmna at the nuclear envelop is more mobile compared to lmnb. in addition, the mobility of progerin is significantly reduced compared to the wild type lmna protein [22, 23 ]. triton treatment of cells disrupts the localization of soluble proteins, and only insoluble proteins and structures that are triton - resistant remain intact. in cells at low passage number, lmna enrichment at the nuclear envelope is significantly lower compared with cells at high passage number (figure 3). the enrichment in lmna accumulation at the nuclear envelope is significantly more pronounced after triton treatment (figure 2). this procedure suggests that lmna at the nuclear envelope is less mobile in senescent cells than in young cells. when zooming into the nuclear envelop with the frap procedure, lower lmna mobility was found at regions with high protein accumulation compared with low fluorescence intensity regions. as the high fluorescence regions are mainly localized at bent structures, this suggests that folding of the nuclear lamina is associated with local accumulation of lmna. quantitative analyses of the nuclear lamina show that the mean normalized intensity of lmna decreased from the young to the old and senescent cells. this indicates changes in the lmna distribution in the nuclear lamina during aging and senescence. changes in the average curvature between young cells and senescent and old cells are smaller compared to the intensity (figure 4). the average curvature increases, however, significantly more in apoptotic cells compared to young or senescent cells. since cell apoptosis is increased in a population of senescent cells, this suggests that a major increase in folding of the nuclear lamina is secondary to an increase in local protein accumulation. regulatory functions of the nuclear lamins have been widely studied, and a dynamic multifunctional role is emerging. lamins directly bind to the chromatin, and chromatin recruitment of the nuclear envelope alters gene expression. how changes in the structure of the lamina contribute to nuclear function in aging cells recent technical advances in monitoring and measuring the dynamics of the nuclear lamina structure [15, 24, 25 ] and lamina - chromatic dynamics on a single cell resolution will open new opportunities to further understand how the structure of the nuclear lamina contributes to regulation of nuclear function. based on the quantification of the biophysical features of the nuclear lamina, the following mechanical models are suggested : initially protein mobility decreases, leading to confined and localized lmna accumulation in the nuclear envelop. the local lmna accumulation could result in a mechanical pressure that subsequently results in bending of the structure. when structural pressure of the nuclear lamina would be too large to maintain, the smooth ellipsoidal shape would deform, and the nuclear envelope structure would form an irregular shape. advances in molecular imaging at the single cell level, in combination with high resolution studies of chromatin dynamics, enable the possibility to study how spatial changes of the nuclear lamina affect nuclear function in aging and senescent cells.	the nuclear lamina is the structural scaffold of the nuclear envelope that plays multiple regulatory roles in chromatin organization and gene expression as well as a structural role in nuclear stability. the lamina proteins, also referred to as lamins, determine nuclear lamina organization and define the nuclear shape and the structural integrity of the cell nucleus. in addition, lamins are connected with both nuclear and cytoplasmic structures forming a dynamic cellular structure whose shape changes upon external and internal signals. when bound to the nuclear lamina, the lamins are mobile, have an impact on the nuclear envelop structure, and may induce changes in their regulatory functions. changes in the nuclear lamina shape cause changes in cellular functions. a quantitative description of these structural changes could provide an unbiased description of changes in cellular function. in this review, we describe how changes in the nuclear lamina can be measured from three - dimensional images of lamins at the nuclear envelope, and we discuss how structural changes of the nuclear lamina can be used for cell classification.
a major innovation in endodontics has been the introduction of nickel - titanium (niti) alloy to manufacture root canal instruments, mainly due to the superelasticity of the niti alloy, which provides increased flexibility and allows the instruments to effectively follow the original path of the root canal.1 - 5 thermal treatment of the alloy is known to produce a better arrangement of the crystal structure, thereby leading to improved flexibility (superelastic behavior) as well as changes in the phase percentages (different grain structure) of the alloy, leading to improved resistance or plastic behavior. proprietary processes are highly influenced by temperature and time intervals, and each small change makes every manufacturing process unique.2 hyflex cm rotary instruments (coltene - whaledent, allstetten, switzerland) are made from a new type of niti wire, namely cm wire that has been subjected to proprietary thermomechanical processing. it is manufactured by a unique process that controls the material s memory, making the files extremely flexible but unlike conventional files, which exhibit a stress - induced phase transformation.6,7 micro - computed tomography (ct) has been shown to be accurate for experimental endodontology and allows nondestructive quantitative analyses of different variables such as volume, surface area, cross - sectional shape, taper, and the proportion of the prepared surface.8 - 10 the manufacturer reports that the shape and strength of files with straightened spirals can be restored during autoclaving,11 meaning that the files appear to regain their shape after sterilization and reuse. the purpose of this study was to analyze the surface defects of hyflex instruments after use and to use ct to determine their ability to return to their original shape after autoclaving.11 twenty - four new hyflex files with different size and taper (0.8/25, 0.4/20, 0.4/25, 0.6/20, 0.4/30, 0.4/40) were scanned before use, after use, and after autoclaving. before the initial scans were performed, a customized jig was fabricated, and each file was mounted such that it could be placed in the same position before and after use. the new files were scanned (figure 1) using a ct scanner (1172 scanner ; skyscan, kontich, belgium) at 100 kv and 100 a, with a resolution of 57 m and using an al + 0.5 mm thick aluminum filter and 54% beam - hardening reduction. the acquired images were reconstructed using nrecon software (skyscan, kontich, belgium), producing two - dimensional cross - sectional slices of the file structure. access cavities were prepared on 12 plastic teeth using # 2 and # 4 high - speed round carbide burs (dentsply - maillefer, ballaigues, switzerland). the working length was determined for each of the 24 canals, and a new hyflex file was used for each canal preparation (dentsply - maillefer, ballaigues, switzerland), using a handpiece at 500 rpm with a torque of 2.5 n / cm. the files were scanned (figure 2) after deformation using the same protocol as described above, ct analyzer software (skyscan, kontich, belgium) was used to obtain all variables. surface analysis was performed for each file to determine the changes in the files after deformation ; defects were recorded as unwinding and curving associated with unwinding (figure 2). these changes were categorized according to the severity as follows : severe : more than 2 flutes showed unwinding, with or without curving moderate : only 2 flutes showed unwinding, no curving minor : only one flute showed unwinding, no curving. file scanned image after use showed visible defects, combined defects (unwinding and curving). used files were wiped and placed in file blocks, to avoid any contact between instruments. all file blocks were placed in a sterilization wrap and steam sterilized at 134c/273f for 6 min and 18 min for inactivation. all files were scanned (figure 3) again using the same protocol as described above, and the initial and final scans of the files were superimposed using nrecon software (skyscan, kontich, belgium) to, illustrate the recovery of the files to their original shape. file scanned image after autoclaving (restored the original shape). data were analyzed by using chi - square and student test, where appropriate, in software (spss for windows 8 ; spss, chicago, il, usa). the t - test of student s newman keuls revealed a significant difference (p < 0.05) between unused and used files. almost all of the used files exhibited significant visible defects without fractures, including unwinding and curving associated with unwinding. the most frequently observed defect was unwinding of the spirals (100%), whereas curving associated with unwinding was infrequent and combined with unwinding, it was observed with 20% of the samples (figure 2). a chi - square test showed that the deformation (curve : p = 0.000 and unwinding : p = 0.011), incidence depended on the size of the files. the greatest frequency of curving occurred in the small files # 20/0.04 and # 20/0.06 ; the difference was statistically significant from all file sizes (p < 0.05). the majority of the files (79%) were able to restore the unwinding and regain their original shape after autoclaving (figure 3). among these, the files with minimum to moderate changes could be restored, whereas the files with severe changes (25%) could not be restored. the highest rate of unrestored files was observed in the small files # 20/0.04 and # 20/0.06 instruments, and the difference was statistically significant from all file sizes (p < 0.05). all files that exhibited changes at the tip were able to be restored after autoclaving. at the beginning of 2000, a series of studies12 - 15 found that changes in the transformation behavior via heat treatment were effective in increasing the flexibility of niti endodontic instruments. since then, heat - induced or heat - altering manipulations were used to influence or alter the properties of niti endodontic instruments. a series of proprietary thermomechanical processing procedures has been developed with the objective of producing se niti wire blanks that contain substantially stable martensite phase under clinical conditions.16 enhancements in these areas of material management have led to the development of the next generation of endodontic instruments. tn) is a novel niti alloy with flexible properties that was introduced in 2010. cm niti files have been manufactured using a special thermomechanical process that controls the memory of the material, making the files extremely flexible but without the shape memory of other niti. cm niti files are unlike conventional files that exhibit a stress - induced phase transformation ; instead, these files show behavior that is similar to what is termed martensitic - active or shape memory, which produces remarkable fatigue resistance.16 it was observed that most of the files showed defects that appeared as lengthening of the spirals due to the unwinding without fractures. this occurrence was explained by the manufacturer : these files respond to pressure, torque, and resistance with a lengthening of the spirals, and, therefore, increases the fracture resistance. this could be explained by the fact that instruments made from cm were nearly 300 - 800% more resistant to fatigue failure than instruments made from conventional niti wire with the same design.4,17 a majority (79%) of the files were able to be restored after sterilization, which supports the company s claim that a characteristic of hyflex is its ability to return back to the original shape after autoclaving. this is consistent with the view that niti files do not present the rebound effect after unloading, and their original shape is restored only after autoclaving.18 this phenomenon was explained in a previous study, which showed that instrument in the stable martensite phase is known for exhibiting the shape memory effect, where it can easily be deformed, yet they have the capacity to recover its shape with heating above the transformation temperature. the explanation for this phenomenon may be that heating transforms the metal temporarily into the austenitic phase, which makes it possible for the file to regain its original shape before cooling down again.4,17 although the exact thermomechanical treatment of cm wires remains unknown, the mechanical deformation behavior is closely related to the phase transformation temperature, which is sensitive to the material s thermomechanical history.19 most of the unrestored files were the small files size (20/0.04 or 20/0.06), this trend of the inability of small files to recover corroborated the findings of other studies, which showed a trend toward a high incidence of distortion and separation in small niti instruments.20 - 23 an analysis of used hyflex instruments indicated that most of the files showed visible plastic deformation such as unwinding and curving. in addition, a recent study by capar.24 revealed that the hyflex files had the highest fatigue resistance compared to protaper next and revo - s. furthermore, many studies showed the new cm - wire manufacturing process to produce niti rotary instruments that were more flexible and more resistant to cyclic fatigue than instruments produced by a traditional manufacturing process or a thermally treated niti alloy (m - wire).4,16 - 18 therefore, with the limitations of this study, it can be concluded that the shape of files with unwound spirals can be restored during autoclaving. this finding means that hyflex cm niti files appear to regain their shape after sterilization and reuse. however, the finding of this study suggest that hyflex size 20 files should be considered as single - use, disposable instruments because of the likelihood of being distorted. hyflex cm niti files appear to regain their shape after sterilization and can be reused, however, further studies are needed to determine the properties of these restored files.	background : the multiple uses of hyflex controlled memory (cm) rotary instruments (coltene - whaledent, allstetten, switzerland) have been recommended after sterilization. the purpose of this study was to analyze the surface defects on hyflex instruments after use and to determine their ability to return to their original shape after autoclaving.materials and methods : twenty - four new hyflex files were scanned using micro - computed tomography before the initial use, after use until defects were visible and after autoclaving. surface analysis was performed on each file to determine the changes in the files after deformation ; defects were recorded as unwinding and curving. these changes were categorized according to the severity, and the initial and final scans of the files were superimposed to illustrate the recovery of the files to their original shape. the data were analyzed using chi - square and t-test.results:the t - test of student s newman keuls revealed a significant difference (p < 0.05) between unused and used files, almost all files exhibited visible defects without fractures, including unwinding and curving associated with unwinding. the most frequently observed defect was unwinding of the spirals (100%), whereas curving associated with unwinding was infrequently observed. a chi - square test showed that the deformation (curve : p = 0.000, and unwinding : p = 0.011, incidence depended on the size of the files. the greatest frequency of curving occurred in the small files # 20/0.04 and # 20/0.06, the difference was statistically significant from all file sizes (p < 0.05). the majority of the files (79%) were able to restore the unwinding and regain their original shape after autoclaving. the highest rate of unrestored files was observed on # 20/0.04 and # 20/0.06 instruments, and the difference was statistically significant from all file sizes (p < 0.05).conclusion : hyflex cm nickel - titanium files appear to regain their original shape after sterilization.
central venous catheters (cvcs) are needed for monitoring patients in special conditions and as a route for parenteral nutrition. depending on the catheter type, cannulation is performed at different sites such as the jugular, subclavian, femoral, and brachial veins. complications associated with the cvcs are infection, failure to place the catheter, arterial puncture, improper catheter position, pneumothorax, hematoma, hemothorax, asystolic cardiac arrest of unknown etiology, and inferior vena cava (ivc) trauma. infection is the most common complication in subclavian vein route ; though arrhythmias and air embolism can occur. one of these extremely rare complications is intravascular loss of a guide wire, which is usually recognized immediately or sometimes with delay. here, we elucidate a case of retained guide wire during cannulation of the right external iliac vein, which was successfully treated with exploration of right iliac vessels. a 60-year - old woman presented to the emergency department with complaints of generalized abdominal pain, constipation, and fever. patient was hospitalized with primary diagnosis of intestinal obstruction. due to patient 's history of heart disease, the central venous catheterization was done for fluid resuscitation via the right internal jugular (ij) vein using the seldinger technique. the catheter was inserted by a 1-year surgical resident with no supervision by senior residents. the catheter tray was checked for the guide wire after the procedure which was missing. after the patient was stabilized, she underwent chest x - ray, plain abdominal radiography, and ultrasonography and the patient was admitted in surgical intensive care unit. the radiographs showed that the catheter had entered in the ivc via the right ij vein and then, had remained in the right iliac vein at the point of bifurcation into the internal and external iliac veins [figures 1 and 2 ]. based on the consultation with the vascular surgeons and radiologists, the patient underwent exploration of right iliac vein. the method was successful and the retained guide wire was removed from right external iliac vein. chest x - ray showing guide wire of catheter had entered in the inferior vena cava (ivc) via the right internal jugular vein x - ray pelvis showing guide wire catheter had remained in the right iliac vein at the point of bifurcation into the internal and external iliac veins central venous catheterization has many complications such as infection, failure to place the catheter, arterial puncture, improper catheter position, pneumothorax, and hematoma. this study reports a case of retained guide wire and its treatment. in our case, the catheter was retained in the right iliac vein at the point of bifurcation. an important point in preventing this rare complication is that the guide wire should be held at least 18 cm distant from the vein. the diagnosis is very simple, which is often established incidentally during routine radiographic exams. the guide wire should be held at the tip at all times to prevent passage into or out of the vessel. predisposing factors for an intravascular placement of the guide wire include : inattentioninexperienced operator- either in method (i.e. seldinger technique) or actually central venous cannulationinadequate supervision of traineesovertired staff. inexperienced operator- either in method (i.e. seldinger technique) or actually central venous cannulation inadequate supervision of trainees the signs of guide wire loss include : the guide wire is missingresistance to injection via the distal lumenpoor venous backflow from the distal lumenguide wire is visible on a radiograph. the guide wire is missing resistance to injection via the distal lumen poor venous backflow from the distal lumen guide wire is visible on a radiograph. the most commonly reported guide wire related complications are listed and will be discussed below. cardiac dysrhythmiascardiac conduction abnormalitiesperforation of vessels or heart chamberkinking, looping, or knotting of wireentanglement of previously placed intravascular devicesbreakage of the distal tip of the guide wire with subsequent embolizationcomplete loss of guide wire within the vascular system. cardiac conduction abnormalities perforation of vessels or heart chamber kinking, looping, or knotting of wire entanglement of previously placed intravascular devices breakage of the distal tip of the guide wire with subsequent embolization complete loss of guide wire within the vascular system. cardiac dysrhythmias most often premature atrial or ventricular contractions are occasionally reported during subclavian or ij cvc insertion. the arrhythmias are typically short lived, resulting from the guide wire touching the endocardium, and resolve when the tip is pulled backa few centimeters. the most common cardiac conduction abnormalities seen during cvc placement are right bundle branch blocks, new left anterior and posterior fascicular blocks, and rarely asystole. the cause of these conduction problems, as described in the case of cardiac dysrhythmias, is also the overzealous advancement of the guide wire. the ease with which a right bundle branch block can be induced is probably related to the bundle branch 's superficial position in the right ventricular endocardium, just inferior to the tricuspid valve. however, in a patient with an underlying left bundle branch block, the induction of further conduction defects may lead to a life - threatening complete heart block requiring temporary pacing. the mentioned arrhythmias and conduction problems are essentially avoidable during central venous catheterization since placement should not involve entry into the heart by the guide wire or by the subsequently placed catheter. in clinical practice, it is often difficult to ascertain what caused the venous perforation ; the introducer needle, the guide wire, or the dilator. nevertheless, the literature reports cases of guide wire related perforation of the great vessels including the brachiocephalic and subclavian veins. this important complication occurs when excessive force is applied against resistance when introducing the guide wire, especially if the straight or angle tip wire, rather than j - tip style wire, is used. in most instances, bleeding from a small penetrating hole in a vein however, serious cases of hemothorax, including fatalities, due to the above complication have been reported. making a timely diagnosis in such cases requires maintaining a high index of suspicion when there is an unexplained drop in hemoglobin or the development of unilateral pleural effusion ipsilateral to a recently placed or attempted central venous catheterization. treatment of a serious perforation may necessitate the insertion of a chest tube or an emergent thoracotomy. perforation of the heart may occur at the time of catheter insertion or any time the catheter tip is placed within the heart chambers. there are at least two reported cases in the literature of heart perforation attributed to the guide wire itself. both of these complications occurred during the insertion of hd catheters : the first during a subclavian approach leading to a life - threatening cardiac tamponade and the second during an ij approach leading to a fatal tamponade. cardiac tamponade usually results from perforation of the right atrium, or less frequently, the right ventricle. tamponade has also been reported after superior vena cava (svc) perforation within the pericardium. the possibility of tamponade should be considered when a patient collapses during, or shortly after placement of a cvc. other diagnoses to consider in that scenario include tension pneumothorax and air embolism. an emergent chest x - ray or bedside echocardiogram followed by pericardiocentesis can be life - saving in such situations. another occasional guide wire complication is kinking or looping of the wire itself. applying force to thread a guide wire through the introducer needle despite significant resistance kinking can also result if the dilator is forced in a direction that diverges from the original path of the wire. if a clinician does not recognize this scenario there is potential for cutting through the vein with possible fatal complications. this type of complication can be avoided by intermittently moving the wire gently in and out as the dilator is being advanced through the subcutaneous tissue. it is almost exclusively described following the subclavian approach which may be due to the curved path the vein takes as it loops over the first rib to descend into the svc. this complication should be suspected when the guide wire can not be pulled out after successful catheter insertion. in this situation, no force should be used to pull the catheter and wire out, and an immediate x - ray should be ordered. once the diagnosis is established, interventional radiology should be consulted, and sometimes surgical intervention is necessary. entanglement of a guide wire with an existing intravascular apparatus is another reported complication of cvc placement. special attention is needed in patients with ivc filters since there have been numerous reports of entrapment of guide wires in these filters. it results from over advancement of the guide wire leading to hooking of the j - tip to the filter. this complication should be suspected when the guide wire can not be retrieved after catheter placement in a patient with an ivc filter already deployed. in such circumstances, no excessive force should be used to free the wire since this could lead to filter dislodgment and cava perforation. x - ray or examination under fluoroscopy should be ordered promptly followed by interventional radiology consultation once the diagnosis is made. shearing and breakage of the wire usually results from pulling the wire back through the needle after it has passed the bevel. hence, if a guide wire fails to pass freely from the introducer needle into the vessel, the careful retraction of the wire through the needle is an option, but it is much safer to withdraw the wire and needle as a single unit. the inadvertent intravascular insertion of the entire guide wire, as in our case, is a completely avoidable complication. although the loss of a complete guide wire might cause arrhythmias, vascular damage, and thrombosis ; it is usually asymptomatic and is often incidentally found on a routine x - ray done up to several months after the procedure. holding on to the proximal tip of the wire at all times is fundamental in preventing this mistake. if this complication happens, use of interventional radiology techniques is the preferred method for retrieval and removal. guide wire fragment, or entire guide wire should be possible to be removed in most cases. during the intervention the patient should be heparinized. usually, the foreign body (e.g. guide wire) is caught by a gooseneck snare passed via the femoral vein using radiographic control. the use of endovascular forceps or a dormier basket increases the risk of endovascular trauma. if the foreign body is captured, it is usually necessary to remove it along with the vascular sheath. if the vascular sheath is twice the size of the lost catheter or the lost guide wire may be possible to withdraw the foreign body through it. if heparinization is contraindicated, extraction should be attempted by careful surgical exploration. during central venous catheterization, guide wire - related complications are uncommon and essentially preventable. the following precautions should be taken : inspect the wire for defects before insertionconsider a guide wire to be a delicate and fragile instrumentwhen resistance to insertion is met, remove and inspect the wire for damage, reposition the introducer so that no resistance to its passage is feltparticular caution should be used when attempting central catheter placement in patients who are predisposed to thrombosis or have had repeated catheterizations of a particular vesselif multiple manipulations are needed, reinspect the wire and replace it if necessarypass catheter over wire into the veinmake sure that the wire is visible at the proximal end, before the catheter is advancedthe catheter should be railroaded over the guide wire into the vein, holding the wire, and not pushing catheter and wire together into the veinalways inspect the wire for complete removal at the end of the procedurehold onto the wire at all times until removal from the vessel. inspect the wire for defects before insertion consider a guide wire to be a delicate and fragile instrument when resistance to insertion is met, remove and inspect the wire for damage, reposition the introducer so that no resistance to its passage is felt particular caution should be used when attempting central catheter placement in patients who are predisposed to thrombosis or have had repeated catheterizations of a particular vessel if multiple manipulations are needed, reinspect the wire and replace it if necessary pass catheter over wire into the vein make sure that the wire is visible at the proximal end, before the catheter is advanced the catheter should be railroaded over the guide wire into the vein, holding the wire, and not pushing catheter and wire together into the vein always inspect the wire for complete removal at the end of the procedure hold onto the wire at all times until removal from the vessel.	catheterization of central veins is a routine technique which is widely used in emergency department and medical intensive care units. seldinger 's technique is widely used to place central venous and arterial catheters and is generally considered safe. the technique does have multiple potential risks. guide wire - related complications are rare but potentially serious. we describe a case of a lost guide wire during central venous catheter (cvc) insertion followed by a review of the literature of this topic. measures which can be taken to prevent such complications are explained in detail as well as recommended steps to remedy errors should they occur.
	plants are more susceptible to the toxic effects of nitrogen dioxide when exposure takes place in the dark. beta - carotene and other common carotenoids react with nitrogen dioxide in the dark to yield intermediate nitrosating agents consistent with the formation of nitrate esters. simultaneous exposure of carotenoids to no2 and light significantly reduced formation of nitrosating intermediates and resulted in the release of nitric oxide (no) into the gas phase. light - mediated reduction of no2 to no by carotenoids may be an important mechanism for preventing damage in plants exposed to no2. the formation of nitrosating agents from the reaction of carotenoids with no2 suggests that their ability to prevent nirosative damage associated with no2 exposure in both plants and animals may be limited in the absence of light.imagesfigure 1.figure 2.figure 3.figure 4.
ganglioneuroma (gn) is a rare (0% to 6% of incidentalomas), differentiated, benign, slow - growing tumor that commonly arises from sympathetic ganglion cells and is composed of mature schwann cells, ganglion cells, and nerve fibres. the family of tumors originating from ganglion cells includes gn which is a benign lesion, ganglioneuroblastoma which is of intermediate differentiation, and neuroblastoma which is a highly malignant lesion. neuroblastoma and ganglioneuroblastoma most often occur in infants and children, whereas gn tends to occur in adolescents and young adults. gns may arise anywhere along the paravertebral sympathetic plexus and occasionally from the adrenal medulla. their localization is the retroperitoneal (32% to 52%), mediastinal (39% to 43%), or cervical region (8% to 9%). when such tumors arise from the adrenal medulla, their assessment and management are similar to that of other adrenal tumors. abdominal pain or the palpation of an abdominal mass, or both of these, are among the most common presenting features. occasionally, symptoms like hypertension, diarrhea, and virilization may develop as a result of mixed hormonally active tumors secreting catecholamines, vasointestinal peptide (vip), androgens, or all of these. the standard treatment suggested for benign adrenal tumors is laparoscopic surgery. at present, relative contraindications to laparoscopic adrenalectomy are a definitive or presumed diagnosis of invasive adrenal cortical carcinoma or circumstances that make a minimally invasive approach technically difficult, such as large tumors. we present herein a case of a large, incidentally diagnosed adrenal ganglioneuroma that was removed completely by the laparoscopic approach. a 23-year - old woman was admitted to the endocrine ward of our hospital, for investigation and management of a 13-cm mass arising from the right adrenal. this mass was found incidentally following ultrasonography (us) of the abdomen while the patient was being investigated for oligomenorrhea. on admission, she was in good health and asymptomatic. blood pressure was within the normal range. on physical examination, a mild discomfort at deep palpation of the right quadrant general endocrinology data synacthen test (august 12, 2004) overnight 1 mg dexamethasone (dex) test (august 14, 2004) computed tomography (ct) of the abdomen showed a 1278-cm homogenous mass of the right adrenal (figure 1). magnetic resonance imaging (mri) showed a solid mass measuring 1377.5 cm arising from the right adrenal. on t2-weighted mr images, after intravenous injection of gadolinium, the mass showed a progressive, heterogeneous, and delayed enhancement (figure 2). abdominal computed tomographic scan indicating the large benign tumor. magnetic resonance imaging. after intravenous injection of gadolinium a laparoscopic approach was decided on, despite the large size of the tumor, because of the low risk of malignancy based on radiology findings. we prefer the transperitoneal lateral decubitus approach, as the best for maximal exposure of the gland and major vessels. using the hasson technique, laparoscopic exploration of the abdominal cavity was normal, and the tumor was easily identified. the right triangular ligament and the retroperitoneal liver attachments were cauterized and divided to allow liver retraction. after dividing the retroperitoneum, the inferior periadrenal fat was carefully dissected from the upper pole of the right kidney and the renal vein identified. the adrenal vein was subsequently identified, dissected, double - clipped, and divided. the specimen was extracted through an extension of the incision done for the hasson technique. the surgical specimen was oval and elastic in consistency, measuring 139.56 cm and weighing 390 g. the patient was discharged on the second postoperative day with bowel function returned to normal and able to resume normal physical activity. by gross inspection, a large encapsulated tumor mass of 139.56 cm, weighed 390 g. on the surface of the tumor, small foci of flattened adrenal tissue were recognized. by sectioning, the tumor had a solid homogenous appearance and was grayish - white to yellowish on the cut surface. mature ganglion cells scattered or arranged in small clusters were also noted (figure 3). two years after the procedure, there is no evidence of recurrence on abdominal ct scan, and the cosmetic result is satisfactory. top : low magnification shows ganglioneuroma (right) with adjacent normal adrenal cortex (left) (h&e 10). we prefer the transperitoneal lateral decubitus approach, as the best for maximal exposure of the gland and major vessels. using the hasson technique, laparoscopic exploration of the abdominal cavity was normal, and the tumor was easily identified. the right triangular ligament and the retroperitoneal liver attachments were cauterized and divided to allow liver retraction. after dividing the retroperitoneum, the inferior periadrenal fat was carefully dissected from the upper pole of the right kidney and the renal vein identified. the adrenal vein was subsequently identified, dissected, double - clipped, and divided. the specimen was extracted through an extension of the incision done for the hasson technique. the surgical specimen was oval and elastic in consistency, measuring 139.56 cm and weighing 390 g. the patient was discharged on the second postoperative day with bowel function returned to normal and able to resume normal physical activity. by gross inspection, a large encapsulated tumor mass of 139.56 cm, weighed 390 g. on the surface of the tumor, small foci of flattened adrenal tissue were recognized. by sectioning, the tumor had a solid homogenous appearance and was grayish - white to yellowish on the cut surface. mature ganglion cells scattered or arranged in small clusters were also noted (figure 3). two years after the procedure, there is no evidence of recurrence on abdominal ct scan, and the cosmetic result is satisfactory. top : low magnification shows ganglioneuroma (right) with adjacent normal adrenal cortex (left) (h&e 10). ganglioneuromas are rare tumors, and there is a relative paucity of information in the literature concerning their exact incidence. this is clinically relevant as the incidence of all adrenal tumors is increasing due to the availability of sophisticated imaging techniques and expertise ; approximately, 1% to 10% of abdominal ct scans report incidentally an adrenal tumor. it is encountered rather more frequently in the literature when comparing its incidence among other large tumors of greater than 6 cm in maximum diameter. approximately 100 cases of adrenal gn and 112 cases of extra adrenal gn have been reported in the japanese literature. the imaging based differential diagnosis of an asymptomatic, nonfunctioning giant adrenal mass without evidence of systematic disease is broad and includes besides gn, neuroblastoma, ganglioneuroblastoma, myelolipoma and angiomyolipoma. in the present case, the differential diagnoses suggested on mri findings were benign tumor of the cns (gn) and myelolipoma. both ct scan and mri are superior to us in detecting and characterizing an adrenal mass. following the availability of studies comparing radiologically found and histologically proven dimensions of adrenal lesions, it was shown that ct scanning might underestimate the tumor size by 16% to 47%, whereas mri does so by 20%. retroperitoneal gns appear as well - defined masses that are oval, crescentic, or lobulated. they tend to surround major blood vessels ; the result is absent or mild compromise of the lumen. intratumoral calcifications are present in 2.4% to 40% of cases ; however, no calcifications were observed in our case. areas of low attenuation that do not enhance are common, although such an enhancement is usually of low intensity. an interesting pattern consists of delayed heterogeneous uptake of contrast media, which was observed on the mri of the present case. adrenal myelolipoma is a rare benign tumor composed of mature fat tissue associated with proliferating hematopoietic cells. ct features are characteristic if lipid content is demonstrated, which was not observed in our case. hemorrhagic adrenal adenoma, adrenocortical carcinoma, metastatic tumors to the adrenal gland, and angiomyolipoma of the upper renal pole may also simulate gn or myelolipoma. the other tumors of the cns (neuroblastoma and ganglioneuroblastoma) develop at the same sites as the gn, but they occur in much younger patients and particularly in children. such tumors are usually quite aggressive, having developed metastases in bone, liver, and lung in approximately 50% of cases. it has recently been demonstrated that scintigraphy and positron emission tomography (pet) can complement ct and mri in diagnosing gns. laparoscopic procedures are associated with less postoperative discomfort, shorter hospital stay, less postoperative disability, and a lower rate of complications. there are few absolute contraindications for laparoscopic adrenalectomy, and most of them are not specific to adrenal surgery. large but well encapsulated adrenal masses without evidence of local invasion can be removed laparoscopically. the lateral transabdominal approach offers the best visualization of major vessels adjacent to the adrenals. the possibility of malignancy should be appreciated preoperatively (using ct, mri, or both) as well as intraoperatively. invasion of periadrenal tissues or organs is not well suited to laparoscopic techniques and needs conversion to an open procedure. several authors limit the laparoscopic adrenalectomy to lesions less than 6 cm in size, whereas others have performed laparoscopic adrenalectomy on tumors up to 13 cm in diameter without any significant morbidity. extensive experience in advanced laparoscopic techniques and open adrenal surgery are mandatory to manipulate and laparoscopically excise large tumors. in summary, adrenal ganglioneuroma is a rare benign adrenal tumor that needs careful evaluation and surgical treatment.	we report on a 23-year - old woman with a right adrenal tumor 13 cm in diameter who was treated by laparoscopy. the patient was asymptomatic, and the tumor was incidentally diagnosed on abdominal ultrasonography. a subsequent computed tomography (ct) of the abdomen confirmed a 1278-cm homogenous mass of the right adrenal. magnetic resonance imaging (mri) showed a solid mass measuring 1377.5 cm arising from the right adrenal. laparoscopic complete excision of the mass was accomplished through a transabdominal lateral approach. the postoperative period was uneventful, and the patient was discharged on the second postoperative day. histology was consistent with an adrenal ganglioneuroma. two years later, there is no evidence of recurrence on abdominal ct scan.
the cyclic amp (camp) pathway plays fundamental roles in the nervous system, where it is prominently involved in synaptic plasticity and memory formation (kahsai and zars, 2011, kandel., 2014). previous studies in vertebrate and invertebrate models have shown that camp can propagate from dendrites to the cell body of neurons (bacskai., 1993, hempel., 1996), in line with the properties of a small diffusible molecule. however, a local mode of action for camp has also been proposed, whereby camp signals are localized to the periphery of neurons namely, dendrites creating a camp microdomain (castro., 2010, li., 2015, neves., 2008, nicol., 2011, nicoll and schmitz, 2005, tomchik and davis, 2009). while the existence of camp microdomains in neuronal dendrites is disputed based on the experimental and theoretical data (calebiro and maiellaro, 2014, gervasi., 2010, 2014), very little is known about possible camp compartmentation in axons and how this may exert local effects at the presynaptic site. in particular, it is unclear how biochemical signals may spread from presynaptic boutons through the axon. to investigate this question in vivo, we used the neuromuscular junction (nmj) of drosophila melanogaster, which displays different forms of synaptic plasticity (collins and diantonio, 2007, frank, 2013, koon., 2011, 2013), many of which are dependent on camp signaling (griffith and budnik, 2006, olsen and keshishian, 2012). both structural and functional properties of larval neuromuscular synapses are heterogeneous, varying between boutons belonging to the same motor neuron (paul., 2015, how such site - specific synaptic differentiation may be achieved at high spatial resolution is currently unknown, though it is tempting to speculate that local camp signals play a role. thus, the developing drosophila nmj is a powerful model to investigate the role of camp in synaptic plasticity under physiological conditions. in this study, we focused on glutamatergic type ib motor neurons (hoang and chiba, 2001), which are structurally regulated via g - protein - coupled receptors (gpcrs) for octopamine (koon. stimulation of these receptors has been shown to induce synaptic bouton outgrowth via a camp- and creb - dependent pathway (koon., 2011). therefore, we set out to measure spatiotemporal patterns of octopamine - induced camp signals in these neurons. to this end, we transgenically expressed a fret (frster resonance energy transfer)-based sensor for camp (epac1-camps ; nikolaev., 2004), which has previously been used to image camp levels in central drosophila neurons (shafer., 2008). our results reveal that camp signals are confined to their initiation site, the individual synaptic bouton, and suggest a highly efficient local mechanism for controlling site - specific synaptic plasticity. to investigate camp changes in vivo and in real time upon octopamine stimulation, we expressed epac1-camps (nikolaev., 2004) in third instar drosophila larvae using a motor - neuron - specific promoter (figures 1a and 1h). the whole larvae were perfused with increasing concentrations of octopamine applied to the bath with a pipette. for normalization at the end of each experiment, the preparations were stimulated with the broad phosphodiesterase (pde) inhibitor ibmx, followed by the direct adenylyl cyclase (ac) activator forskolin to maximally activate the epac1-camps sensor (figures 1b and 1i). in particular, a 10 m concentration of octopamine produced a clear response in the boutons but only a minor effect in the distal axon, while stimulation with higher concentrations (10 m) elicited robust camp responses in both compartments (figures 1b and 1c). the potency of octopamine in eliciting camp fret signals showed a 3-fold higher sensitivity in the boutons compared to the distal axon (figure 1c). next, we aimed to understand whether camp fret signals generated in the periphery of the motor neuron could reach the cell body. to this end, we analyzed the extent of the camp fret signals, in space and time, evoked by 10 m octopamine, a concentration that induces structural neuronal plasticity (koon., 2011). we found that, upon generalized application of octopamine, the amplitude of the camp response was similar in the different synaptic boutons analyzed (figure 1d, blue region). however, in the axon, the amplitude of the camp response decreased nearly exponentially with distance, reaching virtually undetectable levels at 50 m from the most proximal bouton (figure 1d, green region). similar to the signal amplitude, the speed of the camp response was higher in the boutons and in the distal portion of the axon than in more proximal regions (figures 1e and 1f). to better illustrate the propagation of camp, instead of showing the direct fret change at different time points after stimulation, we calculated the first time derivative of the fret data. with this alternative method, the color and intensity of each pixel indicate the rate at which camp levels were increasing (red) or decreasing (blue) at a given time point (figure 1 g ; movie s1). the analysis clearly showed that the production of camp began in the boutons and only later reached a peak in the nearby portion of the axon (figure 1 g ; movie s1). finally, we monitored the octopamine - induced camp fret signals directly in the cell body localized at the level of the ventral nerve cord. stimulation of transgenic larval preparations with a concentration of 10 m octopamine produced no detectable camp increases in the cell body, unless pdes were inhibited with ibmx (figure 1i). moreover, the concentration - response curve showed that the cell body was 100-fold less sensitive toward octopamine than the boutons (figure 1j). control experiments excluded the possibility that the intracellular biosensor concentration or differences in the saturation of the sensor at rest might affect the amplitude and the kinetics of the camp response in the different intracellular compartments (figures s1a s1d). in order to determine whether camp fret signals in the cell body induced by high concentrations of octopamine might be caused by camp generated in the bouton, we repeated the camp measurements in neurons with a severed axon. the resulting concentration - response curve of octopamine was indistinguishable from that obtained in intact neurons, suggesting that the camp in the cell body did not originate from the periphery (figure 1j, dashed line). taken together, our data reveal the formation of a steep camp gradient upon generalized stimulation with octopamine, where camp fret signals triggered in the boutons propagated only into the first portion of the axon but did not reach the cell body. to investigate the degree of camp signal compartmentalization, we performed a series of experiments in which octopamine was applied locally by means of iontophoresis (figure 2a). the delivery of octopamine with increasing ejection currents was well within the linear range of the iontophoresis system (figure s2a). for our experiments we chose a short stimulation (1 s) to minimize the extracellular diffusion of octopamine (figure s2b). such stimuli produced full activation of the receptors located on a single bouton but no equilibrium in camp levels (figures 2b and 2c). stimulation of the most distal bouton with a saturating octopamine pulse (100 na, 1 s) resulted in a rapid and confined increase of camp within the targeted bouton (figures 2c2e), which reverted to baseline within approximately 20 s (figures 2c and 2e). the very high sensitivity of the sensor allowed us to fully detect the physiologically relevant range in camp (figures s2c s2e), which can lead to activation of downstream targets. remarkably, the response to such localized stimuli was confined to the stimulated bouton and spread no further than the adjacent one (figures 2c2e), where a minor response was seen which could be attributed to the diffusion of octopamine (figure s2b). overall, the camp gradient extended to no more than 15 m from the stimulated bouton (figure 2d). the time - derivative analysis of the fret data reveals the very local increase of camp (indicated in red) in the stimulated bouton (figure 2e ; movie s2), which was followed by a slower return to basal levels (indicated in blue in figure 2e). taken together, our results provide direct evidence that camp can be confined to a single synaptic bouton of a neuronal axon and that, in these circumstances, camp diffusion in vivo must be significantly hindered. the data presented earlier show that the boutons, axon, and cell body are independent camp signaling compartments that can, at least under certain circumstances, signal differentially. it has been suggested that cell morphology and local differences in the surface - to - volume ratio lead to the formation of camp gradients between different parts of a cell, with higher camp concentrations in finer structures (neves.,, we found larger camp fret signals in the boutons than in the cell body (figure 1j). however, we observed a stronger camp response in boutons, which have a larger diameter than the axon (3- versus 1 m, respectively ; figures 1b and 1d). furthermore, we monitored a greater camp response in big (> 2 m) than in small (< 2 m) boutons (figure 3a). this indicates that, in our system, camp fret signals are potentiated in larger boutons. we then evaluated the contribution of the proteins responsible for the formation and degradation of camp signals, i.e., receptors, acs, and pdes (calebiro and maiellaro, 2014). first, we assessed the contribution of octopamine receptors to the camp gradient induced by generalized octopamine stimulation. to do so, we monitored camp changes in bouton, axon, and cell body to increasing concentrations of forskolin ; hence, downstream of octopamine receptors. bypassing receptor activation led to the formation of a gradient between bouton and cell body (figure 3b) that was similar to the one seen by octopamine activation, suggesting that mechanisms other than the localization of the receptors are responsible for the differences observed between these two compartments. however, forskolin induced similar changes in bouton and axon (figure 3b), implying that receptor localization plays a role in the octopamine - induced camp gradient formation between these regions. to further corroborate this hypothesis, we compared the camp response induced by iontophoretic delivery of octopamine to the bouton or to the axon. the axon showed virtually no local camp response (figure 3c) ; therefore, we can infer the absence of functional octopamine receptors in this compartment. next, we analyzed the contribution of pdes to the octopamine - induced camp gradient. although it is undisputed that pdes play a critical role in the dynamics of the camp signal, their contribution to diffusion and generation of camp gradients or compartments remains less clear (conti., 2014). when pde activity was blocked with ibmx in the presence of octopamine, the camp fret signals were uniformly elevated along the motor neurons (figure 3d ; for comparison, see figures 1e and 1i). the camp signal in the cell body was of the same magnitude as in the periphery, regardless of the applied octopamine concentration (figure s3). furthermore, we compared the initial rate of octopamine - induced camp accumulation in the presence of ibmx in the bouton versus that in the cell body as a readout of ac activity. no significant differences were observed in the rate of camp accumulation in the cell body and bouton, indicating that octopamine - induced ac activity was similar in the analyzed compartments (figure 3e). additionally, we analyzed the expression pattern of the pdes. to achieve this, we took advantage of a newly developed drosophila line in which the endogenous pde dunce is tagged with egfp (dnc - egfp - dnc ; nagarkar - jaiswal., staining against horseradish peroxidase (hrp) was used to visualize presynaptic arborizations, and anti - gfp staining showed the expression of the dnc - egfp - dnc protein (figure 3f). we found that pdes appeared largely concentrated at the plasma membrane of the synaptic boutons. interestingly, pdes were also localized between two neighboring boutons, reminiscent of a fence, which could obstruct the diffusion of camp (figure 3f, yellow arrows). the dnc - egfp - dnc protein was undetectable in the axons (figure 3f, white arrow), while it showed a strong signal in the cell bodies (figure s4a). together, these data indicate that high pde activity contributes to a low camp concentration in the cell body and that the pde distribution at the nmj hinders camp diffusion from one bouton to the next. at the drosophila nmj, glutamate release from motor neurons is regulated at the level of individual active zones, the specialized presynaptic sites of vesicle exocytosis (ehmann., 2014,, 2013, peled and isacoff, 2011, schmid., 2008). several studies have described that active - zone properties are not homogeneously distributed across motor neurons. average ultrastructural and functional active - zone features differ between boutons (ehmann., 2014, paul., 2015, peled and isacoff, 2011) and functionally similar active zones can cluster within boutons (melom., 2013) the confinement of camp fret signals to a single synaptic bouton might explain how structural and functional diversity is achieved among different boutons. to test this hypothesis, we evaluated whether disturbing the camp metabolism could, consequently, perturb the structural gradient observed along the type ib motor neuron on muscle 6/7. this gradient is manifested as an increase in size and active - zone number of terminal boutons compared to more proximal boutons (paul., we compared wild - type (wt) and pde mutant flies (dunce) (davis and kiger, 1981). it hydrolyzes camp at a significantly slower rate than the wt and should, therefore, be less efficient in segregating camp signals. mutant animals show impaired learning, memory deficits, and altered motor neuron terminal growth (zhong., 1992, zhong and wu, 2004). however, whether structural differentiation of synaptic boutons is preserved in the mutants has, to our knowledge, so far not been addressed. indeed, in dunce mutants, the wt gradient in active - zone numbers per bouton was diminished, and the difference in bouton area was reduced (figures 4a and 4b). these data demonstrate that less efficient degradation of camp, which will lead to a spreading out of local camp increases, perturbs the structural heterogeneity of synaptic boutons. thus, these results are consistent with a site - specific control of synaptic plasticity by local camp. in this study, we genetically expressed the camp sensor, epac1-camps, at the drosophila nmj to monitor and quantify spatiotemporal camp dynamics induced by octopamine stimulation. our results reveal an unexpectedly high degree of camp compartmentalization in motor neurons, which may serve as a basis for local synaptic plasticity, with camp fret signals being ultimately limited to single synaptic boutons. we identify three camp signaling compartments within the motor neuron : boutons, axon, and cell body. for each cellular compartment specifically, we find that boutons constitute the most reactive compartment of the motor neuron in terms of camp accumulation. our results demonstrate that the production of camp is heterogeneous among boutons, with stronger responses to octopamine in large boutons than in smaller ones, possibly related to the increased synaptic strength measured at large boutons (paul., 2015). moreover, the activity and the specific localization of pdes within the synaptic bouton prevent the propagation of camp to the cell body and its diffusion from one bouton to the next. in contrast to the boutons, octopamine receptors seem to be absent or inaccessible in the axon, and pdes were not detected. accordingly, camp fret signals recorded in the axon differ from those in boutons in terms of amplitude and kinetics. hence, the axon emerges as the second independent, but not isolated, camp signaling compartment. interestingly, and in contrast to dendrites (bacskai., 1993, hempel., 1996, it remains to be clarified whether this may relate to functional differences between axons and dendrites, between species, or between specific neuron types. finally, we determined the cell body as the third camp signaling compartment within the motor neuron. we show that the cell body has a very low sensitivity toward octopamine and demonstrate that camp fret signals generated in the cell body are not affected by its physical isolation from the axon. high pde activity in the cell body contributes to this local suppression of camp signaling and may prevent spillover activation of camp effectors in the cell body and in the nucleus. our evidence of camp microdomains restricted to single boutons provides a biophysical basis for the local control of synaptic plasticity. we show that spatially constrained camp changes help to establish differences in morphology and synaptic content of boutons, suggesting that local camp, bouton structure, and synapse formation are intimately linked (ehmann., 2014, the observed confinement of camp supports the notion that individual synaptic boutons may represent largely autonomous signaling units, which can receive and integrate signals independently of the other. the concept that camp could act as a local messenger was postulated almost 40 years ago (corbin., 1977). the existence of camp microdomains has been demonstrated in other cell types (e.g., cardiomyocytes) (zaccolo, 2011). however, in neurons, there are contradicting experimental lines of evidence and simulations concerning the existence of camp microdomains (calebiro and maiellaro, 2014). our data clearly show that camp can act as a local messenger upon physiological stimulation of a neuron. the detailed spatiotemporal analysis of the dynamics of camp reveals that this messenger can be restricted at the micrometer level to induce highly localized physiological responses. a dna fragment containing the whole epac1-camps coding sequence flanked by an agei site at the 5 end and a kpni site at the 3 end was generated by pcr amplification using epac1-camps in pcdna3 (invitrogen) as a template and the following primers:5 acataccggtgacactatagaatagggcc ; 3 aagagggtaccaatcgaaattaatacgactcac. the amplified dna fragment was then cloned downstream of five copies of the upstream activation sequence (uas) in the pjfrc7-derived ptl412 vector, by inserting it between the kpni and agei sites. the obtained plasmid was sent to bestgene to generate the 20xuas - epac1-camps flies by targeted germline transformation. flies were reared on cornmeal / agar media supplemented with yeast and kept at 25c. to obtain selective expression of epac1-camps in motor neurons, we crossed flies expressing gal4 under the control of the ok6 promoter (sanyal, 2009). the dnc - egfp - dnc (ywmi{pt - gfstf.2}dnc) line was obtained from the bloomington drosophila stock center. dunce and canton - s stocks (figure 4) were provided by andreas thum. in order to expose the nmj, third instar larvae were dissected in ice - cold calcium - free hl-3 solution (stewart., 1994). the larvae were immobilized, dorsal side up, on a sylgard 184 (sigma - aldrich) pad with two sharp pins. a cut was made along the dorsal midline, and after pinning down the body wall, the internal organs were carefully removed while severing the tracheal connections to the muscles. larvae were placed in a recording chamber and imaged at room temperature in 2 ml hl-3 solution supplemented with 10 mm l - glutamate to desensitize postsynaptic receptors and thus prevent movement of the larvae. agonists were applied in the bath with a pipette. unless otherwise noted, the experiments were performed on type ib motor neurons innervating muscle 13 in hemisegments a2 and a3 of male third instar larvae. the time constant () of camp accumulation was calculated using a non - linear regression (monoexponential phase decay). ratiometric fret imaging was performed using an upright epifluorescence microscope (axio observer, zeiss) equipped with a water - immersion objective (63/1.1 numerical aperture) ; a xenon lamp coupled to a monochromator (visiview, visichrome) ; filters for cfp (436/20, 455lp dichroic) and yellow fluorescent protein (yfp ; 500/20, 515lp dichroic) excitation ; a beam splitter (dualview, photometrics) with a 505lp dichroic mirror and emission filters for cfp (480/30) and yfp (535/40) ; and an electron - multiplied charge - coupled device (emccd) camera (evolve 512, photometrics). cfp and yfp images upon cfp excitation were captured every 5 s or every 300 ms with 80 ms of illumination time. fret was monitored in real time with the metafluor 5.0 software (molecular devices) as the ratio between yfp and cfp emissions. the yfp emission was corrected for direct excitation of yfp at 436 nm and the bleedthrough of cfp emission into the yfp channel, as previously described (brner., 2011). local delivery of octopamine and rhodamine was performed using a high - speed iontophoresis system with capacitance compensation (mvcs-02c, npi electronic). the headstage was mounted on a micromanipulator (nmn-21, narishige), allowing precise positioning of the glass microelectrode. high - resistance (7090 m) microelectrodes were filled with 10 m octopamine or rhodamine dissolved in 150 mm na2hpo4 (ph 7.2), supplemented with 5% bsa. to allow visualization of the microelectrode tip during fret imaging, the tip of the glass microelectrode was coated with alexa fluor 488-labeled bsa as previously described (sasaki., 2012). during the experiments, larvae were continuously superfused with hl-3 solution supplemented with 10 mm glutamate in the opposite direction of the iontophoretic delivery, using a custom - made perfusion system. for the visualization of bouton structure and active zones (1:1,000 ; life technologies, # a11122) and mouse monoclonal antibody (mab) brp (1:250 ; wagh., 2006). secondary antibodies used were cy3-conjugated goat -mouse (invitrogen), af488-conjugated goat -rabbit (1:500 ; life technologies, # a11008), and cy3- or alexa fluor 488-conjugated goat -horseradish peroxidase (-hrp) (1:250 ; jackson immunoresearch laboratories antibodies). bruchpilot (brp) puncta per bouton were quantified manually using the four terminal boutons of type ib branches. distal boutons were located at the end of bouton chains, whereas proximal boutons were closer to the entry site of the motor neuron at the nmj. different genotypes were stained under the same conditions in the same vial and were analyzed by blinded observers. confocal image stacks were obtained with a line - scanning confocal sp5 system (leica) equipped with a 1.2 numerical aperture 63 water - immersion objective. statistical analyses and curve fitting differences between means were assessed by a two - tailed student s t test (for two groups) and one- or two - way anova, followed by a bonferroni s post hoc test (for three or more groups).	summarythe second messenger cyclic amp (camp) plays an important role in synaptic plasticity. although there is evidence for local control of synaptic transmission and plasticity, it is less clear whether a similar spatial confinement of camp signaling exists. here, we suggest a possible biophysical basis for the site - specific regulation of synaptic plasticity by camp, a highly diffusible small molecule that transforms the physiology of synapses in a local and specific manner. by exploiting the octopaminergic system of drosophila, which mediates structural synaptic plasticity via a camp - dependent pathway, we demonstrate the existence of local camp signaling compartments of micrometer dimensions within single motor neurons. in addition, we provide evidence that heterogeneous octopamine receptor localization, coupled with local differences in phosphodiesterase activity, underlies the observed differences in camp signaling in the axon, cell body, and boutons.
it is complicated to treat patients with composite bone non - union and soft tissue defect caused by high - energy injuries, and the period of the treatment is long. due to the lack of soft tissue surrounding it, the injured bone possesses worse ability to heal and fight infection than normal bone does. in order to solve the problem associated with soft tissue and bone healing in such injuries the technique grafts autogenous cancellous bone matched in shape to the bone defect after debridement, and, while maintaining the wound open, the wound is covered with petroleum jelly gauze and drained. however, advancements in technology have led some researchers to combine vacuum - assisted closure (vac) with open bone grafting to treat patients with osteomyelitis or bone non - union, and a better curative effect has been attained. vac technology uses a medical - grade porous foam material (aperture size 400600 m), called vacuseal, packed into a drainage tube with several holes. vacuseal is placed on the wound, and an adherent semipermeable membrane covering makes the wound airtight. this apparatus is then linked to a vacuum through a drainage bottle to create negative pressure. in this study, we propose to verify the impact of vac combined with open bone grafting in promoting rabbit bone graft vascularization ; we also seek to provide a theoretical basis for its clinical application. twenty - four male new zealand white rabbits weighing 2.8 to 3.2 kg aged four to five months (from the wuhan wanqian jiaxing bio - technology co., ltd, china) were used in this study. the rabbits were fed with standard feed and housed in an ambient environment at constant temperature for at least one week prior to surgery. the rabbits were randomly divided into two groups : the experimental group and the control group. rabbits in the experimental group were grafted with allogeneic bone (made from rabbit cancellous bone of the lower limbs provided by the second clinical college of wuhan university) onto the proximal femur. then, the rabbits were singly placed in a cage and fed in such a way that they could not turn around to bite the tube. animals in the control group underwent a similar surgical procedure other than the fact that the surgical wound was closed by primary closure rather than with vacuseal. animals in the experimental group were anesthetized by injecting a 3% solution of sodium pentobarbital via the ear vein (30 mg / kg). following confirmation that the animals were anesthetized fur in the surgical area (hind leg) was removed and the skin was prepared using 8% na2s solution. after regular disinfection and draping, a 3 cm long incision was performed posterior to the midline on the thigh to expose the proximal femur. three allogeneic bones were grafted and banded with the proximal femur with a suture (figure 1a). china) was placed to cover the allogeneic bones (figure 1b) and was fixed to the wound with sutures. then, the semipermeable membrane (smith & nephew co., england) was adhered to cover the vacuseal outside the wound (figure 1c), and the dressing was linked to a negative pressure aspirator to maintain continuous vacuum aspiration at a pressure of about 60 mmhg to 75 mmhg (figure 1d). the animals were housed in a special cage and an intramuscular injection of penicillin (400,000 u / ci) was given for three days after surgery. the difference in the surgical procedure between the control and experimental groups was that the wound was sutured with vac in the experimental group but not in the control group. four rabbits from each group were sacrificed with air embolism separately at the 4, 8, and 12 week after surgery. then, we estimated the state of bone allograft vascularization in the callus based on the imaging. all the animals were injected with tetracycline hydrochloride (aladdin reagent co., ltd, china) (35 mg / kg) 15 d prior to sacrifice, and they were injected with calcein (sinopharm chemical reagent co., ltd, china) (10 mg / kg) 3 d prior to sacrifice. after sacrificing the animals, the bone allografts of the two groups the allografts were embedded with methyl methacrylate without decalcification over 24 h, and then the specimens were serial sectioned with slices of 5 micron thickness using a microtome (jung type). the bone cortex and callus fluorescence staining for each slice was assessed by leica tcs sp2 confocal microscopy (leica microsystems ir gmbh., germany), and the distance between the two marking lines comprised of tetracycline hydrochloride and calcein was measured by aim image examiner (carl zeiss microimaging gmbh. the bone allografts were accurately weighed after being rinsed, defatted, and dried off, then the specimens were decalcified with 0.1 g / ml trichloroacetic acid, and the calcium content of each specimen was determined using back titration. total protein was extracted from cancellous bone by hammer grinding and all protein samples were stored at 80c. the protein of each group was separated by 12% sodium dodecyl sulfate - polyacrylamide gel electrophoresis (sds - page) and blotted to a polyvinylidene difluoride (pvdf) membrane. the membrane was blocked in tris - buffered saline with tween (tbst) with 5% skim milk for 2 h and incubated overnight at 4c with anti - fgf-2 antibody (boster reagent co., ltd, china) (1:200 dilution). after washing in tbst for 5 times, the membrane was incubated with horseradish - peroxidase (hrp)-coupled secondary antibody (1:50000 dilution) at room temperature for 2 h. subsequently, the samples were washed with tbst again, and the membranes were incubated with an ecl detection kit (beijing comwin biotech co., ltd., china) for 5 min and exposed to medical x - ray film. bandscan was used to analyze the grey value of the film. to compare the difference in each test between the groups, spss17.0 for windows measurement data were presented as the mean sd and analyzed by student s t - test. enumeration data were presented as percentages and were analyzed by the chi - square test. in all cases, a significance level of p<0.05 was used. twenty - four male new zealand white rabbits weighing 2.8 to 3.2 kg aged four to five months (from the wuhan wanqian jiaxing bio - technology co., ltd, china) were used in this study. the rabbits were fed with standard feed and housed in an ambient environment at constant temperature for at least one week prior to surgery. the rabbits were randomly divided into two groups : the experimental group and the control group. rabbits in the experimental group were grafted with allogeneic bone (made from rabbit cancellous bone of the lower limbs provided by the second clinical college of wuhan university) onto the proximal femur. then, the rabbits were singly placed in a cage and fed in such a way that they could not turn around to bite the tube. animals in the control group underwent a similar surgical procedure other than the fact that the surgical wound was closed by primary closure rather than with vacuseal. animals in the experimental group were anesthetized by injecting a 3% solution of sodium pentobarbital via the ear vein (30 mg / kg). following confirmation that the animals were anesthetized fur in the surgical area (hind leg) was removed and the skin was prepared using 8% na2s solution. after regular disinfection and draping, a 3 cm long incision was performed posterior to the midline on the thigh to expose the proximal femur. three allogeneic bones were grafted and banded with the proximal femur with a suture (figure 1a). china) was placed to cover the allogeneic bones (figure 1b) and was fixed to the wound with sutures. then, the semipermeable membrane (smith & nephew co., england) was adhered to cover the vacuseal outside the wound (figure 1c), and the dressing was linked to a negative pressure aspirator to maintain continuous vacuum aspiration at a pressure of about 60 mmhg to 75 mmhg (figure 1d). the animals were housed in a special cage and an intramuscular injection of penicillin (400,000 u / ci) was given for three days after surgery. the difference in the surgical procedure between the control and experimental groups was that the wound was sutured with vac in the experimental group but not in the control group. four rabbits from each group were sacrificed with air embolism separately at the 4, 8, and 12 week after surgery. the femurs of the two groups were assessed by lateral x - ray imaging. then, we estimated the state of bone allograft vascularization in the callus based on the imaging. all the animals were injected with tetracycline hydrochloride (aladdin reagent co., ltd, china) (35 mg / kg) 15 d prior to sacrifice, and they were injected with calcein (sinopharm chemical reagent co., ltd, china) (10 mg / kg) 3 d prior to sacrifice. after sacrificing the animals, the bone allografts of the two groups were obtained and were fixed in 10% paraformaldehyde, dehydrated, and defatted. the allografts were embedded with methyl methacrylate without decalcification over 24 h, and then the specimens were serial sectioned with slices of 5 micron thickness using a microtome (jung type). the bone cortex and callus fluorescence staining for each slice was assessed by leica tcs sp2 confocal microscopy (leica microsystems ir gmbh., germany), and the distance between the two marking lines comprised of tetracycline hydrochloride and calcein was measured by aim image examiner (carl zeiss microimaging gmbh. the bone allografts were accurately weighed after being rinsed, defatted, and dried off, then the specimens were decalcified with 0.1 g / ml trichloroacetic acid, and the calcium content of each specimen was determined using back titration. total protein was extracted from cancellous bone by hammer grinding and all protein samples were stored at 80c. the protein of each group was separated by 12% sodium dodecyl sulfate - polyacrylamide gel electrophoresis (sds - page) and blotted to a polyvinylidene difluoride (pvdf) membrane. the membrane was blocked in tris - buffered saline with tween (tbst) with 5% skim milk for 2 h and incubated overnight at 4c with anti - fgf-2 antibody (boster reagent co., ltd, china) (1:200 dilution). after washing in tbst for 5 times, the membrane was incubated with horseradish - peroxidase (hrp)-coupled secondary antibody (1:50000 dilution) at room temperature for 2 h. subsequently, the samples were washed with tbst again, and the membranes were incubated with an ecl detection kit (beijing comwin biotech co., ltd., china) for 5 min and exposed to medical x - ray film. to compare the difference in each test between the groups, spss17.0 for windows statistical software analysis was performed. measurement data were presented as the mean sd and analyzed by student s t - test. enumeration data were presented as percentages and were analyzed by the chi - square test. in all cases, a significance level of p<0.05 was used. by general observation and x - ray imaging (figure 2a2f), some callus growths crawling from the femur to the bone graft were observed, and the dividing line could be seen at the 4 week after surgery. the femur connected a little more to the bone graft in the experimental group than in the control group. at the 8 week after surgery, more callus growth was observed around the bone graft, and the femur attached to the bone graft solidly in both groups. the dividing line was blurred in the experimental group while a part of the line was still visible in control group. at the 12 week after surgery, the bone graft connected to the femur entirely and a peripheral callus was beginning to form in both groups. fluorescent bone labeling demonstrated a discrete yellow fluorescent line (tetracycline hydrochloride) arranged around the bone graft along with scattered punctiform green fluorescence (calcein) present in the experimental group, while there was only scattered punctiform yellow fluorescence in the control group at the 4 week after surgery (figure 3a, 3b). at the 8 week after surgery, two fluorescent ribbons with clear boundaries could be observed around the bone graft in the experimental group, while the corresponding ribbons were discontinuous and uneven in thickness in the control group (figure 3c, 3d). at the 12 week after surgery, two annular - shaped fluorescent ribbons could be found in both groups, but the distance between the colored ribbons was significantly shorter in the control group than in the experimental group (p<0.05) (figure 3e, 3f, table 1). the calcium content in the callus at the 4, 8, and 12 week after surgery was significantly different between the control and experimental group (p<0.05). the calcium content in the callus in the experimental group was significantly higher than in the control group at each time point after surgery (p<0.05). typical imaging of the expression of fgf-2 by western blot analysis in each group is shown in figure 5. the grey value of each film in the experimental group was significantly higher than in the control group (p<0.05) (figure 6). by general observation and x - ray imaging (figure 2a2f), some callus growths crawling from the femur to the bone graft were observed, and the dividing line could be seen at the 4 week after surgery. the femur connected a little more to the bone graft in the experimental group than in the control group. at the 8 week after surgery, more callus growth was observed around the bone graft, and the femur attached to the bone graft solidly in both groups. the dividing line was blurred in the experimental group while a part of the line was still visible in control group. at the 12 week after surgery, the bone graft connected to the femur entirely and a peripheral callus was beginning to form in both groups. fluorescent bone labeling demonstrated a discrete yellow fluorescent line (tetracycline hydrochloride) arranged around the bone graft along with scattered punctiform green fluorescence (calcein) present in the experimental group, while there was only scattered punctiform yellow fluorescence in the control group at the 4 week after surgery (figure 3a, 3b). at the 8 week after surgery, two fluorescent ribbons with clear boundaries could be observed around the bone graft in the experimental group, while the corresponding ribbons were discontinuous and uneven in thickness in the control group (figure 3c, 3d). at the 12 week after surgery, two annular - shaped fluorescent ribbons could be found in both groups, but the distance between the colored ribbons was significantly shorter in the control group than in the experimental group (p<0.05) (figure 3e, 3f, table 1). the calcium content in the callus at the 4, 8, and 12 week after surgery was significantly different between the control and experimental group (p<0.05). the calcium content in the callus in the experimental group was significantly higher than in the control group at each time point after surgery (p<0.05). typical imaging of the expression of fgf-2 by western blot analysis in each group is shown in figure 5. the grey value of each film in the experimental group was significantly higher than in the control group (p<0.05) (figure 6). for the high operation failure rates of former treatments of open, non - union bone fractures [410 ], papineau introduced open bone graft technology to solve this problem. open bone grafting is also called the rhinelander - papineau or the papineau technique, and its characteristic benefits are conferred by the facts that the microporous structure of cancellous bone is advantageous to osteoblast agglomeration and growth, and that osteoblasts can obtain nutrition from surrounding tissue exudates for survival. new vessels grow easily and quickly to establish a blood supply because of the unique clearance of each bone graft and such vessels bring more nutrients including osteogenesis factors [1116 ]. draining wound exudates through gauze is better at preventing infection when applied with open bone grafts compared with traditional bone grafts [1719 ]. furthermore, the blood from the soft tissue bed or cancellous bone marrow cavity can seep out through the gap and solidify. afterwards, granulation tissue forms and covers the bone graft, and this is followed by capillary ingrowth, which provides the foundation for free skin grafting. all of these characteristics confirm that open bone grafting is applicable to patients with a poor state of local soft tissue and infectious non - union bone ; however, potential problems include needing treatment more frequently, and spending a longer period of time in hospital. fortunately, the emergence of vac greatly mitigates the said problems of open bone grafting. the basic working principle of vac is to use a vacuseal connected to a drainage tube between the wounds, which is collectively connected to negative pressure, enabling for the whole wound to drain and allow for clearance of exudate expeditiously. vac also contacts the wound closely and uses sustained active force provided by the negative pressure, enabling clearance of not only wound exudate, but also of pus and necrotic tissue in a complete and timely manner. vacuseal containing a porous structure has increased water permeability and ability to adsorb solutions better than petroleum jelly gauze. in addition, when exudate is eliminated, vac can inhibit bacterial growth and prevent the absorption of toxins and spread of infection. as the study of vac increases, more advantages to this technology have been discovered [3033 ] : (1) vac can promote vascular endothelial growth factor (vegf) generation in damaged tissue ; (2) the expression of cd34 significantly increases after vac treatment ; therefore, one can deduce that vac apparently promotes the proliferation of vascular endothelial cells and fibroblasts in the tissue around the wound and also increase microvascular density, thus promoting wound granulation tissue growth ; (3) the concentration of transforming growth factor - beta 1 (tgf-1) and platelet - derived growth factor (pdgf) in wounds is significantly increased by vac ; (4) vac can decrease the expression of matrix metalloproteinase (mmp)-1, 2, 9, 13 mrna in wounds, and inhibit the degradation of gelatin and collagen by adjusting the balance of mmps to tissue inhibitors of metalloproteinase (timp) to promote wound healing. the role of active substances, including vegf and cd34, tgf-1, pdgf, and timp-1 in promoting bone revascularization has been confirmed [3438 ]. fgf2 is a member of the fibroblast growth factor family, which is widely distributed within human tissue, and it plays an important role in the repair of bone defects or fractures [3941 ]. in this experiment, we detected fgf2 in allografts by western blot analysis and found that the expression in the experimental group was significantly higher, which indicated that vac can promote the expression of fgf2, and this may suggest that heightened fgf2 is probably one of the reasons behind vac promoting bone vascularization. vac in combination with open bone grafting is currently used in clinical settings mainly for complex open tibial bone non - union with soft tissue defects. because it can shorten fracture healing time, and the time for granulation tissue to cover the wound, it is recognized by a vast number of scholars. we had successfully treated 15 cases of complex tibial bone non - union using this technology since 2007 in our hospital, and the treatment efficacy has been satisfactory. in this experiment, the results of x - ray imaging, fluorescent bone labeling and calcium content in the callus indicated that vac plays a role in promoting bone allografts vascularization in rabbit, while the increased expression of fgf-2 in bone allografts may be the reason for this effect. we could not replace vacuseal every five to seven days strictly, but rather deciding on replacement time based on practical circumstances. in addition, this study only confirmed that vac could promote bone vascularization, but the effect was not quantified.	backgroundpatients with composite bone non - union and soft tissue defects are difficult to treat. vacuum - assisted closure (vac) combined with open bone grafting is one of the most effective treatments at present. the aim of the present study was to preliminarily investigate the effect and mechanism of vac combined with open bone grafting to promote rabbit bone graft vascularization, and to propose a theoretical basis for clinical work.material/methodstwenty-four new zealand white rabbits were randomly divided into an experimental and a control group. allogeneic bones were grafted and banded with the proximal femur with a suture. the experimental group had vac whereas the control group had normal wound closure. the bone vascularization rate was compared based on x - ray imaging, fluorescent bone labeling (labeled tetracycline hydrochloride and calcein), calcium content in the callus, and expression of fibroblast growth factor-2 (fgf-2) in bone allografts by western blot analysis at the 4th, 8th, and 12th week after surgery.resultsat the 4th, 8th, and 12th week after surgery, the results of the tests demonstrated that the callus was larger, contained more calcium (p<0.05), and expressed fgf-2 at higher levels (p<0.05) in the experimental group than in the control group. fluorescent bone labeling showed the distance between the two fluorescent ribbons was significantly shorter in the control group than in the experimental group at the 8th and 12th week after surgery.conclusionsvac combined with open bone grafting promoted rabbit bone graft vascularization.
surface sampling was conducted by using swabs, wipes, and hepa vacuum socks. to compare the sampling techniques, we selected locations where the three types of samples could be collected adjacent to each other on nonporous surfaces, with an emphasis on locations believed to be still contaminated with b. anthracis spores. 17), return air ducts, tops of the window boxes along the postal inspector walkways, and the tops of mail sorting bins in a secure area approximately 23 m from dbcs machine no. 17. the order in which the samples were collected varied in a randomized fashion from location to location ; each site was assigned a location number and sampled according to a predetermined, randomized plan. we used the randomized sampling plan to reduce sampling biases that might be caused by nonuniform distribution of spores across surfaces and affected by the order in which samples were collected. seven swab, six wipe, and five hepa vacuum sock samples were collected as control samples ; that is, these samples were handled in the same way as others but not used to sample any surfaces. the purpose of these control samples was to evaluate the potential contamination of sample media, unrelated to actual sample collection. nine additional blank hepa vacuum sock samples were collected to estimate cross - contamination by inserting them into the vacuum nozzle after a hepa vacuum sock sample had been collected and the nozzle cleaned ; these socks were then withdrawn and placed in a sterile conical tube for laboratory analysis. investigators were given written instructions for collecting samples at each selected location (figure 1). the surface areas sampled by each technique were intended to be comparable, but not necessarily equal. in particularly dirty areas, swabs and wipes could not cover as large a surface area as the hepa vacuum sock samples without becoming overloaded ; investigators were instructed to avoid overloading the samples by adjusting the size of the surface sampled. sample instructions for collection of swab, wipe, and hepa vacuum sock samples, brentwood mail processing and distribution center, 2001. for specific location divide the selected space into three sections where each of the three types of surface samples (swab, wipe, hepa vacuum sock) may be collected. follow the random key above to designate which section will be sampled by each method and in which order the samples will be collected (follow top to bottom). the surface areas need not be equal, but should be sufficient to provide adequate sample collection for each method. sample order for location was : 1) collect the hepa vacuum sock sample first and record surface area. after sampling, clean vacuum nozzle with alcohol and insert clean vacuum sock ; remove this sock without sampling to serve as contamination blank. 4) collect an additional wipe sample across the entire area which had been sampled by hepa vacuum sock. 5).collect an additional hepa vacuum sock sample across the entire which had been sampled by wipe. the following procedures, used to collect the three types of surface samples, were recommended for collecting surface environmental samples for culturing b. anthracis (9). the surface samples were all collected after investigators had donned nonpowdered gloves over two pairs of nitrile protective gloves, as part of the personal protective gear. the area of the surface sampled was measured with a tape measure and recorded in square centimeters. swab samples were collected by removing a sterile, rayon (noncotton) swab (environmental swab kit, cdc, atlanta, ga) from a sterile tube, moistening it by inserting it into a second tube which contained a sponge soaked with sterile 1.5 ml of phosphate - buffered saline (pbs) at ph 7.2, and then swabbing the selected surface by moving the swab back and forth across the surface with several horizontal strokes, then several vertical strokes. the swab was rotated during sampling to ensure that the entire surface of the swab was used. after sampling, the swab was returned to its original, prelabeled sampling tube for submission to the laboratory. at every selected location, approximately half the sites were also sampled again with unmoistened dry swabs to compare the sampling efficiency of dry swabs to wet swabs and other techniques. wipe samples were collected on selected surfaces with a 7.62 x 7.62 cm sterile rayon gauze pad (dukal corp., syosset, ny) premoistened with approximately 5 ml sterile water (baxter healthcare corp., deerfield, il). the surface was thoroughly wiped back and forth by using several vertical strokes, folding the exposed side of the pad, and making several horizontal strokes over the same area with the other side of the wipe. the pad was then placed in a prelabeled, 50-ml sterile conical tube and sealed with a cap. hepa vacuum sock samples were collected by inserting a cone - shaped filtering trap (dust collection filter sock ; midwest filtration co., fairfield, oh) into the nozzle of a hepa vacuum cleaner (atrix international inc., the vacuum had an electric motor (120 v, 6.6 a, 1 hp) to provide suction of 28 cubic feet (792.4 l) per min through the vacuum nozzle (figure 2). the plastic sleeve of the dust collection trap was folded over the outside of the nozzle and held in place by hand while the vacuum nozzle was moved slowly back and forth across the sampled surface. the dust collection trap was removed from the vacuum nozzle, placed in a prelabeled, 50-ml sterile conical tube, and sealed with a cap. before inserting a clean sock into the vacuum nozzle and collecting a subsequent sample, the investigator put on a new pair of gloves and wiped the inside of the vacuum nozzle thoroughly with an alcohol wipe, to physically remove contamination from the nozzle surface (not to sterilize the surface because alcohol does not effectively kill b. anthracis spores). to determine whether cross - contamination of subsequent vacuum samples might occur through contamination of the vacuum nozzle during sampling, occasionally a filter sock was inserted into the vacuum nozzle after a sample had been collected and the nozzle cleaned, but the sock was then simply withdrawn and placed in a sterile conical tube for laboratory analysis. swab and wipe samples were extracted in a laboratory operated by the usps contractor at the brentwood facility. the samples were extracted by adding 2030 ml 0.3% tween 20 in pbs to a 50-ml blue falcon screw - top tube (becton dickinson labware, franklin lakes, nj) and vortexing the tube for 3 min. the contents of the tube were allowed to settle for 5 min, and swabs and wipes were removed. the tube was centrifuged at 3,0004,500 rpm, 1530 min at 10c, the supernatant removed by decanting, and the pellet was resuspended in 2 ml 0.3% tween 20 in pbs solution. approximately half the resuspended extract was shipped to cdc bioterrorism surge capacity and anthrax laboratories for culture and confirmatory analysis. the remaining half of the resuspended extract was retained at the laboratory at the brentwood facility for polymerase chain reaction (pcr) analysis (unpub. 0.1 ml of the suspension (approximately 10% of the extract solution) was plated to trypticase soy agar with 5% sheep blood and streaked for quantification. the plates were incubated at 35c37c in ambient air and examined after 24 h and 48 h. suspect colonies were screened by level a procedures for identification of b. anthracis (11). identification of all strains was confirmed by standard microbiologic procedures and the laboratory response network (lrn) testing algorithm (12,13). results of these samples were reported as number of cfus per plate. to estimate cfus per sampled surface area, the number of cfus per plate was multiplied by 20 (2 ml extract solution divided by 0.1 ml plated solution) and divided by the recorded surface area in square centimeters. when the number of colonies on the culture plates exceeded approximately 300, they were reported as too numerous to count. we used the average weight of five unused sock samples to estimate the presampling weight of the vacuum socks ; the average weight of the unused socks was 0.70 g (standard deviation 0.02 g). the average weight was subtracted from the postsampling weight of each sock sample to determine the weight of its contents. approximately 2030 ml 0.3% tween 20 in pbs was added to a 50-ml cup containing the sock and its contents and placed on a shaker for 30 min. the contents of the cup were allowed to settle for 5 min ; the supernatant then was poured into a 50-ml blue falcon screw - top tube (becton dickinson labware). the tube was centrifuged at 3,0004,500 rpm, for 1530 min at 10c ; approximately 90% of the starting volume was then removed. the pellet in the bottom of the tube was resuspended in approximately 2 ml 0.3% tween 20 in pbs, and 0.1 ml (two drops from pasteur pipette) and 0.01 ml (added by using a calibrated loop) of the suspension were plated to two trypticase soy agar plates with 5% sheep blood and streaked for quantification. the plates were incubated and screened, and suspect colonies were identified by using the same laboratory methods used for the swab and wipe samples. results of these samples were reported as cfu / g of material collected ; the estimated weights of the sock contents were also reported. to estimate cfu per sampled surface area, the reported cfu / g were multiplied by the reported weight of the sock contents and divided by the recorded surface area in square centimeters. to evaluate the effectiveness of the wipe and hepa vacuum samples for removing spores from surfaces, at some locations we collected wipe samples over the same surface area previously vacuumed, as well as hepa vacuum samples over the same surface area previously wiped. we compared the relative difference in cfu / cm reported for the two methods to evaluate the removal efficiency of the wipe and hepa vacuum sock samples. operations to decontaminate the brentwood facility had been done since late october 2001 by using hepa vacuums and sodium hypochlorite solutions. swab, wipe, and hepa vacuum sock samples of dbcs machine surfaces that had been cleaned were collected to evaluate the effectiveness of clean - up operations. sample results (positive vs. negative) were analyzed by using simple descriptive statistics, including counts and percents. agreement between paired sampling methods was assessed by using cohen s kappa, a statistical method that measures agreement beyond what would be expected based on chance alone (15). kappa scores 0.75 indicated excellent agreement ; kappa scores between these values indicated fair to good agreement. sample levels (cfu / cm) were analyzed by simple descriptive statistics, including sample median and range. spearman s rank correlation coefficient significance tests based on ranks that do not assume normality were used as a measure of the association between two paired sampling methods (16). agreement between paired sampling methods with respect to ordered categories (0, 0.11.6, 1.715.5, and > 15.5 cfu / cm) was assessed by using kendall s tau - b statistic, which measures ordinal association (17). descriptive statistics for the culture analysis of the dry and wet swab, wipe, and hepa vacuum sock samples are shown in table 1. b. anthracis was cultured from 4 (14%) of 28 dry swab samples, while 36 (54%) of 67 wet swabs were culture positive. fifty - eight (87%) of 67 of the wipe samples and 51 (80%) of 64 of hepa vacuum sock samples were culture positive. although cfus / cm were reported for each positive sample, these results should only be considered semiquantitative ; absolute concentrations can not be directly compared across the sampling methods. however, the calculated concentrations of b. anthracis spores in the culture - positive hepa vacuum sock samples tended to be greater than in the other types of samples. level of b. anthracis (cfu / cm) : negative = 0, low = 0.11.6, medium=1.715.5, and high=>15.5. positive samples only. 232.5 cfu / cm is the maximum value considered too numerous to count for a concentration ; 300 cfu is the maximum value considered too numerous to count for a culture. of the nine blank hepa vacuum samples collected from the vacuum nozzle to estimate cross - contamination, eight were culture negative ; one b. anthracis cfu was detected in one sock. the results of the dry swab samples are compared with results obtained by using the other types of samples (table 2). these results indicate that when corresponding wipe and hepa vacuum sock samples were culture positive for b. anthracis spores, the dry swab samples detected b. anthracis 4 of 23 times. when the corresponding wet swabs were positive for b. anthracis spores, the dry swabs detected b. anthracis 4 of 13 times. at no time were the dry swabs positive while the other types of corresponding samples did not detect spores. two samples from the same location are concordant if both positive or both negative for bacillus anthracis spores. two samples from the same location are discordant if one is positive and the other negative for b. anthracis spores. rs denotes spearman s correlation coefficient between level of b. anthracis (cfu / cm) obtained by using the dry swab method and the level of b. anthracis obtained by using the comparison sampling method.. a total of 58 sets of wet swab, wipe, and hepa vacuum sock samples collected side - by - side were available for comparison, and 67 sets of wet swab and wipe samples collected side - by - side were also available for comparison (table 3). results of wet swab and wipe sample analysis were concordant in 64% of the sample comparisons ; 23 wipe samples were reported as culture positive when the wet swab samples failed to detect spores, and 1 culture - positive wet swab sample was reported when the corresponding wipe sample was culture negative. results of the wet swab and hepa vacuum sock samples were also concordant on 64% of the sample comparisons with similar results as the wet swab and wipe comparison. twenty - one (36%) hepa vacuum samples were reported as culture positive when the wet swab samples were negative, but no culture - positive wet swab samples were ever reported when the corresponding hepa vacuum sock samples were negative. results of hepa vacuum sock and wipe samples were concordant 84% of the time ; when they were discordant, the corresponding hepa vacuum sock and wipe samples did not detect b. anthracis spores about the same number of times (five negative for hepa vacuum sock and four negative wipe samples). only the comparison of hepa vacuum sock versus wipe sample had a cohen s kappa score > 0.4, indicating fair to good agreement (table 3). two samples from the same location are concordant if both positive or both negative for presence of bacillus anthracis spores. two samples from the same location are discordant if one is positive and the other negative for presence of b. anthracis spores. the hepa vacuum sock samples typically collected higher concentrations of b. anthracis spores than both the wet swab and wipe samples, and the wipe samples collected higher concentrations of spores than the wet swab samples (table 4). these comparisons indicate good agreement between the hepa vacuum sock samples and the wipe samples (kendall s tau - b 0.66 ; spearman s rank correlation coefficient 0.81). although wet swabs were correlated with both the hepa vacuum samples and the wipe samples, the agreement was not as strong. level of b. anthracis (cfu / cm) : negative = 0, low = 011.6, medium=1.715.5, and high=>15.5. two samples from the same location agree if they are concordant and are both in the same grouping. the randomly selected surface areas where 13 hepa vacuum sock samples had been collected were immediately sampled again with wipe samples. all the hepa samples were positive for b. anthracis spores ranging in concentrations from 0.5 to 310 cfu / cm. the spore concentrations collected by the subsequent wipe samples (0 to 16 cfu / cm) were usually lower than the original vacuum samples ; only two of the subsequent wipe samples were negative for b. anthracis spores. the surface areas where 12 wipe samples were collected, corresponding to 12 of 13 hepa vacuum sock samples, were immediately sampled again with hepa vacuum sock samples. all the wipe samples were positive for b. anthracis spores, ranging in concentrations from 1.4 to 233 cfu / cm. only one of the subsequent hepa vacuum samples was negative for spores and the concentrations in nine of the hepa vacuum sock samples were virtually the same as on the original wipe samples. the results of the side - by - side comparison of swab, wipe, and hepa vacuum sock samples on nonporous surfaces indicated good agreement between the hepa vacuum sock and wipe samples. however, the hepa vacuum sock and wipe samples agreed poorly with the swab samples. the wet swabs did not detect spores > 33% of the time when spores were detected by the wipe and vacuum sock samples. the dry swabs performed especially poorly, failing to detect spores > 66% of the time when spores were detected by wipe and vacuum sock samples. based on these results, dry swabs should not be used to sample for b. anthracis environmental contamination. applying wet swabs in certain circumstances may be useful, for example, to sample crevices, inside machinery, and places difficult to reach by wipe and hepa vacuum samples ; however, dry swabs should not be used to sample surfaces where wipe and hepa vacuum samples are likely to yield superior results. sampling with wipes and hepa vacuum socks is likely to yield very similar results on nonporous surfaces ; wipes are preferable for sampling surfaces with relatively light dust, while hepa vacuum socks should be selected to sample surfaces with heavy dust. wipes may become quickly overloaded on dusty surfaces and thus unable to cover a large surface area. the sampling sensitivity of hepa vacuum socks may be greater because they can collect large dust loads over much larger surface areas than wipes. the relative difference between the wipe samples and the subsequent hepa vacuum sock samples was not influenced by the initial concentration of spores collected by the wipe samples. after especially dirty areas were sampled with both wipes and hepa vacuum sock samples, residual dirt was often still visible. the samples were collected side by side so that the exact same surface area was not sampled by all methods. because of nonuniform distribution, spore concentrations may have varied across the surfaces sampled by each method. however, we also set the order of sampling as random, making it unlikely that any particular method consistently encountered fewer spores than the other methods. strong differences in these particular results more likely resulted from the sampling technique and not to nonuniform distribution of spores on these highly contaminated surfaces, where the different types of samples were collected very close to each other. in areas likely to have been contaminated over a broad surface at high concentrations (such as dbcs machine no. 17), an adequate number of spores for detection was likely available for all three sampling techniques, but in other, less - contaminated areas, fewer spores were available for detection. if investigators are careful to avoid contamination of the samples, the number of false - positive samples is reduced. however, sampling all surfaces within a building is not practical, and some surfaces containing b. anthracis spores might be missed. the measurements collected in this study were not adequate to evaluate the sampling efficiencies of wipe and hepa vacuum sock samples, particularly since the initial concentrations of spores on the sampled surfaces were unknown. however, sequential hepa vacuum sock samples indicated better collection efficiency on nonporous surfaces than wipe samples. this efficiency is evident because wipe samples collected following vacuum samples were much lower than the initial vacuum samples, while the vacuum samples collected after wipe samples often collected a similar concentration of spores as the initial wipe samples. care was taken after sampling to stay within the previously sampled area, but spores from outside the previously sampled area may have been inadvertently collected by the hepa vacuum samples (e.g., spores from surrounding unsampled areas may have been drawn into the hepa vacuum sock). to avoid contamination of the vacuum when collecting samples, using disposable inserts may be more appropriate, such as cardboard sleeves, which can be placed inside the vacuum nozzle ; the sampling sock can then be inserted into the sleeve and discarded after sampling. disposable inserts may prevent cross - contamination of the vacuum nozzle or subsequent sock samples. care must be taken to prevent contamination of the inserts before they are used for sampling. while vacuum nozzles may not always be completely cleaned after sampling, our investigation indicated that cross - contamination could not be the reason for the high concentrations of spores detected on the numerous hepa vacuum sock samples. the results of this investigation may be used to guide future sampling efforts and serve as a baseline for follow - up measurements after the building has been cleaned further. the sampling and analytical techniques used in our study may provide useful reference for evaluations of other situations in the future. this study provides additional evidence for the need to quantify sampling efficiency to develop the type of limit - of - detection data normally created for other types of sampling and analytical methods. the collection efficiency (removing spores from the surface) and recovery efficiency (removing spores from the sampling media) need to be further evaluated for these methods. our study focused on sampling nonporous surfaces ; under these circumstances, hepa vacuum sock samples and wipe samples performed similarly. however, this level of agreement may be difficult to achieve in sampling porous materials such as carpet and furniture, and the collection efficiency of sampling methods on other surfaces needs to be evaluated. understanding the sampling efficiency of these methods on various types of surfaces is a critical requirement for future efforts to develop numerical criteria for surface contamination and potential exposures to humans. lack of understanding about the efficiency of various sampling methods limits our ability to determine whether an environment has been adequately cleaned.	during an investigation conducted december 1720, 2001, we collected environmental samples from a u.s. postal facility in washington, d.c., known to be extensively contaminated with bacillus anthracis spores. because methods for collecting and analyzing b. anthracis spores have not yet been validated, our objective was to compare the relative effectiveness of sampling methods used for collecting spores from contaminated surfaces. comparison of wipe, wet and dry swab, and hepa vacuum sock samples on nonporous surfaces indicated good agreement between results with hepa vacuum and wipe samples. however, results from hepa vacuum sock and wipe samples agreed poorly with the swab samples. dry swabs failed to detect spores > 75% of the time they were detected by wipe and hepa vacuum samples. wipe samples collected after hepa vacuum samples and hepa vacuum samples after wipe samples indicated that neither method completely removed spores from the sampled surfaces.
in most high - income countries access to good quality, evidence - based health - care continues to improve. because of the overwhelming burden of disability caused by mental health problems throughout the world, many high - income countries have begun to address the inequity of access to mental health care with improvements in policies ; training ; work - force ; primary, secondary, and tertiary services ; programmes ; and public education. as a recent who report concludes, people with mental health conditions are among the most marginalized and vulnerable groups. it is important that they be empowered to reach their goals and participate fully in society. governments in many developed countries are recognizing the importance of integrating mental health into primary care services and general hospitals, as well as providing public education, and protecting and promoting the human rights of people with mental disabilities. however for those in many low- and middle - income countries (lamics), globalization, urbanization, and rapid social change are part of a slowly worsening profile for mental health and the resources for the management of these problems do not match the increased need. experience in china, india, and eastern europe has alerted health professionals to the importance of preparing the health workforce in countries where the traditional culture is being replaced by a more modern westernised culture. for many lamics there are many barriers to making good quality mental health care available and until this is addressed the disability and suicide rates due to mental health problems are likely to increase. in some lamics there has been an increase in tertiary - level mental health facilities which will only be available to very few people because of access and affordability. making mental health care available to all should also involve primary health care practitioners, traditional healers, teachers, police, corrections, and community leaders a whole - of - community approach that reflects the complex nature of mental health aetiology and management. vanuatu, with a population of about 220,000 people, has 22 doctors and 450 nurses spread over 83 islands. this very small health workforce mostly operates at a primary care level ; there are no full - time publicly available mental health professionals and only a few people with any mental health training at all. building up, supporting and mentoring a mental health workforce, reducing stigma in the community, training the informal mental health workforce, and advocating with government and ngos are all important aims of the pacific islands mental health network (pimhnet), a who initiative funded by nzaid. jb and dp, both general practitioners (family physicians) in australia, have developed and implemented intensive 3-week mental health training programmes to doctors, nurse practitioners, nurses, ngo workers, teachers, police, corrections, public health officers, and nursing lecturers over 2009 and 2010. the training has included case studies drawn from the experience of the group, an emphasis on primary health care skills, supervised visits to local villages, and the hospital and simple research techniques. in 2010 jb visited many of these professionals in their place of work, often on remote islands, to supervise, mentor, and further train them. further visits in 2011 will aim to consolidate skills so that more health professionals can be trained and there is a strong network in the country of support for each other. poverty, lack of education, unemployment, conflict, disasters, and gender imbalance are well known as the social determinants of mental health [1, 3 ]. mental disorders account for 8.8% of the total burden of disease in lamics and the additional risks of urbanization, loss of tradition, loss of subsistence farming activity, and loss of extended family mean that those who live in countries in economic transition such as the pacific islands are likely to have a worsening mental health profile [2, 3 ]. any figures on mental health do not take into account the disability experienced within the family unit, their community, or their country because of the mental illness [3, 5 ]. the treatment gap for serious mental health disorders in lamic is 7685%, and even for those who are treated, it is often sporadic or with inadequate doses of outdated medication. as deaths decrease from infectious diseases such as measles, pneumonia, gastroenteritis, tuberculosis and malaria, there has been an increase in suicides and alcohol - related deaths in countries such as eastern europe, india, south america, and china [2, 3, 6 ]. the few resources that are available are often spent on tertiary institutions that are inaccessible for the majority of the population. ngos and governments need to work together to ensure that mental health is given priority and that this is embedded in the primary care workforce. one - off western - style training has not been found to be successful, whereas it is both efficient and cost - effective to train primary care workers in a culturally appropriate manner in psychological and pharmacological interventions and then to ensure that they are supervised and supported [7, 8 ]. such a strategy will not only decrease morbidity, comorbidity, and mortality but can also encourage resilience, problem - solving, and social support and hence improve the prevention of mental health disorders and decrease referral to high - cost tertiary institutions. including ngos, community organizations and traditional resources such as chiefs, traditional healers, and religious pastors as part of mental health training will ensure a more holistic and sustainable approach for addressing mental health problems [1, 5 ]. the world health organization (who) and world organization of family doctors (wonca) recently collaborated to write a report entitled integrating mental health into primary care. a global perspective that sets out seven good reasons for integrating mental health into primary care and 10 principles for integrating mental health into primary care (see boxes herein after). box 1seven good reasons for integrating mental health into primary care are as follows.the burden of mental disorders is great.mental and physical health problems are interwoven.the treatment gap for mental disorders is enormous.primary care for mental health enhances access.primary care for mental health promotes respect of human rights.primary care for mental health is affordable and cost effective.primary care for mental health generates good health outcomes. seven good reasons for integrating mental health into primary care are as follows.the burden of mental disorders is great.mental and physical health problems are interwoven.the treatment gap for mental disorders is enormous.primary care for mental health enhances access.primary care for mental health promotes respect of human rights.primary care for mental health is affordable and cost effective.primary care for mental health generates good health outcomes. primary care for mental health generates good health outcomes. box 2 10 principles for integrating mental health into primary care are as follows.policy and plans need to incorporate primary care for mental health.advocacy is required to shift attitudes and behaviour.adequate training of primary care workers is required.primary care tasks must be limited and doable.specialist mental health professionals and facilities must be available to support primary care.patients must have access to essential psychotropic medications in primary care.integration is a process, not an event.a mental health service coordinator is crucial.collaboration with other government nonhealth sectors, nongovernmental organizations, village and community health workers, and volunteers is required.financial and human resources are needed. 10 principles for integrating mental health into primary care are as follows.policy and plans need to incorporate primary care for mental health.advocacy is required to shift attitudes and behaviour.adequate training of primary care workers is required.primary care tasks must be limited and doable.specialist mental health professionals and facilities must be available to support primary care.patients must have access to essential psychotropic medications in primary care.integration is a process, not an event.a mental health service coordinator is crucial.collaboration with other government nonhealth sectors, nongovernmental organizations, village and community health workers, and volunteers is required.financial and human resources are needed. policy and plans need to incorporate primary care for mental health. collaboration with other government nonhealth sectors, nongovernmental organizations, village and community health workers, and volunteers is required. the need for more mental health services in the pacific region grew out of a situational analysis conducted by who which showed that most pacific nations had growing rates of mental health problems (including suicide) but had few resources to tackle the burden. pimhnet was formally launched in march 2007 in response to requests by ministers of health of pacific island countries (meeting of pacific islands ministers of health march 2005 samoa). highest standards of mental health and well - being through access to effective, appropriate, and quality mental health services and care. its mission is to facilitate and support cooperative and coordinated activities within and among member countries that contribute to sustainable national and subregional capacity in relation to mental health. in consultation with its 18 pacific island members, several priority areas were identified which needed to be addressed : human resource development and training, service development focused on primary care, since its inception three countries have mental health policies in place with another nine having draft policies. thirteen countries have developed plans for human resource development and training and seven of these are already implementing them. these processes are not sequential and so, for instance, in vanuatu, the implementation of the human resource development and training plan proceeded prior to the formal adoption of the mental health policy and strategic plan vanuatu is defined by the un as a least developed country with approximately 75% of the population living in rural areas. accessing health services is very difficult for most people because most of the health services are in cities and large villages and there is a wide distribution of the population and often difficult terrain. as with many lamics there is a migration of healthcare workers out of the country, primarily to australia and new zealand, and training in any specialty area including mental health is generally conducted overseas with the assistance of fellowships. the health services in vanuatu are divided between the northern health group which has a main referral hospital at luganville on espiritu santo and the southern healthcare group with vila central hospital on efate island. there are 5 smaller provincial hospitals ; 29 health centres, each with a nurse or nurse practitioner ; 74 dispensaries, each with a nurse ; and 123 health aid posts and 6 mobile clinics, each with a community worker who does health promotion. vanuatu has been a subsistence society with a culture of traditional problem - solving through chiefs in each village. there are over 110 different mother - tongues spoken in the country and these are the languages where the words for mood, philosophical concepts, and deeper traditional ideas are found. the common language of bislama is a trade language with no direct translations of any of the western words for mental health such as depression or anxiety and only a limited mood vocabulary. social problems have always been taken to the chief to resolve, mostly in a fairly autocratic manner but each chief has had a different style and collaborative approach. human rights issues for people with severe psychosis depended on the support of the community, the family, how dangerous they were, and what contact they had had with the criminal justice system. people with severe untreated schizophrenia, bipolar disorder and brief psychotic disorder have been given traditional medication and religious exorcism and cared for by their family and community. women tended to move to their husband 's village and look after the extended family, usually sharing gardening tasks with their husband. life has revolved around traditional ceremonies, tending the garden, caring for the houses and village and family issues. as subsistence farming has decreased, young men have moved to the cities to drive taxis and try to get jobs. this urbanization and selling of land has broken many of the family traditions, increased poverty, increased unemployment, decreased social support, and so forth. young people in the cities are bored and have increased substance misuse and interactions with police. women are now more educated and want to choose between education, career, marriage, and children. the chiefs themselves are often feeling overwhelmed with the stress of their positions and the change in their status and are at risk of depression. the church and pastors have always had a very important role in counseling and problem - solving for those in their congregation and have mostly been the only source of mental health care available for many people. however a recent survey by a psychiatrist from australia found that there was a great deal of misunderstanding about mental illness both in the church leaders and in the congregations. 45% of those surveyed thought that mental illness was a sin, 34% due to demon possession, and 38% due to a curse. as the country changes, the health system must change with it to take into account the new illnesses that are likely to emerge. based on prevalence rates from the world mental health survey in 2004, it is estimated that 13% of the adult population are likely to have a mental health disorder, 3% of these severe. however in 2007 only 48 people in vanuatu were recorded to have received treatment for a mental disorder indicating a treatment gap of over 99%. jb and dp, two general practitioners from australia who have an interest in mental health, were asked by the who to develop and present a programme for pimhnet over 3 years. this involved developing a culturally appropriate mental health training programme, training key health, and social service personnel ; supervising those who have been trained in their places of work ; mentoring and setting up support networks for them ; presenting more advanced training ; and training local doctors and nurses to be mental health trainers and mentors themselves. jb and dp also spent time networking with the formal and informal mental health stakeholders in vanuatu, including hospital administration, government, church organization, and nongovernment organizations. box 3training visits for health workers and the community are as follows : first visit : nurses from vila central hospital (4), ngo workers (3), nursing lecturers from vila (2), military medic (1), government health administrators (2), and doctors from vila central hospital (2);second visit : 8 nurses, nurse practitioners and nurse aides from outlying islands (three nurses from santo, one nurse from pentecost, one nurse practitioner from tanna, one nurse practitioner from malekula, one nurse from torba, one nurse 's aid from the local village of erakor), and 5 social services personnel (one nurse who works in administration in the department of health, a chief from an ngo on santo, a senior education department administrator, a correctional services officer, and the senior sergeant of police) ; in every session there was someone trained during the first visit to assist with the training ; they had done a two - day train the trainer course before the start of the training;third visit : visiting all the doctors, nurses, and social services personnel in their workplaces ; discussing cases, giving additional training, mentoring, seeing difficult patients, and training those people the nurses had trained. training visits for health workers and the community are as follows : first visit : nurses from vila central hospital (4), ngo workers (3), nursing lecturers from vila (2), military medic (1), government health administrators (2), and doctors from vila central hospital (2);second visit : 8 nurses, nurse practitioners and nurse aides from outlying islands (three nurses from santo, one nurse from pentecost, one nurse practitioner from tanna, one nurse practitioner from malekula, one nurse from torba, one nurse 's aid from the local village of erakor), and 5 social services personnel (one nurse who works in administration in the department of health, a chief from an ngo on santo, a senior education department administrator, a correctional services officer, and the senior sergeant of police) ; in every session there was someone trained during the first visit to assist with the training ; they had done a two - day train the trainer course before the start of the training;third visit : visiting all the doctors, nurses, and social services personnel in their workplaces ; discussing cases, giving additional training, mentoring, seeing difficult patients, and training those people the nurses had trained. first visit : nurses from vila central hospital (4), ngo workers (3), nursing lecturers from vila (2), military medic (1), government health administrators (2), and doctors from vila central hospital (2) ; second visit : 8 nurses, nurse practitioners and nurse aides from outlying islands (three nurses from santo, one nurse from pentecost, one nurse practitioner from tanna, one nurse practitioner from malekula, one nurse from torba, one nurse 's aid from the local village of erakor), and 5 social services personnel (one nurse who works in administration in the department of health, a chief from an ngo on santo, a senior education department administrator, a correctional services officer, and the senior sergeant of police) ; in every session there was someone trained during the first visit to assist with the training ; they had done a two - day train the trainer course before the start of the training ; third visit : visiting all the doctors, nurses, and social services personnel in their workplaces ; discussing cases, giving additional training, mentoring, seeing difficult patients, and training those people the nurses had trained. box 4programme of presentations for pimhnet in vanuatuare as follows : what is mental illness?depression, anxiety, communication skills, structured problem - solving, cognitive behavioural therapy, narrative and family therapy, alcohol and substance abuse, risk management and crisis intervention, psychosis and bipolar disorder, personality disorders, child mental health, adolescent mental health, mental health and the elderly, research skills. programme of presentations for pimhnet in vanuatuare as follows : what is mental illness?depression, anxiety, communication skills, structured problem - solving, cognitive behavioural therapy, narrative and family therapy, alcohol and substance abuse, risk management and crisis intervention, psychosis and bipolar disorder, personality disorders, child mental health, adolescent mental health, mental health and the elderly, research skills. communication skills, structured problem - solving, cognitive behavioural therapy, narrative and family therapy, alcohol and substance abuse, risk management and crisis intervention, psychosis and bipolar disorder, personality disorders, adolescent mental health, mental health and the elderly, during the first visit to vanuatu for training, a combination of teaching methods was used. the mornings were taught using an interactive didactic format and the afternoons in workshop style. in the afternoons participants brainstormed topics in groups, role - played what they had learnt in the morning, and refined skills in small group discussions. the content of the case studies and role - plays were built up over the course of the training as jb and dp developed an understanding of the problems that caused mental health issues and how they might present in vanuatu. many of these were very different to those in australia for instance, the use of kava causing social disruptions in families, the strong religious culture in the community, the role of chiefs in resolving family and community issues, arranged marriages, the importance of making lap - lap (traditional dish made with ground taro or yam roots), young men coming to port vila to find work, the controversy about the use of marijuana, and so forth. because of the importance of keeping the presentations relevant, culturally appropriate and in - tune with the needs of the participants, most of the sessions were prepared during the first visit to vanuatu. both jb and dp have had about 30 years of experience as general practitioners (family physicians) in australia. many resources were used from the who but other important ones included jb 's book on mental health across cultures, where there is no psychiatrist by patel, and essential skills for mental health carea handbook from ghana. the training sessions during the second visit were more difficult as the group that had been identified for training comprised some of the most highly trained nurse practitioners in vanuatu and others who had no health background or experience at all. the first day was spent discussing the challenge of how to prevent the disability associated with mental health problems becoming so severe that the only avenues available to manage the situation were hospitals, police, corrections, church elders, and so forth. this was a very useful discussion because it enabled this very diverse group to focus on a common goal. even though they came from such different backgrounds, after about the third day, the differences had almost disappeared and everyone seemed to be working at a similar level. one of the differences between the participants was the issue of clinical care, something the nurses were very familiar and comfortable with. the social services personnel brought a different perspective to many of the sessions because they insisted on discussing the philosophy behind strategies such as confidentiality, parenting, the role of spirituality in mental health, and human rights. as with the first training group, the postworkshop questionnaire showed that everyone had enjoyed the course very much, that they thought it was relevant to their current situations, that it had changed the way they related to patients with mental health problems, that their confidence in diagnosing and treating mental health problems had increased because of the course, and that they could help people with mental health problems have better lives. most thought they could make a management plan for people with depression, anxiety, suicidal risk, crisis, or substance abuse but still felt tentative about being able to develop management plans for those with schizophrenia, bipolar disorder, or somatisation. the practical nature of this training was very different to the mental health training that some of the participants had experienced in the past. previous training was theoretical, with an emphasis on written material and dsmiv and icd10 diagnoses, and as a consequence health workers were not able to apply any new knowledge or skills in the clinical setting. as general practitioners, jb and dp were able from the outset to emphasise the acquisition of skills that were practical and relevant. for instance, participants were encouraged to grade patient 's illness on a likert scale from 1 to 10 but also to scale the disability on a scale from 1 to 10. they were then able to ask what would make the patient better or worse, prepare an appropriate risk management plan, and mobilize the patient 's resources. drawings such as the social atom to discuss social structure and family trees for children and the hand exercise that helps access social and personal assets were very popular. the use of stories and humour and discovery of resilience in narrative therapy and the cultural awareness tool which helps a health professional find the meaning of a situation for the patient were thought to be two particularly useful tools. these are tools that help explore the patient 's understanding of their beliefs and access their own resilience. the cultural awareness tool is a series of patient - centred questions, and in narrative therapy the problem is externalized and the patient is more able to access their own resources for dealing with their problems. respect for the diverse traditional, cultural, and religious beliefs in the group was also extremely important but challenging and using differences in beliefs as problem - solving exercises proved the immediate relevance of the use of this tool. slow breathing and relaxation techniques were practiced every day until everyone in the group felt confident that they could teach these techniques to patients with panic and anxiety disorder. as the skills of the groups improved, the cases developed for the training became more realistic and difficult and they were able to revise and consolidate their knowledge and develop and communicate management plans for the role - playing cases. dp spent time with the first group on her second visit, consolidating their skills and exploring new concepts and strategies with them. this deepened their understanding and improved their confidence in using techniques such as cbt. only the nurse practitioners and doctors in the group (4 people) were able to prescribe medication ; so the emphasis in the course was on diagnosis, assessment, and psychotherapy. the third trip involved jb and the main mental health nurse from vila central hospital travelling to five different islands, some of them very remote with limited electricity, accessibility, or support for the few health professionals there. seeing people in their own communities was found to be of the utmost importance. it helped with skill development ; it raised the profile of mental health and encouraged local people and other health professionals to trust the nurses who had been trained ; it gave the nurses confidence that they were on the right track ; it highlighted the importance of mental health being embedded in the primary health care model. the list of mental disorders was almost identical to that which a gp in australia would see, except that most of them had no previous treatment. because there are no words for mental health in bislama, clinics were advertised as sik long bran (brain clinics) and so physical illnesses which can affect mental health were seen such as grand mal seizures, petit mal seizures, temporal lobe epilepsy, parkinsons disease, anaemia, vitamin b12 deficiency, hyponatraemia, liver disease, sequelae of encephalitis, sequelae of cerebral malaria, hypertension, and migraine. all of these except hypertension were clinical diagnoses as there were absolutely no facilities for testing or referral. the rest of the patients had depression, anxiety disorders, bipolar disorder, schizophrenia, substance abuse, personality disorders, and so forth. the only other difference was that of brief psychotic disorder which is rarely seen in developed countries. this is often associated with religious experiences as jb found in vanuatu and lasts less than a month with a return to premorbid functioning. western - based ideas of confidentiality, screening tools, pattern recognition, appropriate places for consultations, management plans, and follow - up all needed to be modified to suit the environment. consultations were held in many diverse places such as in the grass huts that served as remote nurse aid posts, in patient 's houses, in the centre of the village, and in more formal settings in the hospital. almost all patients came with family or community members, with up to ten people accompanying very sick patients with schizophrenia. sometimes two local interpreters were needed as patients did not speak bislama but only the local mother tongue. in every area the focal nurses, and nurse practitioners had trained other nurse practitioners, nurses and nurse aids in basic mental health awareness, diagnostic techniques, and treatment skills. they had also been considerably active in raising awareness about mental health problems in their communities. everywhere the team went there were more people than could possibly be seen during a short visit. many of the patients had been identified by the nurse aid who had been trained by the nurses that jb and dp had trained. in some places the local chief accompanied the patient and identified other patients who needed to be seen, and others responded to signs or announcements in church. all of the untreated patients with very disruptive psychosis had been treated with custom medication and had a traditional understanding of what was causing the problem. one of the most powerful health promotion tools was to say that people all over the world have this same disease with very similar symptoms and that this is an illness and not black magic. untreated psychosis is an enormous burden and worry for families and there is a great deal of stigma associated with the community thinking there is a curse on the person. many of the nurses in remote areas were feeling quite lonely in their quest to diagnose and treat mental health problems and were very pleased to have someone to listen to their successes and problems. working through case studies and actually seeing patients together was a fertile ground for further training and such supervision is invaluable. because the participants of the training on mental health worked in very different workplaces, they found that different skills were useful for them. so, for instance, the nurse in emergency at vila central hospital had enormous success with using the slow breathing techniques for people who were coming in regularly with the chief had used structured problem - solving to change the way he dealt with the people who came to see him. the nurse in the paediatric ward had used family therapy a great deal and wanted to expand her understanding of this skill. one of the doctors had found it very useful to be able to properly assess the risk of suicide and use some cbt techniques. she had also started to use medication for more people, as she felt more confident about it. one of the remote nurses had found simple narrative therapy very useful and had taught the nurses aids in the area basic diagnostic skills, the hand exercise, slow breathing, and narrative therapy skills. one afternoon a week and had the likert scales for disease and disability and the hand exercise in every patient 's notes. he had taught the two other nurses who worked with him so that they were also now doing this. the nurse in vila central hospital who had done further training in australia was seeing a lot of people with schizophrenia and was doing a full mental state examination on all these patients which gave him a clearer picture of the their mental state. the police and corrections people saw a lot of people with substance abuse problems and found the assessment tools and motivational interviewing very useful. all of these practitioners were very enthusiastic about having more training as they were often seeing rapid changes in the attitudes, functionality, health, and social circumstances of patients who had previously been struggling with mental health problems but had not been able to alter the situations they were in. all the participants were eager to teach their colleagues what they had found useful and some of those who had been taught in this way had begun to teach others. as well as the training, jb and dp spent time networking with mental health stakeholders in vanuatu, advocating with the government for more resources in mental health, mental health promotion, liaison with churches, development of templates and questionnaires for use in the communities and in hospital, and so forth. by the second visit in 2009, the vanuatu mental health policy and strategic plan (20092014) had been approved and many of the students were actively using the techniques and instruments to assist people with mental health problems in the community and the hospital. an australian volunteer psychologist had been recruited and based in the community and support had been gained from chiefs, the church, and other ngos to assist with mental health problems in several villages. the hospital was proceeding with delivery of mental health care and is working on a process to facilitate communication between the hospital and the community. education in mental health was proceeding in other areas such as the military, police, and education department. teaching evaluation and basic research skills was seen to be an integral part of the training as well as the importance of evaluating any programme so that its usefulness could be assessed and lessons learned. the groups discussed the importance of ethics and confidentiality and validated the translations of research tools. the kessler 10 (k10) has been translated into many languages but not into bislama. the debate highlighted the value of a formal validation process if the k10 was going to be used as a research tool, the caution needed when using such a tool to screen people for mental health problems, and the importance of using clinical judgement rather than a tool to make a diagnosis of mental illness. patients in the hospital and people in several of the surrounding villages were assessed using these versions of the k10. none had been given a medical diagnosis of mental illness and none were currently being treated for psychological issues, stress, or mental health problems. the collation and analysis of the results was a way of helping health workers understand simple mental health assessment procedures, statistical, and research techniques but lacked the thorough methods required for rigorous research. of the 80 people (40% male, 60% female) interviewed at vila central hospital, 25% had severe mental health distress particularly vulnerable groups were women in the antenatal clinic (30% scored over 30) and men in the 4049 year age group (75% in the severe range). comments from learners included a remark that most people were happy to be interviewed as no one usually asked them about these issues. other participants commented that doing the social atom helped the interviewee stand apart from her problem and look at it, and that doing the hand exercise helped people identify their personal resources. work in vanuatu as part of who pimhnet has shown that equity of access to mental health care is possible when it is integrated into primary care, the workforce is properly trained and mentored, and the government supports the process. as with many lamics, the high need for mental health care in the western pacific region was not being met because of lack of workforce, lack of culturally appropriate and practical training, limited financial resources, stigma, and the isolation of many communities. training the primary health care workforce has begun to address this inequity. with the support of who and nzaid, further visits to vanuatu will support those already trained and begin to target those who can be trainers and mentors themselves. it is the beginning of a new era in mental health care in pacific island countries.	inequity in health - care delivery for those with mental illness is widespread throughout low- and middle - income countries. in the pacific island countries there are many barriers to addressing the growing mental health burden. in an effort to address this problem, the who is coordinating the pacific islands mental health network involving 18 countries in the pacific region with the financial support of new zealand aid (nzaid). jb and dp have developed and presented mental health training to health professionals, community leaders, and social service personnel in an environment in vanuatu that is very different from that of their usual australian - based general practices. they discuss evidence for their work, an outline of the programme, some difficulties working across different cultures, and the enthusiasm with which the training has been greeted. vanuatu is now well on its way to addressing the inequity of access to mental health care with a culturally appropriate and self - sustaining mental health workforce.
the human eye is prone to a number of monochromatic aberrations, including spherical, coma, astigmatism, field - curvature, and image - distortion aberrations, all of which are principally generated in the cornea and the crystalline lens.1,2 the most common aberration (spherical aberration) occurs when light waves which are parallel to the optic axis but at different distances from the optic axis fail to converge to the same point, producing a circular blurred image.3 in contrast, coma aberrations occur when light waves focus either side of the plane producing a tear - drop-shaped image. removal of the crystalline lens followed by insertion of an intraocular lens (iol) during cataract surgery provides an opportunity to modify the spherical aberration of the eye, thereby improving visual function.46 to do this, aspherical iols have been designed that either : 1) nullify the spherical aberration of the whole eye, thereby focusing an image at the plane of focus with good contrast ; 2) reduce the spherical aberration of the whole eye to approximately 0.1 m (pupil diameter 6 mm ; a value typically found in healthy young people) ; or 3) decrease the spherical aberration of the iol to approximately zero, thereby retaining the spherical aberration of the cornea.79 numerous studies have reported the misalignment of iols in postoperative eyes.1013 in these studies, average levels of decentration and tilt were 0.300.16 mm and 2.621.14, respectively.4 given that misalignments can lead to a reduction in visual performance for some, but not all iols, it is important to assess the impact of misalignment on the quality of the retinal image for individual lenses.10,12,14,15 marcos investigated the optical performance of eyes and found that the optical quality with aspherical iols was better but tolerance to defocus was lower.16 the theoretical effect of decentration of aspherical iols (spherical aberration 0.287 m) was reported by wang and koch.17 however, in these studies, they evaluated only one type of aspherical iol. the purpose of the present study was therefore to create a model eye with aligned or misaligned spherical and aspherical iols in order to measure the optical properties (wave front aberration, modulation transfer function [mtf ], point spread function [psf ], and retinal image) of iols with various degrees of spherical aberration correction. the lens for the cornea of the model eye was made from optical glass s - bsl 7 (ohara inc., kanagawa, japan) with a power of 40 d and a spherical aberration of 0.25 m (the corneal spherical aberration of a typical human eye). the aperture was set in front of the iol, and the diameter needed to produce an image of the pupil with a diameter of 6 mm on the cornea was determined (figure 1). the first was without misalignment, the second was decentered downwards by 0.5 mm along the y - axis, and the third was rotated clockwise to produce a tilt of 5.0 around the x - axis. these values were selected to reflect the misalignments reported by clinical studies.18 wave front aberration measurements were performed with a front - open hartmann shack (hs) aberrometer codeveloped by topcon (tokyo, japan) and aston university (birmingham, uk).19 the model eye was fixed to the aberrometer using a specially designed holder, and the axes of both instruments were aligned. the high - order wave front aberration of the model eye was expanded to the sixth order of zernike polynomials with a 6 mm diameter. third- and fourth - order aberrations of zernike terms were also investigated. in order to obtain a clear psf for each iol, measurements were performed three times without removing the iols, and the best psf for each iol was used for analysis. the zernike polynomial is an orthogonal function defined within a unit circle with a radius of 1. defocused mtf and psf measurements were performed using a matrix - plus (unipulse corp., tokyo, japan) in which the fundus had been detached from the model eye. the matric - plus is a piece of lens - testing equipment that measures the mtf, describing the optical performance of lenses and optical systems ; it is accredited by the united kingdom accreditation service. the distance between the pinhole and the model eye was fixed at 50 cm (2 d). the best image surface was set at a position where the mtf value with 30 lines / mm became maximal, and the 1.5 d defocus measurement was performed at a 0.15 d (0.05 mm) step. because the intensity of the psf varied considerably depending on the aberration or the defocus status, the exposure time for the psf image was adjusted so the intensity at the center was approximately constant. simulated landolt rings were obtained using software developed in - house. from the third- and fourth - order zernike coefficients, the position of the best image surface with the highest strehl ratio was obtained. the zernike defocus term (z20) corresponding to 0.5 d from the focused position was used to obtain the defocused image of the landolt ring. four types of three - piece acrylic iols were examined, each with a power of + 20 d. the first of these iols (a) was spherical, whereas iols b, c, and d were aspherical. the corrected values of spherical aberration were 0.27 m for iol b (corneal aberration fully corrected on average), 0.17 m for iol c (whole eye aberration similar to that found in young subjects [0.1 m ]), and 0.04 m for iol d (iol spherical aberration value approximately 0). each iol was fixed in three different types of holder, and wave front aberration, defocused mtf, and defocused psf were measured sequentially. landolt ring simulations were performed based on the high - order aberrations measured by the hartmann the lens for the cornea of the model eye was made from optical glass s - bsl 7 (ohara inc., kanagawa, japan) with a power of 40 d and a spherical aberration of 0.25 m (the corneal spherical aberration of a typical human eye). the aperture was set in front of the iol, and the diameter needed to produce an image of the pupil with a diameter of 6 mm on the cornea was determined (figure 1). the first was without misalignment, the second was decentered downwards by 0.5 mm along the y - axis, and the third was rotated clockwise to produce a tilt of 5.0 around the x - axis. wave front aberration measurements were performed with a front - open hartmann shack (hs) aberrometer codeveloped by topcon (tokyo, japan) and aston university (birmingham, uk).19 the model eye was fixed to the aberrometer using a specially designed holder, and the axes of both instruments were aligned. the high - order wave front aberration of the model eye was expanded to the sixth order of zernike polynomials with a 6 mm diameter. third- and fourth - order aberrations of zernike terms were also investigated. in order to obtain a clear psf for each iol, measurements were performed three times without removing the iols, and the best psf for each iol was used for analysis. the zernike polynomial is an orthogonal function defined within a unit circle with a radius of 1. defocused mtf and psf measurements were performed using a matrix - plus (unipulse corp., tokyo, japan) in which the fundus had been detached from the model eye. the matric - plus is a piece of lens - testing equipment that measures the mtf, describing the optical performance of lenses and optical systems ; it is accredited by the united kingdom accreditation service. the distance between the pinhole and the model eye was fixed at 50 cm (2 d). the best image surface was set at a position where the mtf value with 30 lines / mm became maximal, and the 1.5 d defocus measurement was performed at a 0.15 d (0.05 mm) step. because the intensity of the psf varied considerably depending on the aberration or the defocus status, the exposure time for the psf image was adjusted so the intensity at the center was approximately constant. simulated landolt rings were obtained using software developed in - house. from the third- and fourth - order zernike coefficients, the position of the best image surface with the highest strehl ratio was obtained. the zernike defocus term (z20) corresponding to 0.5 d from the focused position was used to obtain the defocused image of the landolt ring. four types of three - piece acrylic iols were examined, each with a power of + 20 d. the first of these iols (a) was spherical, whereas iols b, c, and d were aspherical. the corrected values of spherical aberration were 0.27 m for iol b (corneal aberration fully corrected on average), 0.17 m for iol c (whole eye aberration similar to that found in young subjects [0.1 m ]), and 0.04 m for iol d (iol spherical aberration value approximately 0). each iol was fixed in three different types of holder, and wave front aberration, defocused mtf, and defocused psf were measured sequentially. landolt ring simulations were performed based on the high - order aberrations measured by the hartmann wave front aberration measurements show that spherical aberrations for iols a, b, c, and d were 0.36, 0.05, 0.11, and 0.28 m, respectively (figure 2, iols a, b, c, and d). peak mtf values increased concomitantly with a decrease in spherical aberration (figure 3a, iols a, b, c, and d). the vertical and horizontal mtf values were nearly equal, which suggests coma aberration was not induced. when the degree of spherical aberration correction was zero or small (ie, the spherical aberration in the eye was large), a small point image was observed (figure 4a, iols a and d). the size of the point image did not change even when defocus was added, which suggests the depth of focus for these iols was large. in contrast, the image point for iols with a high degree of aberration correction enlarged, and the tail became narrower, when defocus was added (figure 4a, iols b and c). landolt ring simulations show that the depth of focus increased when the degree of spherical aberration correction was zero or small (figure 5a, iols a and d). however, images produced by iols with a high degree of spherical aberration correction were clear at the plane of focus. these results are consistent with the observed mtf values (figure 5a, iols b and c). wave front aberration measurements for decentered or tilted iols show that the degree of spherical aberration was similar to that observed for iols without misalignment (figure 2, iols a, b, c, and d). however, vertical coma aberrations for decentered iols (0.12, 0.21, 0.10, and 0.03 m for iols a, b, c, and d, respectively) and tilted iols (0.15, 0.36, 0.25, and 0.03 m for iols a, b, c, and d, respectively) were proportional to the spherical aberration corrective power of the lens. vertical coma aberrations with tilted iols were larger than those observed with decentered iols. for iols that induced a high level of vertical coma aberration, defocused mtf values decreased for both decentered and tilted iols, whereas the differences between vertical and horizontal mtf values increased (figure 3a, iols b and c). characteristic vertical coma aberrations showing an upward tail were observed at the defocused plane of 2.0 d and at 1.0 d (figure 4b, iols b and c and figure 4c, iols b and c). at the plane of focus, landolt ring images produced by decentered iols with a high spherical aberration corrective power were clear even in the presence of vertical coma aberration, whereas vertical coma aberration led to blurred images at the plane of defocus (figure 5b, iols b and c). for the same iols without decentration but with tilt, blurring due to vertical coma aberrations was large at the plane of focus and even larger at the plane of defocus (figure 5c, iols b and c). for misaligned iols with little or no spherical aberration correction, levels of vertical coma aberration were low (figure 2, iols a and d) ; defocused psf (figure 4b and c, iols a and d) and landolt ring simulations (figure 5b and c, iols a and d) were not significantly affected by defocus ; and the depths of focus were high. wave front aberration measurements show that spherical aberrations for iols a, b, c, and d were 0.36, 0.05, 0.11, and 0.28 m, respectively (figure 2, iols a, b, c, and d). peak mtf values increased concomitantly with a decrease in spherical aberration (figure 3a, iols a, b, c, and d). the vertical and horizontal mtf values were nearly equal, which suggests coma aberration was not induced. when the degree of spherical aberration correction was zero or small (ie, the spherical aberration in the eye was large), a small point image was observed (figure 4a, iols a and d). the size of the point image did not change even when defocus was added, which suggests the depth of focus for these iols was large. in contrast, the image point for iols with a high degree of aberration correction enlarged, and the tail became narrower, when defocus was added (figure 4a, iols b and c). landolt ring simulations show that the depth of focus increased when the degree of spherical aberration correction was zero or small (figure 5a, iols a and d). however, images produced by iols with a high degree of spherical aberration correction were clear at the plane of focus. these results are consistent with the observed mtf values (figure 5a, iols b and c). wave front aberration measurements for decentered or tilted iols show that the degree of spherical aberration was similar to that observed for iols without misalignment (figure 2, iols a, b, c, and d). however, vertical coma aberrations for decentered iols (0.12, 0.21, 0.10, and 0.03 m for iols a, b, c, and d, respectively) and tilted iols (0.15, 0.36, 0.25, and 0.03 m for iols a, b, c, and d, respectively) were proportional to the spherical aberration corrective power of the lens. vertical coma aberrations with tilted iols were larger than those observed with decentered iols. for iols that induced a high level of vertical coma aberration, defocused mtf values decreased for both decentered and tilted iols, whereas the differences between vertical and horizontal mtf values increased (figure 3a, iols b and c). characteristic vertical coma aberrations showing an upward tail were observed at the defocused plane of 2.0 d and at 1.0 d (figure 4b, iols b and c and figure 4c, iols b and c). at the plane of focus, landolt ring images produced by decentered iols with a high spherical aberration corrective power were clear even in the presence of vertical coma aberration, whereas vertical coma aberration led to blurred images at the plane of defocus (figure 5b, iols b and c). for the same iols without decentration but with tilt, blurring due to vertical coma aberrations was large at the plane of focus and even larger at the plane of defocus (figure 5c, iols b and c). for misaligned iols with little or no spherical aberration correction, levels of vertical coma aberration were low (figure 2, iols a and d) ; defocused psf (figure 4b and c, iols a and d) and landolt ring simulations (figure 5b and c, iols a and d) were not significantly affected by defocus ; and the depths of focus were high. the relationship between depth of focus and mtf has been studied by marcos.16,20 in their 1999 paper,20 they normalized the peak position of defocused mtf and defined the depth of focus as the range with values of 60% of the peak value or higher. however, in their 2005 paper,16 they did not refer to the specific numerical values as a depth of focus, but instead qualitatively described the depth of focus based on the shapes of graphs and retinal simulation images. based on the results of vertical mtf corresponding to decimal visual acuity of 0.2 without misalignment (figure 3b) and based on the results of landolt ring simulation, our current study has also indicated that the depth of focus increased with the increase of the residual value of spherical aberration. in the absence of misalignment, our results also showed that the difference between vertical and horizontal mtfs for each lens (a measure of coma aberration) was small, irrespective of the degree of spherical aberration (figure 3a). in contrast, the differences between vertical and horizontal mtfs for misaligned lenses increased with the spherical aberration corrective power of the lens (figure 3a). these observations are consistent with our wave front aberration measurements (figure 2), and with theoretical calculations that form the basis of lens - aberration theory.21 the latter states that, when the misalignment of a lens is small, the degree of spherical aberration is independent of the misalignment, whereas the degree of coma aberration is proportional both to the degree of iol misalignment and to the level of spherical aberration. in our study, the iols with low spherical correction power (ie, high levels of spherical aberration in the eye) had the lowest peak mtf values and the broadest tails (figure 3a). these observations support those from previous studies, which suggest that the influence on visual outcome is large for iols with corrected spherical aberration.10,17 however, although psf measurements showed little difference between iols in the quality of the image at the plane of focus, at the plane of defocus the image focus spread as the spherical aberration corrective power increased (figure 4a c). similar results were obtained from landolt ring simulations (figure 5a c). here, the image at the plane of focus was sharpest for iols with a high degree of spherical aberration correction (iols b and c) but images degraded with defocus and misalignment. in contrast, iols with little or no corrective power (iols a and d) produced an image that was slightly blurred at the plane of focus but was hardly affected by defocus or misalignment. these results are supported by lens aberration theory.21 however, since the cornea itself has a coma aberration that increases with age,18,22 further studies are necessary to evaluate the effects of iol misalignment on retinal image in eyes with coma aberration in the cornea. the levels of decentration and tilt selected for this study were based on typical iol misalignments observed in clinical practice (average decentration of 0.300.16 mm and tilt of 2.621.14).10 however, because decentration and tilt are two - dimensional vectors, some studies have reported misalignments, not only in terms of magnitude, but also in terms of direction.23,24 the directionality of misalignment was not investigated in the present study. the present study investigated the actual retinal images generated by misaligned spherical and aspherical iols using a custom - built model eye. we found that, for iols that correct spherical aberration, iol misalignment was associated with degraded retinal images due to increased coma aberration, and that coma aberrations were related to the degree of spherical aberration correction by the iol. this suggests that, in clinical practice, the quality of vision can potentially be improved by reducing the risk of coma aberration using iols that retain, or have little effect on, the spherical aberration of the eye.	purposeto examine the effect of misalignment (decentration and tilt) of intraocular lenses (iols) on retinal image quality using a water - immersed model eye with corneal spherical aberration adjusted to the values found in normal human eyes (spherical aberration 0.25 m ; pupil diameter 6 mm).methodsthree types of iol holders were prepared. the first was without decentration or tilt, the second had a decentration of 0.5 mm, and the third had a tilt of 5.0. one spherical iol and three aspherical iols, each with a power of + 20 d, were set in the holders and their optical properties (wave front aberration, defocused modulation transfer function, defocused point spread function, and landolt ring simulations) were compared.resultscoma aberrations generated by misaligned iols were related to the spherical aberration corrective power of the iols. landolt ring simulations show that the depth of focus increased as spherical aberration increased and that the retinal image quality was degraded by increases in coma aberration.conclusioncoma aberration was generated by iols with a large degree of spherical aberration correction, leading to reduced retinal image quality when the iol was misaligned. this suggests that, in a clinical setting, the quality of vision might be improved by reducing the degree of coma aberration using iols that retain, or minimally correct, spherical aberration.
q fever was first described in 1935 in queensland, australia, during an outbreak of a febrile illness of unknown origin (query fever) among abattoir workers. b critical biological agent by the centre for diseases control and prevention and is considered a potential weapon for bioterrorism. q fever is a public health concern throughout the world. while q fever is an oie notifiable disease, domestic ruminants (cattle, sheep, and goats) are considered as the main reservoir for the pathogen which can infect a large variety of hosts, mammals (humans, ruminants, small rodents, dogs, and cats) and also birds, fish, reptiles, and arthropods [414 ]. it was reported to be a highly infectious disease in guinea pigs during experimental intraperitoneal infections [15, 16 ]. both in animals and humans, however, q fever infections remain poorly understood [17, 18 ] and their prevalence have been underestimated for many years. the causal agent of q fever is coxiella burnetii, an obligate intracellular gram - negative bacterium of the legionellales order, which was first observed as a rickettsia - like organism in the spleen and liver of mice inoculated with the urine of the abattoir workers [1922 ]. its predilected target cells are the macrophages located in body tissues (e.g., lymph nodes, spleen, lungs, and liver) and the monocytes circulating in the blood stream. the difference between phase i and phase ii bacterial forms resides in the variation of the surface lipopolysaccharide (lps) as classically described for enterobacteria. only phase i bacteria have a complete lps on their surface and are virulent bacteria. phase i bacteria can be isolated from a naturally infected individual or from animals infected in a laboratory [27, 28 ]. on the other hand, phase ii bacteria have an incomplete lps due to a spontaneous genetic deletion of 25,992 bp and are nonvirulent. phase ii bacteria occur during serial passage in an immunologically incompetent host, such as cell cultures or fertilized eggs [2729 ]. the deleted chromosomal region comprises a high number of genes that are predicted to function in lps or lipooligosaccharide biosynthesis, as well as in general carbohydrate and sulfur metabolism. however, in australia, the study by thompson. and a later study by denison. on the genome of phase ii human strains by polymerase chain reaction (pcr) reported the absence of truncated genes or of deletions. the institute for genomic research has suggested that at least two other chromosomal regions are implicated in phase transition. indeed, serologically, anti - phase ii antibodies (igg and igm) are found at high levels in acute q fever, whereas anti - phase i antibodies (igg and iga) are found at high levels only during chronic infection. the chromosome varies in size from 1.5 to 2.410 base pairs and was highly variable among different c. burnetii strains. occasionally, a 33 to 42 kb plasmid (depending on the plasmid considered) can be observed but its function remains to be determined. bacterial isolates can be identified by a probe to 16s ribosomal rna (rrna), which is highly conserved. genetic heterogeneity of coxiella burnetii is limited with approximately 30 distinct variants. according to experimental studies, studied the macrophage infectivity potentiator gene (cbmip of 654-base dna) and the sensor - like protein gene (qrsa of 1227-base dna) sequences between eleven strains. their results demonstrated that cbmip and qrsa sequences were highly conserved (> 99%) and did not explain differences in pathogenicity. furthermore, three different plasmids have been identified in coxiella variants : qph1, qprs, and qpdg [42, 43 ]. another plasmid (qpdv). however, several identical genomic sequences are present in all plasmids. in bacteria without plasmids, these sequences are found on the chromosome. generally, plasmids are of little interest for identification of microorganisms because they are not critical for survival and can infect a large variety of organisms. however, coxiella burnetii plasmids have proven to be useful because different strains contain different plasmids. in fact, qph1, qprs, and qpdv were present in different genotypes and were associated with difference in pathogenicity in the study by frazier. moreover, savinelli and mallavia reported that, in human patients, the qph1 and the qprs plasmids (or plasmidless strains containing qprs - related plasmid sequences) were associated with acute and chronic infection, respectively. however, later studies by genomic restriction fragment length polymorphism analysis, plasmid typing, or lipopolysaccharide analysis, on a larger number of strains did not confirm their results. indeed, recent data shows that genetic variation has an apparent closer connection with the geographical source of the isolate than with clinical presentation [33, 46 ]. moreover, host factors seem to be more important than genomic variation for development of acute or chronic infection [47, 48 ]. according to the recent report by the oie, no specific genotype is associated with acute or chronic infection, with a particular clinical outcome, or with a specific host. the most important route of infection is inhalation of bacteria - contaminated dust, while the oral route is considered of secondary importance. once inhaled or ingested, the extracellular form of coxiella burnetii (or scv after small - cell variant) the multiple intracellular phagolysosomes eventually fuse together leading to the formation of a large unique vacuole. coxiella burnetii has adapted to the phagolysosomes of eukaryotic cells and is capable of multiplying in the acidic vacuoles. in fact, acidity is necessary for its metabolism, including nutrients assimilation and synthesis of nucleic acids and amino acids. multiplication of coxiella burnetii can be stopped by raising the phagolysosomal ph using lysosomotropic agents such as chloroquine [52, 53 ]. and akporiaye and baca identified three proteins involved in intracellular survival : a superoxide dismutase, a catalase, and a macrophage infectivity potentiator (cbmip). redd and thompson found that secretion and export of cbmip was triggered by an acid ph in vitro. later, studies by zamboni and rabonovitch and by brennan. demonstrated that growth of coxiella burnetii was reduced by reactive oxygen intermediates (roi) and reactive nitrogen intermediates. hill and they demonstrated experimentally that both a recombinant acid phosphatase (racp) enzyme and coxiella burnetii extracts had a ph - dependent acid phosphatase activity. moreover, racp and bacterial extracts were capable of inhibiting roi response by pmn despite their exogenous stimulation by a strong pmn stimulant. inhibition of the assembly of the nadph oxidase complex was found to be the mechanism involved. in vitro studies on persistently infected cells with phase i and phase ii bacteria reported a similar mitotic rate in infected and noninfected cells. moreover, the authors frequently observed asymmetric cellular divisions in infected cells and suggested that this phenomenon could allow maintenance of persistent infection. the intracellular cycle of coxiella burnetii leads to the formation of two development stages of the bacterium known as small - cell variant (scv) and large - cell variant (lcv) [6164 ]. typically rodshaped, scvs are compact measuring from 0.2 to 0.5 m with an electron - dense core. according to previous studies, scvs are considered to be metabolically inactive and capable of resisting extreme conditions such as heat, desiccation, high or low ph, disinfectants, chemical products, osmotic pressure, and uv rays [19, 22, 62, 64, 65 ]. their resistance in the environment (pseudospores) would enable the bacteria to survive for long periods of time in the absence of a suitable host. indeed, once inhaled or ingested, the scv attaches itself to a cell membrane and is internalized. after phagolysosomal fusion, the acidity of the newly formed vacuole induces activation of scv metabolism and its development into lcv. during the morphogenesis from scv to lcv, no increase in bacterial number intracellular growth is relatively slow with a doubling time of approximately 8 to 12 hours. the endogenous spore - like forms can undergo further development and metabolic changes until finally reaching the scv form. finally, cell lysis, or possibly exocytosis, releases the resistant bacteria into the extracellular media. the physical and biological factors responsible for the sporulation - like process remain unknown. according to rousset., most natural infections by coxiella burnetii are probably due to scv or pseudospores present in the environment. thus, decreasing the prevalence of q fever infections requires a strict limitation of the environmental population of coxiella pseudospores by using hygienic preventive measures. studies on the immune reaction in naturally or experimentally infected individuals have suggested that cellular immunity and the synthesis of ifn are essential for control of coxiella burnetii infection [6870 ]. demonstrated the predominant role of ifn, its level of production determining the outcome of infection. indeed, ifn has been successfully tested to treat q fever in patients not responding to antibiotic treatment [71, 72 ]. a study by shannon. reported that the development of protective antibody - mediated immunity in vivo was found to be independent of the cellular fc receptors and of the complement. the major part of vaccine - derived humoral response consists of igg antibodies directed against proteins [73, 74 ]. several studies report that natural humoral response to coxiella burnetii is directed against both protein and glycolipid fractions [7578 ]. however, all the cd4 + t cell epitopes did not lead to b - cell stimulation and specific antibody production. koster. reported that, in chronic infections, peripheral blood lymphocytes do not proliferate when exposed to coxiella burnetii antigens despite proliferating when exposed to other antigens or mitogens. in addition, shannon. observed that coxiella burnetii phase i cells appeared almost invisible to dendritic cells. hormonal changes during pregnancy cause immunomodulation in the female body causing reactivation of the organism. this immunomodulation has been advanced as an explanation for the increased multiplication of the organism in the placenta. further research is more than necessary to fully understand the complex processes developed by coxiella burnetii to enter and infect a specific host cell, to resist in the intracellular and extracellular environment, and its ability to cause illness. moreover, a better understanding of the immune system reaction to infection would give an insight into the processes developed by the bacterium and by the host that determine the final outcome of disease (asymptomatic, acute, or chronic). inhalation is the most common route of infection in both animals and human [8486 ]. under experimental conditions, however, similar studies have not been done in animals. as mentioned above, domestic animals are considered the main reservoir for the pathogen [8991 ]. infected animals contaminate the environment by shedding coxiella burnetii in milk, feces, urine, saliva [13, 92 ], and very importantly in vaginal secretions, placenta, amniotic fluids, and other products of conception [13, 9299 ]. coxiella burnetii also spreads by wind causing infections at a distance from the initial source of bacteria [93, 96, 100, 101 ]. in domestic ruminants, results of previous studies on the subject are considered inconclusive [7, 103106 ]. further research is required to clarify the probability of infection by oral route. if infection by oral route is proven to be efficient, the sufficient number of pathogens capable of causing q fever should be determined. human - to - human transmission does not usually occur [49, 107 ] although it has been described following contact with parturient women [108, 109 ]. in addition, cases of sexual transmission of q fever have been reported [110112 ]. currently, risk of transmission through blood transfusion is considered negligible [113, 114 ]. transplacental transmission, intradermal inoculation, and postmortem examinations have been associated with sporadic cases of q fever [115118 ]. q fever is widespread in livestock, and its seroprevalence is thought to have increased in recent years. often neglected in the differential diagnosis, q fever can persist in a herd causing great financial losses on the long term. in ruminants, well - known manifestations of q fever are abortion, stillbirth, premature delivery, and delivery of weak offspring. however, these dramatic clinical manifestations are generally only expressed in sheep and goats. in cattle, in addition, c. burnetii was found to be significantly associated with placentitis [119, 120 ]. placental necrosis and fetal bronchopneumonia were also significantly associated with the presence of coxiella burnetii in the trophoblasts. unlike humans and experimentally infected cows, naturally infected ruminants rarely present respiratory or cardiac signs. performed respective studies on shedding of coxiella burnetii by infected cows. in the study by guatteo, the apparent proportion of shedders in clinically infected dairy herds among the cows sampled was 45.5%. milk was the most frequent positive sample for the bacterium compared to feces and vaginal mucus samples. the percentage of positive samples of each type was 24.4, 20.7, and 19%, respectively. 65.4% of sampled cows excreted by one shedding route only, whereas 6.4% shed bacteria in the vaginal mucus, feces, and milk simultaneously. a combined shedding in vaginal mucus and in feces and in vaginal mucus and milk were observed in 14.6% and 10% of cases, respectively. the study by beaudeau. reported, within endemic infected dairy herds, that 85% of their infected cows excreted by one shedding route only. in their study, only 2% of the infected cows shed bacteria in the vaginal mucus, feces and milk simultaneously. when combined shedding occurred, the combination of shedding in vaginal mucus and milk was the most frequently observed. the results of these two french studies are very similar and the slight differences observed could be due to differences in the sampled population of cattle. furthermore, different areas can have variable prevalence of q fever and not only is shedding in milk intermittent and its outset not associated with parturition, but also it differs from one herd to another despite species being identical. with milk being such a major shedding route, bulk tank milk (btm) samples are useful for investigation of the sanitary grade of bovine, ovine, and caprine herds. indeed, polymerase chain reaction (pcr) and enzyme - linked immunosorbent assay (elisa) performed on btm samples, in addition to the analysis of serum samples of at least 10% of the animals in the herd, give rapid, economical, and valuable information on the herd 's status [97, 126 ]. in cows ' milk, vaginal and fecal bacterial discharges seem to have a major impact on environmental contamination as a result of practices at kidding and effluent management [88, 97 ]. indeed, the incidence of q fever has been observed to increase significantly during the lambing period (winter and early spring). a seasonal correlation with spread of goat manure was reported in the netherlands during the outbreaks of 2007 - 2008. epidemiological data has shown that cows are more frequently chronically infected than sheep with persistent shedding of bacteria as a result. the sites of chronic coxiella burnetii infection are the uterus and the mammary glands of females [20, 24 ]. moreover, heifers are less frequently infected than older animals even in infected herds [128, 129 ] making those a preferential group for effective vaccination programs. numerous studies have suggested that epizootics of q fever in goats are related to outbreaks in humans [99, 114, 130, 131 ]. indeed, in many countries, goats are the most common source of human infection due to their extensive raising and close contact with humans. q fever in goats can induce pneumonia, abortions, stillbirth, and delivery of weak kids, the latter two clinical signs being the most frequently observed [98, 99, 132 ]. the frequency of occurrence of q fever abortions in goats is more important than in sheep with up to 90% of females being affected. moreover, a study by berri. reported that pregnancies subsequent to abortion may not be carried to term. these animals also frequently develop chronic infections with persistence of the bacteria in the uterus and mammary glands. shedding of coxiella burnetii by infected goats is discontinuous [98, 99, 134136 ]. in naturally infected goats, however, an experimental study performed by berri. reported that infected animals can shed coxiella during two successive kidding seasons in vaginal mucus and milk. moreover, shedding in milk can be maintained for a long period [98, 136 ]. milk has been considered the major route of bacteria excretion for this species by several authors. de cremoux. in their recent study on bacterial shedding in vaginal mucus observed persistence of shedding at the subsequent kidding season following a q fever outbreak despite a reduced number of clinical signs. similar to cattle, susceptible or nave animals were more common among kids than adults. however, q fever in sheep seldom causes chronic infections [13, 17, 132 ]. infected sheep, like goats, shed coxiella burnetii in vaginal secretions, urine and feces, and to a lesser extent in milk. in naturally infected sheep, the bacterium has been isolated in vaginal discharges long after abortion and can be shed at subsequent pregnancies. in the study by rodokalis some sheep have been found to shed coxiella burnetii during 11 to 18 days postpartum in feces. most ewes shed bacteria by at least two routes, mainly in feces and vaginal mucus. reported a flock where no ewe ever shed by a single route (no exclusive shedding in milk or feces or vaginal mucus). in certain areas (e.g., basque country), garcia - prez. studied the presence of coxiella burnetii dna in btm and its association with seroprevalence. herds with a history of abortion had a higher seroprevalence than other herds, but the difference observed was not significant. flocks with a level of seropositivity superior to 30% were more frequently positive on btm - pcr analysis. however, flocks with a low number of seropositive animals but with a positive result at btm - pcr analysis were also observed in this study. in france, two cases of human q fever were associated with the use of ovine manure as garden fertilizer. the flock of sheep that had provided the manure did not present any clinical signs, despite the presence of seropositive animals and excretion of bacteria in feces. the q fever outbreak that occurred in bulgaria in 2004 was also associated with infected sheep and goats. astobiza. studied the effect of vaccination with a phase i inactivated vaccine (coxevac). after vaccinating 50% of replacement lambs and 75% of ewes in two naturally infected sheep flocks, the number of abortions, shedders and the general bacterial load were significantly diminished. however, this measure did not prevent seroconversion of nonvaccinated lambs and identification of coxiella burnetii in aerosols obtained in the sheep 's environment. this confirms that vaccination must thus be associated with other preventive and control measures in infected environments. natural susceptibility has been demonstrated by the presence of antibodies to coxiella burnetii in their serum. however, the role played by pigs in the epidemiology of q fever remains unknown. dogs and cats can be infected by q fever and have been associated with human infections in rural and urban areas [145155 ]. in 1952, gillepsie and baker performed successful feline experimental infections by subcutaneous inoculation, feeding infected yolk sacs, and by contact with infected cats. despite the presence of pathogen in blood, urine, and a serological response, however, infected cats excrete bacteria in the environment and become a potential source of human infections. in japan, cats are widely considered to be one of the most important reservoirs of coxiella burnetii. the study by komiya. found that seroprevalence was significantly higher in stray cats than in domestic cats. the potential importance of dogs for the transmission of q fever to humans is rarely mentioned. dogs can potentially be infected by inhalation, tick bites, consumption of placentas, or milk from infected ruminants. indeed, q fever in parturient dogs has previously been associated with early death of the pups. similar to cats, a previous study on dogs in california reported a higher prevalence of infection in stray than in domestic dogs. currently, the effect of infection and its clinical presentation remain poorly investigated in felines and canines. in previous studies, horses have been found to be seropositive toward q fever. indeed, the study by willeberg. reported a seroprevalence of 26% (31/121) in horses in california. to our knowledge, no human case of q fever has been associated with equids. moreover, q fever is not investigated routinely in cases of infertility or obstetric complications in this species. at present, wildlife is considered of minor importance for q fever epidemiology. many wild mammals and birds have been found to be hosts to the infectious organism [13, 159163 ]. a few cases of transmission of coxiella burnetii from wild animals to humans have been reported but merit further experimental research [11, 13 ]. among ectoparasites, experimental infections have been obtained in guinea pigs with ixodes holocyclus, haemaphysalis bispinosa, rhipicephalus sanguineus, and dermacentor andersoni. in europe, ixodes ricinus is the most common tick and rhipicephalus sanguineus is frequent on dogs [102, 168, 170 ]. thus, both these species of ticks could be (or become) reservoirs of coxiella burnetii in europe. ticks excrete bacteria in saliva and feces. after multiplying in the cells of the midgut and stomach of an infected tick, the feces are extremely rich in bacteria and may reach a concentration of 10 organisms per gram [20, 89 ]. furthermore, transovarial transmission is suspected as bacteria have been isolated in the ovaries of infected ticks. however, despite all these factors, ticks are not thought to contribute to the maintenance of coxiellosis in endemic areas [13, 17 ]., none of the ticks analyzed were positive when tested by pcr despite the area being endemic for q fever. similarly, in two recent studies in endemic areas, a low percentage of dermacentor spp. and ixodes ricinus were found to be infected. nevertheless, ticks are suspected of having a significant role in the transmission of coxiella burnetii among wild vertebrates, especially rodents, lagomorphs, and wild birds [20, 89, 174 ]. in addition, hirai and to hypothesized that they could play a role for transmission of coxiella from infected wild animals to domestic naive animals. sporadic isolations of coxiella burnetii in chiggers, lice, and flies have been reported [164, 175, 176 ]. the main characteristic of q fever in humans is its clinical polymorphism [1, 15, 20 ]. q fever is therefore considerably underdiagnosed and underreported. after an incubation period of 1 to 3 weeks [33, 107 ] size of the inoculum, geographical area, route, and time of infection, as well as host factors influence the duration of the incubation period [15, 178, 179 ] and may contribute to the clinical expression of acute or chronic infection [20, 127 ]. in the acute form, infections can be totally asymptomatic in 50 to 60% of cases or cause a self - limiting illness associated with fever, fatigue, headache, and myalgia (influenza - like syndrome). when clinically expressed, acute fever is frequently accompanied by atypical pneumonia and/or hepatitis. pneumonia is an important manifestation of q fever in humans [1, 15, 20 ]. it is uncommon in australia and some parts of russia, whereas in north america and europe it is the major manifestation of infection [93, 96 ]. pneumonia is typically mild, but progression to acute distress syndrome can occur [107, 180, 181 ]. endocarditis can exceptionally be associated with acute infection in 0.76% of cases [182, 183 ]. in pregnant woman, q fever can lead to spontaneous abortion, intrauterine fetal death (iufd), premature delivery, or intrauterine growth retardation. transplacental infection of the fetus in utero has also been reported [109, 184, 185 ]. moreover, carcopino. found that q fever was significantly associated with oligoamnios, which is a recognized cause of neonatal morbidity and mortality [187, 188 ]. one case of endocarditis has been reported in a pregnant women with a bioprosthetic aortic valve and led to maternofetal death at 27-week gestation. infection during the first trimester of the pregnancy is particularly associated with a negative outcome [185, 186 ]. after infection mortality rate of acute q fever is estimated at 1 to 2% [107, 114, 189 ]. myocarditis, occurring in less than 1% of cases, is the first cause of death. most commonly, endocarditis is observed in 6070% of cases of chronic infections, but chronic hepatitis, osteomyelitis, septic arthritis, interstitial lung disease, chronic fatigue syndrome, or infection of aneurysm and vascular grafts can also occur [1, 15, 20 ]. q fever - associated endocarditis has been estimated to be responsible for 3 to 5% of all cases of human endocarditis [182, 195 ]. individuals with underlying valvulopathy or other cardiovascular abnormalities are predisposed to the development of endocarditis [182, 183 ]. over recent years, the occurrence of rare clinical manifestations such as osteomyelitis, optic neuritis, pericarditis, lymphadenopathy, and guillain - barre has significantly increased. meningitis, encephalitis, polyradiculoneuritis, peripheral neuropathy, cranial nerve deficiency, optic nevritis, and paralysis of the oculomotor nerve have been reported in 3.5% of infected patients. the main clinical signs in neurological cases are headaches, behavioral problems, cognitive deficiency, or confusion. convulsions and even epileptic fits and aphasia are possible [196198 ]. in pregnant women, chronic q fever can lead to spontaneous abortions in future pregnancies [109, 117, 199 ]. antibiotic treatment is less effective, and the disease is usually long with mortality rates that can reach more than 50%. in children, this could be explained by frequent asymptomatic or nonspecific presentations of infection leading to undiagnosed cases of coxiella infection. cases of hepatitis and pneumonia have however been reported in children and can be fatal [93, 200 ]. in northern ireland, mccaughey. reported a seropositivity rate inferior to 10% in children. the seropositivity rate increased markedly during the late teenage years and especially in young adults. increased exposure with age the study by limonard. on followup of patients after the q fever outbreak in the netherlands reported fatigue in 52% of patients 6 months after illness and in 26% one year after illness. they observed very high level of anti - phase i and anti - phase ii antibodies up to 3 months after the onset of disease, then a gradual decrease in the following 9 months. post - q fever chronic fatigue syndrome has also been reported [114, 194, 203 ]. this syndrome has been attributed to dysregulation of cytokine production, induced by persistent antigens including lps and proteins, rather than persistent latent coxiella. recent experimental data indicates that host factors rather than specific genetic bacterial determinants are the main factors influencing the clinical course of coxiella burnetii infection [14, 47, 195, 204 ]. in humans, q fever is mainly considered an occupational hazard. a study by whitney. on 508 american veterinarians reported a prevalence rate (22.2%) far superior to the prevalence rate in the general adult us population. veterinarians from a mixed or large animal practice were significantly more likely to be seropositive than veterinarians from a small animal practice. furthermore, living on a farm, in the past or present, increased the probability of being seropositive for coxiellosis. absence of protective clothing or mask, occupational risks (accidental cuts and needle sticks), and routine contact with water were also demonstrated as significant risk factors for infection. mcquiston and childs reported a seroprevalence of 7.8% among american veterinarians, farmers, slaughterhouse workers, and tannery workers. furthermore, a northern irish study reported a seropositivity rate significantly higher among farmers than in the general population (p < 0.001). seropositivity was found to be slightly, but significantly, higher in males than in females (p = 0.023). in the netherlands, during the q fever outbreak in may 2010, people living within 2 km of the infected dairy goat farm had a much higher risk for q fever than those living more than 5 km away. on the contrary to countries where cats are the main reservoir of coxiella burnetii where no sex predisposition is reported, men between 30 and 70 years of age are the most frequently affected by clinical q fever in countries where cattle are considered the main reservoir [208214 ]. the study by raoult. reported that being a male and over the age of 15 increased the risk of symptomatic disease. in australia and france, although the precise reason for this difference remains unknown, it has been suggested that sex hormones play a role [195, 204 ]. indeed, leone. reported a protective role of 17beta - oestradiol towards q fever infection rendering females more resistant. surprisingly, in a comparative study on clinical expression and outcome of q fever after the outbreak in chamonix valley in france, pregnant women were found to be significantly less frequently symptomatic than other women and other patients (9.1% of pregnant women were symptomatic versus 90.6% of men, 75% women, and 33.3% children). furthermore, people with heart valve lesions, vascular abnormalities, liver cirrhosis, and cancer are more susceptible to developing chronic q fever [183, 218 ]. in the study by carcopino., 52.8% of the pregnant women with acute q fever developed chronic serologic profiles. in nonpregnant women and other patients with acute q fever, treatment consists in a daily dose of 200 mg doxycycline for 14 to 21 days. hydroxychloroquine increases the ph of the phagolysosomes, and its association with doxycycline has a bactericidal effect [219, 220 ]. rifampin, erythromycin, clarithromycin, and roxithromycin can also be used as an alternative treatment [221, 222 ]. fluoroquinolones are recommended in cases of meningoencephalitis as their penetration into the central nervous system is better compared to doxycycline. as mentioned previously, treatment with ifn treatment of chronically infected patients consists in a daily dose of 200 mg of doxycycline associated with 600 mg of hydroxychloroquine for duration of 18 to 36 months. the dosage of hydroxychloroquine is adjusted three months after initiation of treatment to obtain a plasmatic level of 1 0.2 mg / l. during the treatment, an ophthalmologic followup advised a plasmatic concentration of 5 mg / l of doxycycline for effective treatment. minimal inhibition concentration (mic) for doxycycline against coxiella burnetii varies from 1 to 4 g / ml. bacterial resistance to doxycycline was observed by rolain. thus, serological testing on a regular basis to evaluate response to treatment is advised. it is considered that an anti - phase i igg titer inferior to 200 is predictive of a clinical cure. a decrease of 2 dilutions in antibody titers in one year at the minimum signifies successful evolution. in infected pregnant women, administration of long - term cotrimoxazole (320 mg trimethoprim and 1600 mg sulfamethoxazole for at least 5 weeks), a bacteriostatic antibiotic, after delivery, a daily treatment with 200 mg of doxycycline associated with 600 mg of hydroxychloroquine for 1 year minimum is advised for chronically infected women. reported that long - term cotrimoxazole therapy significantly reduced the number of obstetric complications, the frequency of placentitis, and the development of a chronic serological profile. in their study, no iufd was observed in treated women (0% versus 27% of iufd in nontreated women). guinea pigs are regarded as a model of acute q fever in humans. on the contrary to guinea pigs, mice develop a chronic infection, although endocarditis does not occur in normal adult mice. extrapolations of observations on experimentally infected laboratory rodents to naturally infected humans could prove erroneous in some aspects, and caution is recommended. after infection, the macaques developed fever and pneumonia after 4 to 7 days. their clinical expression of q fever is thus unsurprisingly the closest to human clinical expression. in 2009, infected goats were at the origin of 2,361 human cases of q fever (including six deaths) diagnosed in the netherlands. most cases were diagnosed in the north brabant province. a recent increase in high intensity dairy goat farming has lead to development of very large densely populated farms : from 100,000 goats in 2000 to 230,000 goats on approximately 350 farms in 2009. transport of animals, manure spreading, and wind have been reported as influential factors for human infections. in a study by karagiannis. on the outbreak in 2007 in the netherlands, living east of the area in which a positive goat farm, cattle, or small ruminants were situated, smoking and contact with agriculture products were found to be associated with recent infection. scientists suspected the emergence of a more virulent subtype of coxiella burnetii than the initial subtype of bacterium identified [131, 231 ]. however, an identical subtype was identified in a dairy goat herd suffering from multiple abortions and in several human patients. this particular subtype could thus have a survival and propagation advantage compared to other bacterial subtypes [131, 231 ]. performed multiple locus variable tandem repeat analyses on a large number of q fever positive samples originating from domestic ruminants and associated with the outbreak. their study found that one genotype predominated on all dairy goat farms in the southern part of the netherlands. indeed, on 12 out of 14 dairy goat farms, this genotype was found in 91% of samples, varying from 33% to 100%. nine other genotypes occurred once, each representing only 0.8% of all found genotypes on the farms. the predominant genotype was found on 11 farms in the southern netherlands and on a farm in the eastern part of the country. however, another study by huijmans. found five distinct genotypes (3 in humans and 4 in livestock) implicated in the outbreak and concluded that environmental factors such as animal and human density and climate, rather than one particularly virulent strain favored the dutch outbreak and its spread. vaccination was exclusively performed in areas of the human outbreak. in 2009, vaccination of all dairy sheep and goats on farms with more than 50 animals became mandatory, and area of vaccination was enlarged (north - brabant province and parts of adjacent provinces). movement and breeding were banned for dairy goats and sheep on infected farms. moreover, culling of all pregnant goats and sheep on these farms was also in application until may 2010. direct diagnostic methods identify the presence of the bacterium or of one of its components. direct visualization of coxiella burnetii on smears or frozen tissue is a method of q fever diagnosis. smears are taken from the placenta of aborted ruminants, from the fetus stomach content, or from other body tissues. coxiella burnetii does not stain reliably with gram stain, and gimenez stain is preferentially used. a stamp - macchiavello coloration, commonly called macc staining, or routine giemsa stain can also be performed. the specificity and sensitivity of direct visualization by bacterioscopic examination is poor, due to possible confusion with other pathogens such as brucella spp. it can be utilized for detection of coxiella burnetii in tissues fixed in paraffin or acetone smears. dilbeck and mcelwain developed an avidin - biotin - peroxidase complex ihc staining method for diagnosis and routine screening of ovine and caprine placental tissues after abortion. this technique is rapid and does not necessitate live bacteria or fresh tissues for diagnosis. however, ihc is not useful for large - scale epidemiological studies. in vitro cell culture of the bacteria is the gold standard for diagnosis of bacterial infections. coxiella burnetii can be cultured efficiently in the yolk sac of chicken embryos and also on diverse cellular specimens, such as human embryonic lung fibroblasts, mosquito cells, green monkey kidney (vero) cells, l cells, tick tissue cultures, and so forth. however, technically, culture of coxiella burnetii remains a difficult process and sensitivity of this diagnostic method is low. recently, coxiella burnetii can be grown outside a host cell in a cell - free laboratory medium. the latter cell - free medium has a composition that strictly corresponds to the organism 's metabolic requirements in the phagolysosome (in vivo coxiella is strictly intracellular). another practical limitation to bacterial isolation is that it requires a biosafety laboratory 3 because of its high infectivity. as a result, culture is rarely performed, especially in veterinary medicine. moreover, epidemiological studies based on bacterial isolation are not practical. pcr offers substantial benefits for the identification of coxiella burnetii compared to other laboratory techniques, especially in the early clinical stages of the illness [241, 242 ]. it has been successful in detecting coxiella burnetii dna in various samples, including cell cultures, biopsy samples, blood, arthropods, and serum samples [193, 243 ]. however, the usefulness of conventional pcr is limited by its inability to quantify the bacteria present. the development of real - time quantitative pcr (rtq pcr) not only renders pcr a rapid diagnostic tool but also provides quantifiable information. rtq pcr can be automated and thus can be used in large - scale studies. the qualities of pcr make it very useful for early diagnosis of infection during the period when antibodies are not yet present. a primer originating from the frequently repeated dna sequence is1111 (7 to 120 copies per genome) is commonly used and has proven to allow very sensitive testing [241, 246 ]. in a study by schneeberger., coxiella burnetii dna was detected in 98% of seronegative acute q fever patients and in 90% of anti - phase 2 igm seropositive patients. hou. noticed a significant association between the absence of igm antibodies and a positive pcr result. negative serology was most frequent in q fever cases where sampling had been performed within the two weeks of illness. in such cases, the authors recommend routine pcr testing as well as serology. when pcr was included in the diagnostic procedure primarily based on serology, a marked increase of sensitivity was observed (78% versus 29% when serology was used as unique diagnostic method). pcr and rtq pcr are considered methods of choice for the dna detection in diverse samples. however, pcr and rtq pcr are incapable of distinguishing bacterial dna from live and/or dead bacteria. the advantage is that sampled tissues can be frozen, put into formalin, or fixed with paraffin. the disadvantage is that interpretation of positive results in fresh samples can be difficult [107, 249 ]. analysis of samples by pcr should be performed on sampling day to minimize the risk of obtaining false - negative results. indeed, the study by guatteo. reported that only two - thirds of milk and one - half of vaginal mucus samples that were initially positive on the day of sampling remained positive after storage at 20c during three days. samples found to be persistently positive had a significantly higher estimated initial bacterial titre than those which became negative after storage. indirect diagnostic methods identify specific humoral or cellular immunity in response to coxiella burnetii infection. the diagnostic methods available in human and veterinary medicine differ, and little information is available concerning epidemiological sensitivity and specificity of diagnostic methods, especially in ruminants (table 1). in veterinary medicine, cft was the method of reference for serological diagnosis according to oie [102, 255, 256 ]. it is capable of detecting approximately 65% of infected subjects during the second week after initial clinical signs and 90% during the fourth week. cft is more laborious, less specific, and less sensitive than indirect immunofluorescence assay (ifa) or elisa (described hereafter). a study by rousset. on goats originating from different herds reported that cft results obtained on sera of aborting goats and of nonaborting goats were not significantly different and confirmed the lack of sensitivity of cft compared to elisa. cft, on the contrary to elisa, is incapable of detecting all igg subclasses. in ruminants, only igg1 fixes moreover, igg2, igm and anticomplement substances potentially present in sera are capable of interfering with fixation of igg1 to the complement lowering the titer of igg1 detected by cft. this method is more sensitive and easier to perform, and to standardize than cft. moreover, a strong association was reported between strongly positive elisa results and the occurrence of abortion in goats. as mentioned above, the higher sensitivity of elisa compared to cft could be due to the detection of the igg2 subclass of antibodies, which are incapable of binding to the guinea pig complement. commercially available human elisa kits are frequently coated by phase 1 and phase 2 antigens. the antigens present are of two possible origins : antigens of the american nine mile strain of coxiella burnetii isolated from an endogenous tick or antigens of a strain originating from infected european domestic ruminants. elisa kits coated by the latter antigens are more sensitive and are advised for serological diagnosis. in france, the elisas available for veterinary diagnostic purposes do not differentiate anti - phase 1 and anti - phase 2 antibodies but detects total antibodies. elisa tests can be automated which facilitates large - scale studies. in the study by guatto., persistent shedder cows had mainly a persistently highly seropositive status (20 out of 27 and 17 out of 23 for weekly and monthly samplings, resp.) while these proportions were much lower for the other shedding patterns. in addition, around 50% of persistently highly seropositive cows were found to be persistent shedders, while seropositive cows with other patterns were mainly either non- or sporadic shedders. in human medicine, ifa is commonly considered the reference diagnostic test and is the most frequently used worldwide. the study by rousset. on goats demonstrated an overall good agreement between ifa and elisa results. the study also reported that ifa results obtained on sera of aborting goats and of nonaborting goats were significantly different and were associated with occurrence of abortion. moreover, ifa is capable of detecting the two antigenic variants of coxiella burnetii (phase i and phase ii). as mentioned previously, in acute infections, anti - phase ii antibodies are detected. in chronic infections, anti - phase i and anti - phase ii antibodies are present but anti - phase i antibodies predominate. as anti - phase ii antibodies are present in all stages of infection, screening for epidemiological purposes is based on the detection of anti - phase ii antibodies. cross - reactions with legionella micdadei and bartonella have been reported but do not cause any problem for the interpretation of quantitative results [259, 260 ]. titers of anti - phase ii igg superior or equal to 200 and of anti - phase ii igm superior or equal to 50 correspond to an acute infection with a predictive value of 100%. an isolated high titer of igm (50) can correspond to the beginning of an acute infection as long as the possibility of a false positive can be rejected. a chronic infection is characterized by a high titer in anti - phase i igg (800) with a predictive value of 98% for a titer of 800 and a predictive value of 100% for a titer of 1600. the preferential use of ifa instead of cft in veterinary medicine would be advantageous for diagnosis and control of q fever at animal level. a skin test method was proposed to investigate the cellular response and to improve detection of infected cows at herd level. the skin test consists in an intradermal injection of extremely diluted inactivated vaccine (coxevac, ceva - sant animale, libourne, france). if the animal has previously been infected by q fever, a nodule of variable size will appear at the site of injection. the dependence of cft, ifa, and elisa on the presence of antibodies limits their diagnostic value. indeed, specific antibodies are often absent during the first 2 to 3 weeks of infection, making early diagnosis by serology difficult, if not impossible. in q fever, phase ii antibodies can be detected within two weeks of infection in most cases and within three weeks 90% are seropositive. afssa reports that a definitive diagnosis of human cases by ifa can only be confirmed one month and a half after the initial clinical signs. cellular immunity also necessitates time before becoming detectable but the use of skin testing at herd level prevents from it being a problem as different stages of infection are present simultaneously in the herd. none of these tests are capable of confirming an etiological diagnosis at an individual level. moreover, in acute infections, rheumatic factor, anti - mitochondrial antibodies, anti - nuclear antibodies, anti - smooth muscle antibodies, anticardiolipin and lupus anticoagulant, and other autoantibodies often present a marked increase in their plasmatic levels and can interfere with diagnostic assays. a high prevalence of igm against epstein - barr virus, cytomegalovirus, parvovirus, bordetella pertussis, and mycoplasma pneumonia has also been detected. the global clinical and epidemiological context must be taken into account as well as the limitations of diagnostic assays. bacteriological analyses are necessary to confirm or infirm any suspicion of q fever. in acute cases of hepatitis, the presence of doughnut granulomas can be visualized in histopathology hepatic specimens but they are not pathognomonic of q fever. however, in chronic infections, granulomas are less organized but bacteria can be detected in large vacuoles. as mentioned above, in bovids, placentitis was found to be significantly associated with coxiella burnetii infection. on microscopic examination, an increased number of mononuclear cells (macrophages, lymphocytes, and plasma cells) can be identified in the chorionic stroma. increased stromal collagen is also frequently observed. the cytoplasms of infected cells appear bright red with the macc stain, and their nuclei are commonly eccentric. a modified koster 's acid - fast (maf) staining of fresh placenta smears is considered a good screening test, but confirmation by immunohistochemical techniques remains necessary. in endocarditis lesions, in the case of a q fever outbreak, sanitary and prophylactic measures should be applied at herd and human level, in order to limit disease transmission. human and animal infections must be diagnosed early and treated immediately to prevent the development of chronic infections and secondary complications. with q fever being a zoonosis, prophylaxis at herd level is fundamental. in the netherlands, spread of manure from infected herds based on the results of this study, adding 0.5% of calcium cyanamid to contaminated dung is an effective disinfectant. this measure, or its modified equivalent, should be applied worldwide if its effectiveness is proven. transport of animals in and from infected areas should be strictly controlled and restricted to days without wind. in france, when q fever has been diagnosed in a herd on a cheese - producing farm, milk of the aborted females must be discarded. indeed, sale, transformation, and treatment of this milk are strictly forbidden (afssa, 2007). milk of the remainder of the flock can be used for transformation, unless it is highly suspected that dairy products originating from these animals are dangerous for human consumption. in the latter case, the milk must be pasteurized at 72c during 15 minutes or by an equivalent thermal treatment (afssa, 2007). if q fever is diagnosed on a farm producing raw milk for direct human consumption, sale of milk is forbidden during one year after the initial diagnosis of q fever in an animal (afssa, 2007). in the uk, health protection agency guidelines suggest the use of 2% formaldehyde, 1% lysol, 5% hydrogen peroxide, 70% ethanol, or 5% chloroform for decontamination of surfaces, and spills of contaminated material should be dealt with immediately using hypochlorite, 5% peroxide, or phenol - based solutions (health protection agency, 2010 at http://www.hpa.org.uk/deliberate_accidental_releases/biological). however, the guidelines note than decontaminating moreover, scott and williams found that formaldehyde vapour was ineffective in the absence of a high relative humidity. high - risk material (contaminated bedding, placenta, and aborted fetuses) should be buried with lime or incinerated. treatment of manure with lime or calcium cyanide is also recommended before spreading, and spreading must be performed on a calm day. at human level, prevention of exposure to animals or wearing gloves and masks during manipulation of animals or their litter is advised [102, 205 ]. postexposure prophylaxis guidelines for the general population have been established in the usa [267, 268 ]. doxycycline at a dose of 100 mg a day or 500 mg of tetracycline twice daily started 812 days after exposure is advised. no recommendation is available for pregnant women although cotrimoxazole has been suggested [267, 268 ]. identification of an antibiotic capable of severely diminishing or completely stopping shedding would radically modify the management of coxiella infection at a herd level. it could also prove useful to prevent infection of pregnant women by inhibiting shedding of bacteria by domestic cats and dogs. pasteurization of all milk products should be performed if ingestion is proven an effective route of infection and after determination of the minimal infecting dose of bacteria. awaiting further scientific research, caution is recommended especially for individuals at high risk of chronic infection. suggested a followup of bovine and caprine herds by btm analysis. in herds presenting a pcr - positive btm result, pools of 10 individual milk samples should be tested by pcr to identify the shedding animals. if shedders are not very numerous, they can be eliminated and the other animals should be vaccinated. rodokalis. also suggested vaccination of herds in the proximity of infected herds or flocks. in animals, vaccines considered the most effective are composed of inactivated whole phase 1 bacteria. indeed, in goats, the inactivated phase i vaccine (coxevac, ceva - sant animale, libourne, france) protects efficiently against abortion and has been shown to prevent bacterial shedding in vaginal mucus, feces, and particularly in milk. furthermore, vaccination did not clear infection in previously infected goats [135, 236, 271 ] and cattle. [128, 272 ] on dairy cattle reported that the probability of becoming a shedder for vaccinated nave nonpregnant bovids was 5-fold inferior to the probability for nave bovids receiving a placebo. [128, 272 ] hypothesized that the immunodepression induced by pregnancy was responsible for the lack of effective immune response after vaccination. this explanation seems plausible, but a second study is necessary to reach confirmation of this hypothesis. one abortion was reported in a vaccinated infected cow, but no further investigations were performed on this animal except for a pcr on vaginal mucus at abortion time that gave a negative result. the major problem associated with vaccination with inactivated phase 1 vaccine is the impossibility of distinguishing vaccinated and naturally infected animals using elisa methods. at herd level, the effectiveness of a vaccination program could be evaluated by monitoring bacterial shedding in btm. currently, prophylaxis includes vaccination with the nonfully licensed inactivated phase i vaccine, coxevac (ceva - sant animale, libourne, france), when a focus of q fever is declared. this vaccine contains formalin - inactivated coxiella burnetii (strain rsa 493/nine mile phase i). a recent study by hermans. reported the presence of bacterial dna originating from the coxevax vaccine in goat milk after inoculation. coxiella burnetii dna was detectable until 9 days after vaccination in quantities estimated by pcr up to approximately 100 genome equivalents per ml. after the booster, the duration of vaccine - derived dna excretion was shorter, quantitatively lower, and detectable in fewer animals than after the first inoculation. this finding induced the modification of the dutch control strategy. indeed, after the outbreak, testing btm by pcr on dairy farms with at least 50 sheep or goats every two weeks and vaccination of all dairy goats and sheep with coxevac became compulsory. after this study, a two - week interval between vaccination and btm testing was imposed. the study by de cremoux. demonstrated that vaccination in infected goat herds diminished clinical signs and overall vaginal shedding with vaccination being most effective in young susceptible animals. an inactivated phase ii vaccine, chlamyvax fq, has also been tested on animals but has failed to be effective. several different vaccines have been developed since the discovery of coxiella burnetii. in the soviet union in the 1960s, a live attenuated strain m-44 obtained after repeated passage through guinea pigs and mice was used extensively for vaccination. because of the long - term persistence of the attenuated bacteria in animals and vaccinated human, this vaccine was never used in the west. the americans developed a chloroform - methanol residue vaccine based on the phase i henzerling strain of coxiella burnetii. despite being effective in protecting animals against aerosol challenges, a trichloroacetic acid extract of phase i nine mile strain which comprised proteins and lps was also used for vaccination. however, kazar. reported severe reactions in response to vaccination in humans. vaccinating with phase i nine mile extracts treated with chloroform - methanol prevented severe reactions, but the loss of protective immunogenicity rendered these vaccines ineffective [279282 ]. currently, a formalin - killed whole - cell vaccine prepared from phase i henzerling strain of coxiella burnetii (qvax, csl limited, parkville, victoria, australia) is licensed in australia. this vaccine proved effective in several studies, but vaccination is only possible for individuals that have not previously been in contact with coxiella burnetii to prevent serious reactions [17, 49, 284 ]. screening is thus required before vaccination [285, 286 ], making this preventive measure time consuming and costly. all the studies included in their analysis reported a protective effect of vaccination (average effectiveness after pooling raw data : 97%, ci : 9499%). however, several biases were present in the latter studies. conclude that generalization of the results to the general population or to a specific risk group was not possible. development of recombinant protein subunit vaccines has proven disappointing [76, 288290 ]. before initiating a vaccination program, indeed, in endemic regions, vaccination is impossible for practical reasons (screening) and noneffective as a preventive measure. however, vaccination could be beneficial for at - risk individuals such as pregnant or immunocompromised individuals, farmers, veterinarians, abattoir workers, and research and reference laboratory personnel in contact with coxiella burnetii. availability of vaccines causes a real problem in most countries and vaccination is currently not performed in europe. a better knowledge of q fever would improve diagnostic procedures, control, and prevention of the disease in the future. a better understanding of the bacterium and of its pathogenesis (entry into the cell, sporulation - like phenomenon, metabolism, mechanism of resistance to acidic conditions, infectivity of the bacterium, survival time in the environment under different management practices, etc. the risk factors for illness must be determined (infectious dose, potential danger linked to pets or not, etc.). moreover, studies are required to define more precisely the incidence, clinical spectrum, treatment, morbidity, and mortality associated with q fever in children. further studies on risk factors could confirm or contradict results of previous studies, such as the studies by whitney. and mccaughey. importantly, a high standard of laboratory diagnostic methods should be available in all accredited laboratories and a systematic search for coxiella burnetii should be performed when clinical signs render the individual or animal suspected of q fever. new guidelines for general practitioners and gynecologists should be established to increase the rapidity of diagnosis of clinical q fever. moreover, guidelines for prevention of infection of the medical staff and for prevention of transmission from one patient to another are necessary indeed, the effect of antibiotic treatment on shedding of bacteria in ruminants has been insufficiently studied. antibiotics have been suggested to diminish shedding by infected female animals but the effectiveness of this measure remains hypothetical. the prevalence and potential clinical consequences of q fever in pets, wildlife, and other neglected domestic animals, such as pigs, must also be investigated. veterinarians must include q fever in their differential diagnoses of clinical cases whenever it can not systematically be rejected as a potential diagnosis. inactive whole - cell phase i vaccines have proved to be effective but present several limitations (e.g., effectiveness may not be observed in the short term, current impossibility of distinguishing vaccinated and naturally infected animals). further research in this field and the development of new recombinant vaccines would permit a better management of foci of infection at a herd level but also in human populations in the future. animal vaccination and vaccination of individuals at high risk of exposure or / and of severe clinical disease would diminish significantly the zoonotic risk. the lack of awareness of this disease leads to underdiagnosing and underreporting of q fever cases. q fever is ubiquitous, and the potential hosts for the infectious bacteria are extremely numerous. q fever infections can have a significant economical impact on animal reproduction, animal trade, and the production and commercialization of animal products. in small ruminant flocks, furthermore, with the advances in human medicine, the number of immunosuppressed, premature, elderly, and chronically ill individuals has greatly increased compared to several years ago. thus, the population predisposed to infection, and especially to chronic infection, has also greatly increased. chronic infections can lead to severe consequences that necessitate intense medical treatment increasing public health costs and the patients ' suffering. an early diagnosis and early initiation of treatment are essential to improve prognosis by preventing the development of a chronic infection or other potential complications associated with coxiellosis.	q fever is an ubiquitous zoonosis caused by an resistant intracellular bacterium, coxiella burnetii. in certain areas, q fever can be a severe public health problem, and awareness of the disease must be promoted worldwide. nevertheless, knowledge of coxiella burnetii remains limited to this day. its resistant (intracellular and environmental) and infectious properties have been poorly investigated. further understanding of the interactions between the infected host and the bacteria is necessary. domestic ruminants are considered as the main reservoir of bacteria. infected animals shed highly infectious organisms in milk, feces, urine, vaginal mucus, and, very importantly, birth products. inhalation is the main route of infection. frequently asymptomatic in humans and animals, q fever can cause acute or chronic infections. financial consequences of infection can be dramatic at herd level. vaccination with inactive whole - cell bacteria has been performed and proved effective in humans and animals. however, inactive whole - cell vaccines present several defects. recombinant vaccines have been developed in experimental conditions and have great potential for the future. q fever is a challenging disease for scientists as significant further investigations are necessary. great research opportunities are available to reach a better understanding and thus a better prevention and control of the infection.
families are a fundamental component of the fabric of society. however, across the world, we observe important distinctions in how societies arrange familial relations., children are brought up within varying gendered, class - based, or age - based hierarchies in the setting of their natal home. but how can we measure these differences in family organization ? and what factors can we discern to offer a tentative explanation for where these differences come from ? in this article, we introduce a new global dataset on historical family characteristics at the level of the ethnic group. using these data, we construct a composite measure of the family constraints on women s agency (fcowa) and explore the geographical factors that may drive differences in such constraints in an attempt to explain the patterns that can be observed. in recent years, a growing interest in what is often called new institutional economics has led to a wave of research in economics and economic history geared toward capturing facets of societies that go well beyond classic economic data (guiso, sapienza, & zingales, 2006). the original formulation of new institutional economics argues that the way that transaction costs and property rights are arranged is crucial to growth and development (north, 1990). in this model, secure property rights and low transaction costs will lead to the efficient allocation of resources through investment in physical and human capital. however, new institutional economics has also spawned an interest in informal institutions which were long excluded from standard economic analysis. one element of this is the attempt to incorporate culture into economic models (guiso., cultural variation is challenging to capture empirically in large part due to its multifaceted nature. as such, it had largely disappeared from economic models as an explanatory variable in the middle decades of the 20th century. however, the recent interest of economists in culture has resulted in research claiming societies norms and values as well as their institutions may persist, rather than simply following in line with the development process (overviews in alesina & giuliano, 2015 ; nunn, 2009 ; spolaore & wacziarg, 2013 ; examples include alesina, giuliano, & nunn, 2013 ; branisa, klasen, & ziegler, 2013 ; nunn & wantchekon, 2011). in much of the economics literature, topics of culture and the institutional determinants of development are tackled at the national level. however, this ignores the wealth of variation that can often be found at the subnational level, particularly in ethnolinguistically diverse countries (alesina, devleeschauwer, easterly, kurlat, & wacziarg, 2003 ; michalopoulos, 2012 ; szotysek, gruber, klsener, & goldstein, 2014, on the historical case). in this article, we explore the possibilities for developing a global dataset of the distribution of family characteristics at the regional level to get a sense of how certain institutions cluster at the subnational level (see also work in a similar vein michalopoulos & papaioannou, 2014 and michalopoulos & papaioannou, 2013). one of our two aims in this article is therefore to introduce a global, geo - referenced dataset of societal characteristics with a focus on family organization. our second objective is to explore how the global distribution of family organization, specifically the existence of fcowa, relates to a set of geographical variables : ruggedness, elevation, distance to coast, and distance to the centers of the neolithic revolution. we hope that this will contribute to further understanding of why certain features of family organization occur in one place but not in another, and by doing so also give insight into the wider determinants and consequences of cultural differences. here, we are interested in one particular element of culture, that of family organization and in particular how family organization impinges upon or empowers women. why focus on women and family organization ? for a start, the family is the key nexus of socialization. this means that family organization and gender roles are particularly likely to be passed on from one generation to the next. moreover, family life and the household are central to many societies and cultures, which means that they are likely to have effects well beyond the realm of the family. this has even been argued to mean that what children learn about authority and equality within their families can be seen reflected at the level of the polity (aristotle, 2014 ; todd, 1985, p. 6). an important element of such power relations within familial arrangements is that of men versus women. in situations where women have greater freedoms, rights, and access to resources, this will likely be reflected in better development outcomes, broadly construed, for the society at large (currie & moretti, 2003 ; dollar & gatti, 1999 ; eswaran, 2014 ; food and agriculture organization of the united nations, 2011 ; king, peterson, adioetomo, domingo, & syed, 1986 ; schultz, 1988 ; strauss & thomas, 1995). the position of women is integrally entwined with culture. in defining a gender system, karen mason (2001) writes the following : a set of beliefs and norms, common practices, and associated sanctions through which the meaning of being male and female and the rights and obligations of males and females of different ages and social statuses are defined. (p.161) a set of beliefs and norms, common practices, and associated sanctions through which the meaning of being male and female and the rights and obligations of males and females of different ages and social statuses are defined. (p.161) the beliefs, norms, and common practices in mason s definition are all part of what may be called culture. these gender systems may thus be influenced by norms and values which, in turn, are historically determined. in other work, we have addressed how the position of women within family systems affects current day outcomes (carmichael, dilli, & rijpma, 2014 ; dilli, rijpma, & carmichael, 2015 ; rijpma & carmichael, 2016), and this is generally also the focus of the interest in culture from the new institutional economics (alesina & giuliano, 2015). however, an important question remains : why do certain family organization arrangements occur in one region but not another ? szotysek (2014) distinguishes a number of explanations for variation in family systems in eurasia. important for our purposes is the distinction between functionalist, developmental explanations on one hand, and a number of slow - moving, persistent explanations on the other. the former view basically argues that family systems change as societies develop, become wealthier, and change demographically (e.g., ruggles, 2009). szotysek also discusses a number of explanations of family organizations that one can expect to be persistent over a long period of time : ecology, institutions, religion, and kinship. although we will also try to include developmental explanations in our analyses, our main focus is on explanations that have an important role in the persistence of family organization. although kinship, for example, is also very important for female autonomy (dyson & moore, 1983 ; whyte, 1978), our outcome variable (discussed below) already includes a number of aspects of kinship (e.g., descent). this limits our set of explanatory variables to geographical variables that go back centuries or even millennia. several ideas have been put forward as to the deep causes of the distribution of female - friendly family arrangements. a number of scholars have identified the agricultural system of a given region as one of the underlying determinants of family organization. goody (1976) argues that societies practicing hoe agriculture tend to transmit property (land) to members of the same gender in a descent group, whereas societies practicing plow agriculture transmit to descendants of both sexes. this, in turn, affects other outcomes such as the prevalence of dowries or control of premarital sex. others have argued that plow agriculture is detrimental to the position of women (alesina., 2013 ; boserup, 1970). similarly, the very introduction of agriculture since the neolithic revolution is argued to have had a negative impact on female freedom (hansen, jensen, & skovsgaard, 2015, todd, 2011). for historical europe, mitterauer (2010, see also mitterauer & sieder, 1982) suggests that cropping regimes shaped inheritance practices and family organization. fauve - chamoux (2006) indicates that stem families are associated with pastoral, mountainous regions. finally, brunnbauer (2002) argues that political legal environs could lead to diversity in family organization within similar geographical settings although he does not dismiss mitterauer s hypothesis. many of these approaches boil down to geography as a fundamental explanation for societal developments related to family and women. in this article, we take a first exploratory look at a number of geographical characteristics which are suggested by the literature to have an effect on family systems particularly as they pertain to the position of women. these characteristics are distance to coast, ruggedness, elevation, and distance to the nearest center of the neolithic revolution. in the next section, we discuss briefly the motivation for the inclusion of each variable. the rest of the article proceeds by sketching the method of matching the family characteristics to locations and the measurement of the geographic variables. the subsequent results section is split in two with the first part presenting the patterns we find for the family systems variables and the second section demonstrating the correlations we find with the various geographic features. the underlying idea is that the position of women is better in coastal areas where men are often away at sea for long periods of time as fishermen or sailors. polnia (2009), in her work on early modern portugal, shows that the phenomenon of absent men had positive results for women ; mobility and multiple male absences led to a growing demand for female labor and economic participation (polnia, 2009, p. 710 ; see also polonia, 2006). a similar type of relationship has been proposed linking the north sea culture, male absence, and female autonomy for north - western europe (pye, 2014, pp. distance to the nearest center of the neolithic revolution is used as a proxy for the timing of the neolithic revolution. the argument for the effect on gender relations runs as follows ; as societies shifted from a nomadic existence to one of settled agriculture more rigid hierarchies emerged between classes, generations, and men and women. the suggestion is that societal organization prior to the neolithic revolution (for instance, in hunter gatherer groups) was relatively female friendly, with goddess worship and associated fertility ritual paramount. gatherer groups in the congo and the philippines (dyble., 2015). they demonstrate that in these modern day hunter gatherer groups, equality between the sexes is the norm, with men participating in child care and unanimous decision making the norm. these findings hark back to the work of morgan (1877/1985) and engels (1884/2010). engels argued that the subordinate position of women follows as a direct result of the rise of alienable property rights which together with monogamy underpin modern civilization. similarly, goody argues that the differences in family organization that can be observed within eurasia were small compared with the differences between eurasia and sub - saharan africa. this he ascribes to the presence of intensive agriculture in eurasia and the associated importance of landownership, inheritance, and social stratification (goody, 1972, 1996 ; nimkoff & middleton, 1960). two more variables that we explore are ruggedness and elevation. both of which represent, to some extent, an inhospitable environment which could result in societies being isolated from larger cultural developments. cultivation is also more difficult (nunn & puga, 2010). in a way, these are therefore used as measures of a later advent of such events as the introduction of agriculture or the incorporation into states (scott, 2009). harsher environments are likely to mean that women s labor is required outside the household and therefore possibly improve the position of women. however, the effect of these variables could cut both ways as isolation from wider societal security networks could force people to rely more heavily on family connections and drive the formation of joint families. in addition, ruggedness and elevation could leave regions beyond the influence of state authority and wider societal developments. this article is built around a dataset which uses two sources : the ethnographic atlas compiled by george murdock (1969) and the atlas narodov mira (anm). in the ethnographic atlas, murdock brought together as many ethnographies as possible in such a way as to make cross - cultural comparison possible. to this end, he created more than 100 variables which were coded from the various monographs, manuscripts, and articles consulted to allow researchers to examine how societies compared with one another in a consistent manner. the anm (bruk & alenenko, 1964), meanwhile, was a project undertaken in the 1960s by soviet academics to map and quantify the location of each ethnic group globally. by using a geo - referenced version of the anm, the geo - referencing of ethnic groups (greg) dataset (weidmann, rd, & cederman, 2010), we are able to link the cultural traits described per group in the ethnographic atlas (from hereon ea) to the location of the ethnic groups. the two sources were combined by matching the ethnic groups coded in murdock with a list of names of ethnic groups listed in the anm (bolt, 2010, 2012). the matching was done using a string - distance algorithm (van der loo, 2014) which provided a list of the most likely candidates in the anm database based on the ea names. we then selected by hand the correct group, either on the basis of the same names or close matches. by doing this, we were able to match 597 societies, 88% of the ethnic groups available in bolt s dataset. this amounts to 47% of the 1,267 societies in the ethnographic atlas and 53% of the 1,071 principal ethnic groups in the anm. the unmatched percentage can be attributed to changing naming practices as well as the ethnographies covering a different set of societies than covered in the anm. figure 1 shows which societies from murdock we were able to match to the anm. first, there is europe for which coverage in the ethnographic atlas is poor (rijpma & carmichael, 2016). australia and the western part of latin america are also frequently unmatched. besides these specific regions with low matching rates, missing societies are fairly evenly spread across the continents. an important exception, however, is africa, for which coverage is very good., we create a composite variable to measure fcowa (this measure is described in more detail in carmichael and rijpma, in press). our choice for these variables is based on an overview of fcowa written by jan kok (in press). in it, he discusses a number of family situations limiting women s ability to make their own life - course decisions. of course, we were also dependent on the data available in the ethnographic atlas. for instance, this meant kok s argument for the inclusion of a variable measuring the right of women to initiate divorce could not be addressed. first of all, we look at descent systems as patrilineal descent could deny women status, power, and claims on inheritance. we also look at whether women have access to property, specifically inheritance rules and property transfers at marriage. we consider patrilocal residence arrangements after marriage, arguing that the absence of nearby kin could remove the support network of the spouse in the new marriage. nonmonogamous marriage is also included as it could weaken women s bargaining position in the household. we look at a preference for cousin marriage to capture whether women have a choice in their marriage partner which would again mean a stronger bargaining position (ideally, we would use a direct measure such as the practice of arranged marriages, but this is not in the ethnographic atlas). again, we use an indirect measure to capture this : the hierarchies present in complex (extended) households. their expected effect is ambivalent : they could either provide support and resources, or they could constrain the decision - making freedom. for all these variables, we created dummy variables indicative of low women s agency (e.g., patrilineal rather than bilateral or matrilineal descent). these 8 variables are brought together into a composite indicator which we call the fcowa measure. it tries to find a latent variable that accounts for the variation and correlation among the multiple variables. the resulting index is a linear combination of the eight variables, using weights that ensure that the index explains as much of the variation in the eight variables as possible. more details about the procedure can be found in the appendix and in carmichael and rijpma (in press). besides geographical coverage, it is also important to be aware of other limitations of the ethnographic atlas. in other work, we have detailed some of the issues with murdock s data that surface if one checks it with external datasets and if one looks at the original source material (rijpma & carmichael, 2016). comparison with external datasets (todd, 1985) on a selection of family - related variables showed that murdock s data could be corroborated for about 70% of the countries. in cases where there was a mismatch, likewise, it was also found that substantial variation could exist within the ethnic groups described. this was especially likely for ethnic groups that were spread across a large geographical area or were observed during times of great social change. because our starting point is the murdock dataset and we are not able to redo his work, these quality issues can not be fully addressed. in the analyses below, we will try to control for them indirectly. the murdock s data are frequently claimed to be preindustrial or precolonial (e.g., nunn & wantchekon, 2011), but the truth is more complicated. most observations were made between 1860 and 1960 with the median in 1920. because geography will largely be constant over time, the heterogeneity in time of observation should not cause a discrepancy between our outcome and explanations. however, it is important to remember that some societies are observed much later than others and, accordingly, some will have been influenced by global trends in family practices. in our analysis below, we try to correct for this by including a variable for the moment of observation. because greg allows us to geospatially locate all societies as polygons on a world map, we are able to use a number of geographical datasets. to calculate the distance to the coast of a society, we used the 1:50 m coastline map from natural earth. the shortest (as the crow flies) distance on the world geodetic system (wgs) ellipsoid between the central point of the society s polygon and the coastline was calculated (hijmans, 2015). the distribution is as expected, though it is important to note that in our procedure, societies that are spread over a large geographical area have large polygons and that their center is therefore far removed from the coast. to the extent that these societies are concentrated near the coast may introduce a bias in our data. distance (as the crow flies) from the society s spatial central point to the coast. we calculate the grid cell size weighted average elevation for each society s polygons (nunn & puga, 2010 : 22 on the need for weighting ; hijmans, 2016, for the computations). ruggedness is a measure of local variation in terrain and makes cultivation and transportation more difficult (nunn & puga, 2010). it was calculated as the average of the absolute differences of a grid cell with its neighbors (wilson, oconnell, brown, guinan, & grehan, 2007). again, we took a grid cell size weighted average for each society s polygon. figures 3 and 4 present our two measures of inhospitability for each ethnic group s polygon. the distribution is as expected, with the himalayas and the andes standing out as particularly inhospitable regions. the distance to the centers of the neolithic revolution was based on a map produced for a supplementary issue to current anthropology which provides 11 major regions of domestication of plants and animals (price & bar - yosef, 2011). this includes one region in the middle east, one in present - day india, two in china, one in new guinea, one in africa, and five in the americas. the map also roughly dates the major domestication sites, and we use this below to correct distance measures for the time since domestication. one is the absolute distance on the wgs ellipsoid from the society s spatial center to the closest center of domestication (figure 5). distance as the crow flies from closest major center of plant / animal domestication. however, the as the crow flies measure has some shortcomings. at distances of many 1,000s of kilometers for this reason, we also calculated the least - cost distance, which takes into account these characteristics to calculate a distance as the wolf runs the gtopo30 elevation data were combined with the major domestication sites to make this calculation. large bodies of water were set to be very costly to traverse, though the grid had been smoothed at the coastline to allow crossing of small bodies of water such as the strait of dover. figure 6 shows an example distance for the afghans, and figure 7 shows the global distribution of this measure. the following maps present first the composite index as well as three of its eight components to give an idea of the geographic spread of the family systems variables we look at. note that to be able to calculate the score on this index, all eight of the variables discussed earlier had to be present. the map shows high family constraints on female agency in western and southern asia, as well as china. africa, both north and south of the sahara, also shows high constraints, especially around the equator. some features are familiar : female agency was lower in the middle east than it was in western europe, for instance (carmichael., 2014). moreover, though there are many ethnic groups unaccounted for in india, we confirm that family constraints on female agency were higher in the north than they were in the south, especially sri lanka (dyson & moore, 1983). in figure 9, the prevalence of extended family is mapped. the variable here is extended (joint) families versus nuclear and stem families which tended to dominate european family systems. we see that extended households are a phenomenon occurring in a belt across the central region of the eurasian continent, as well as in the east of china and in parts of africa. the areas west of china, south - east asia, and southern africa are not characterized by extended families., we see that a large part of asia, north africa, and sub - saharan africa have family systems organized around patrilineal descent, while europe and the americas tend not to have patrilineal descent systems (the alternatives are matrilineal or bilateral). finally, turning to preferred cousin marriage, we see that this appears in the central parts of eurasia, the east of india, canada, and in large parts of north africa (figure 11). preferred cousin marriage. in this section, we check the correlation of the geographical data with the fcowa measure. for this, we use linear regression models. in an exploratory paper such as this, scatterplots and simple correlation coefficients might be more apt. however, we think it is important to at least control for the year of observation in murdock (henderson & whatley, 2014). moreover, we also want to ascertain that the distribution of our fcowa measure is not better explained by very broad geographical or developmental characteristics (e.g., the fact that sub - saharan africa is poorer than other parts of the world). in short, some basic control variables are necessary even at this exploratory stage. research has shown that even at close proximity, there can exist substantial variation in household structure (szotysek., 2014). our own detailed reading of some of the source material used in the ethnographic atlas also revealed that substantial variation can lie behind murdock s coding of ethnic groups (rijpma & carmichael, 2016). this issue can not be truly solved with these data : it is after all reported at the level of the ethnic group. we can, however, try to include a number of proxy variables to control for the issue. one such variable is the area inhabited by the ethnic group, based on the intuition that an ethnic group spread over a larger area (e.g., the russians) would be more likely to display heterogeneity. the area of each polygon is directly available from the digitized anm (weidmann., 2010). for similar reasons, we include population size in c. 1920, the average year of observation in murdock s atlas (data from klein goldewijk, beusen, van drecht, & de vos, 2011). finally, we include the number of polygons that an ethnic group was assigned to in the narodov mira atlas. again, this can be interpreted as a sign of substantial dispersion of the population over a diverse terrain (e.g., islands) or living among other ethnic groups, which would make it more likely for the ethnic group to display diversity in its family structures. it is likely that economic activities and government policies could have a substantial impact on family organization. examples of these ideas include ruggles (2009) work on the economic and demographic determinant of family systems in historical western europe and nimkoff and middleton s (1960) work on the relation between subsistence patterns and the prevalence of extended families. although the ethnographic atlas is a very rich source providing a lot of potential variables, two seem particularly pertinent : jurisdictional hierarchy beyond local community and subsistence economy. the former is a variable coding whether the ethnic group was part of a larger state structure which may have been able to influence family customs. it can take the values ranging from none to four levels of political authority beyond the local community (four levels would usually mean being part of a large state). subsistence economy is an additional variable in the extended version of the ethnographic atlas (gray, 1998). it codes which economic activity contributed most to the society s subsistence : gathering, hunting, fishing, or one of a number of types of agricultural activity. many ethnic groups are represented by more than one polygon. because these observations all share the same score on the outcome variable, the data have been aggregated at the ethnic group level by calculating the mean of the geographic characteristics for each ethnic group (taking the population or area - weighted mean gave qualitatively similar results). although this does discard some important variation in the model, it also prevents shrinking the standard errors too much in the regressions. missing observations for certain variables for some polygons or ethnic groups means the aggregation procedure discards additional observations, meaning we end up with fewer than the 597 societies we were able to match. the alternative was to estimate a full multilevel model which should give similar results (bates, maechler, bolker, & walker, 2015 ; primo, jacobsmeier, & milyo, 2007 ; snijders & bosker, 1999). although we tried to estimate such a model, the models gave substantial convergence problems (c.f. given the fact that our motivation is exploratory, we did not pursue this option further, but we do think that a full analysis of these data would require thorough exploration of the possibilities of fitting a full multilevel model. overall, societies that are further away from a major center of plant and animal domestication display lower family constraints on female agency. however, the mixed results on our two measures (absolute distance and distance over land), and especially the fact our preferred measure (distance by land) gives less precise results, means these results are not very strong. moreover, correcting the measure for the time span since the start of agriculture in the relevant site reversed the relationship. nr = neolithic revolution ; ols = ordinary least squares ; fcowa = family constraints on women s agency ; rmse = root mean square error. p <.05. p <.01. a greater distance to coast was associated with a higher fcowa score, meaning that societies close to the coast had more female - friendly family characteristics. 0.003 lower score on the fcowa measure. with the fcowa measure ranging from 1.8 to 3.4 and with distance to coast ranging from less than a 100 m to more than 2,000 km ruggedness has a negative effect, though the parameter estimate is only significant at the 10% level. elevation has a positive effect, which means that higher, less accessible and less hospitable regions had polities that typically scored higher on the fcowa index, that is, there were more fcowa. the magnitude of the effect is similar to those discussed above : 1% higher mean elevation in a region was associated with a 0.002 higher score on the fcowa measure. with mean regional elevation ranging from sea level to more than 5,000 meters this includes the indicator variables for the macroregions of the world as crude controls for unobserved developmental and geographical effects. here, we also add the subsistence economy and jurisdictional hierarchy variables. because we are not primarily interested in the correlation of these variables with the fcowa index, we report the full regression tables in the appendix (tables a1 and a2). nr = neolithic revolution ; ols = ordinary least squares ; fcowa = family constraints on women s agency ; rmse = root mean square error., we observe that including these controls changes the effects of our variables of interest. notably, the association of the fcowa measure with the variables measuring the distance to the neolithic revolution and elevation is stronger and more accurately estimated. moreover, the contradictory effect of distance corrected by duration since the neolithic revolution has disappeared. although we remain cautious about the relation between the sites of origins of agriculture and the existence of fcowa, it is a factor worth further investigation. evidently, part of the global variation in fcowa can be explained by other factors than geography. the first was to present a new subnational dataset on family characteristics to supplement our earlier country - level dataset. at the same time, we explored a number of ideas about the spread of family institutions that constrain women s agency. concretely, we investigated the role of the spread of agriculture, the role of being a coastal community or landlocked, and the role of terrain inaccessibility. the choice for these factors was driven by a literature which links the position of women to agricultural developments which themselves are largely driven by geography. we were able to create such a database based on a combination of murdock s ethnographic atlas and a digitized, geo - referenced version of the anm. although we focused on a number of family characteristics we have previously investigated (carmichael & rijpma, in press), this procedure allows us to spatially place any ethnographic variable from murdock s atlas. we think that being able to account for this regional diversity is an improvement over the frequent country - level analyses of murdock s data. in our investigation of the distribution of fcowa, we found some support for the role of the distance to the major centers of plant and animal domestication, with societies further removed showing fewer constraints. likewise, we found support for an impact of distance to coast, with societies closely located to the sea displaying less fcowa. however, our measures of terrain inhospitability, elevation, and ruggedness did not show a consistent relation to our measure of fcowa. regions with a higher mean elevation showed less female - friendly family institutions, and there was no discernable relationship with ruggedness. some caveats to our dataset and these explorative analyses are called for. in previous work, we have critiqued some of the codings in murdock s data (rijpma & carmichael, 2016). although our dataset tries to solve the issue of geographic variation at the subnational level, it can not address this shortcoming. although our matching procedure gave us reasonable coverage, future effort to extend it is likely worthwhile. furthermore, better measures of the timing and location of the agricultural transition for each society are possible. distance to the major centers of plant and animal domestication is of course a very crude measure. it would be useful to have a more fine - grained measure that, for example, incorporates actual migration patterns in humanity s early history (pagani., 2016). finally, the possibility exists to use the extensive work done on european household and family history to alleviate the poor coverage for europe in murdock s ethnographic atlas. especially promising from this perspective are projects systematically collecting data on households such as the north atlantic population project and the mosaic project (ruggles, roberts, sarkar, & sobek, 2011 ; szotysek & gruber, 2016). although some parts of the fcowa index can probably be measured in this way (polygamy, the existence of extended households and perhaps consanguineous marriage), others such as descent or the presence of clans would have to be reconstructed from the literature. updating the ethnographic atlas in this way has revealed interesting correlations but future research could do more to revisit and revise murdock s work to improve our understanding of the cross - cultural patterns of developments we can still observe today. to construct our fcowa index, we have tried a number of relevant latent variable models. here, further details of this procedure when applied to country - level data can be found in carmichael and rijpma (in press). we have performed a principal components analysis, a factor analysis (r core team, 2015), and a polychoric factor analysis (revelle, 2015). in each case, we estimate for one factor. the polychoric model is designed to accommodate variables that are not continuous (flora & curran, 2004). the loadings which would determine the weight of each variable in the fcowa index are presented in table a1. generally speaking, they are similar (principal components analysis is rotation invariant, so the sign on the principal components analysis - pca - can be ignored). however, the polychoric factor analysis gives more weight to the variables coding marital payments to the family of the bride, preferences for consanguineous marriage, and polygamous households. as our variables are all dichotomous, we have used the results of the polychoric factor analysis. fa = factor analysis ; poly fa = polychoric factor analysis ; pca = principal components analysis. ols regressions of the fcowa measure on geographical characteristics and socioeconomic and political variables. note. reference category jurisdictional hierarchy : no levels (no political authority beyond community). ols = ordinary least squares ; fcowa = family constraints on women s agency ; rmse = root mean square error. p <.1. p <.05. p <.01.	this article introduces a new dataset of historical family characteristics based on ethnographic literature. the novelty of the dataset lies in the fact that it is constructed at the level of the ethnic group. to test the possibilities of the dataset, we construct a measure of family constraints on women s agency from it and explore its correlation to a number of geographical factors.
intramedullary spinal cord tumors (imscts) compromise about 2 - 4% of all central nervous system neoplasm and about 20 - 25% of all spinal tumors2,12,35). surgery of imscts is very challenging to neurosurgeons, which may result of devastating neurologic deficit. recently, surgical outcomes of patients with imscts have been improved due to advances of diagnostic imaging, microsurgical technique, surgical equipment, and neurophysiologic monitoring. the purposes of our study were to evaluate the recent surgical outcomes of patients with imsct considering recent advances of diagnostic imaging and surgical technologies. in addition, we analyzed the prognostic factors affecting neurological outcome after surgical resection of imscts. between 1998 and 2013, 69 patients with an imsct were admitted and underwent surgical treatment at one institution. the post - operative follow up periods were ranged from 6 to 171 month (mean 34.8 months). basic demographic data, clinical presentation and radiologic exams were retrospectively reviewed in each patient. the neurological state was classified according to american spinal injury association (asia) grade in order to achieve a grading of functional disturbance of daily life activities and gait disturbances. the neurologic outcome was classified as poor (a+b), fair (c) and good (d+e) according to the asia grade. patient 's age at diagnosis, sex, histological origin and grade, tumor location, level of tumor extension, pre - operative neurologic state, initial presenting symptoms and extend of tumor resection were reviewed to analyzed prognostic factors. neuroepithelial tumors were classified as low - grade tumors (gradei+ii) and high - grade tumors (grade iii+iv) by world health organization (who) classification. level of tumor extension was classified into less than four involved segments and four or more segments. extend of tumor removals were classified as total resection (tr), subtotal resection (str) or biopsy. the tr was defined as complete removal of tumor by microscopic surgical finding and postoperative mri finding. all patients had taken magnetic resonance imaging (mri) pre- and post - operatively. few patients were monitored intraoperatively with somatosensory evoked potential (ssep) and motor evoked potential (mep). the steroid was prescribed preoperatively in cases with acute neurological deterioration or edematous signs of the surrounding spinal cord tissue in mri. conus medullaris (n=19, 27.5%) and cervical tumors (n=18, 26.1%) were followed. in 38 patients (55.1%), less than 4 segments were involved and in 31 cases (44.9%) four or more segments were involved. there were forty cases of neuroepithelial tumors (58%) and 29 cases of non - neuroepithelial tumors (42%). seven patients were diagnosed as a high - grade tumor (10.1%) whereas 62 patients (89.9%) showed low - grade histology. the most common histological origins of imscts was ependymoma (n=26, 37.7%) and astrocytoma (n=41, 20.3%) was followed. the hemangioblastoma (n=7, 10.1%) was most common histological type in non - neuroepithelial tumors (table 1). in all patients, the neurological outcomes were good in 52 patients (75.4%), fair in 10 (14.5%), and poor in 7 (10.1%). forty seven patients (68.1%) showed the same final neurological states at last follow - up compared to the preoperative neurological state. sixteen patients (23.2%) showed neurological improvement, whereas six patient 's (8.7%) neurological states were aggravated. one patient showed neurological improvement of asia grade a to b, asia grade c to d in four patients and asia grade d to e in 11 patients. however, one patient 's neurological state was aggravated asia grade c to a and 5 patients showed neurological worsening asia grade d to c (fig. postoperative complications were postoperative hematoma (2), csf leakage (1), laminoplasty impingement (1), and aspiration pneumonia (1). in 8 patients, most strong predictive factor for good neurologic outcome after surgery for imscts is a patient 's preoperative neurologic state. patients who can gait dependently or independently (asia grade d and e) preoperatively resulted 90.4% of good outcome and 9.6% of fair and poor outcomes. however, patients who can not gait (asia grade a, b and c) resulted only 29.4% of good outcome and 70.6% of fair and poor outcomes (p=0.000). similarly, patient 's with motor weakness showed poor neurological outcome than other symptoms (p=0.029). relations between pre - operative clinical predictive factors and post - operative neurological outcomes are summarized in table 2. other predictive factors that affect the neurological outcome are location of tumor (p=0.011) and high grade tumor (p=0.03). tumors in cervical (94.4%) and conus medullaris (88.9%) lesion result better neurological outcomes than in thoracic (59.4%) lesion. in contrast, poor outcome in higher in thoracic (18.8%) than in cervical (0%) and conus medullaris (5.3%). high grade tumor shows 42.9% of good outcome and 28.6% of poor outcome, whereas 79% of patients showed good outcome and 8.1% showed poor outcome in low grade tumor. patient 's sex, age (less or more than 40 years), histological origin (neuroepithelial versus non - neuroepithelial), level of tumor extension, and extend of tumor resection (total resection, subtotal resection or biopsy) were not associated with postoperative neurological outcome. conus medullaris (n=19, 27.5%) and cervical tumors (n=18, 26.1%) were followed. in 38 patients (55.1%), less than 4 segments were involved and in 31 cases (44.9%) four or more segments were involved. there were forty cases of neuroepithelial tumors (58%) and 29 cases of non - neuroepithelial tumors (42%). seven patients were diagnosed as a high - grade tumor (10.1%) whereas 62 patients (89.9%) showed low - grade histology. the most common histological origins of imscts was ependymoma (n=26, 37.7%) and astrocytoma (n=41, 20.3%) was followed. the hemangioblastoma (n=7, 10.1%) was most common histological type in non - neuroepithelial tumors (table 1). in all patients, the neurological outcomes were good in 52 patients (75.4%), fair in 10 (14.5%), and poor in 7 (10.1%). forty seven patients (68.1%) showed the same final neurological states at last follow - up compared to the preoperative neurological state. sixteen patients (23.2%) showed neurological improvement, whereas six patient 's (8.7%) neurological states were aggravated. one patient showed neurological improvement of asia grade a to b, asia grade c to d in four patients and asia grade d to e in 11 patients. however, one patient 's neurological state was aggravated asia grade c to a and 5 patients showed neurological worsening asia grade d to c (fig. postoperative complications were postoperative hematoma (2), csf leakage (1), laminoplasty impingement (1), and aspiration pneumonia (1). in 8 patients, most strong predictive factor for good neurologic outcome after surgery for imscts is a patient 's preoperative neurologic state. patients who can gait dependently or independently (asia grade d and e) preoperatively resulted 90.4% of good outcome and 9.6% of fair and poor outcomes. however, patients who can not gait (asia grade a, b and c) resulted only 29.4% of good outcome and 70.6% of fair and poor outcomes (p=0.000). similarly, patient 's with motor weakness showed poor neurological outcome than other symptoms (p=0.029). relations between pre - operative clinical predictive factors and post - operative neurological outcomes are summarized in table 2. other predictive factors that affect the neurological outcome are location of tumor (p=0.011) and high grade tumor (p=0.03). tumors in cervical (94.4%) and conus medullaris (88.9%) lesion result better neurological outcomes than in thoracic (59.4%) lesion. in contrast, poor outcome in higher in thoracic (18.8%) than in cervical (0%) and conus medullaris (5.3%). high grade tumor shows 42.9% of good outcome and 28.6% of poor outcome, whereas 79% of patients showed good outcome and 8.1% showed poor outcome in low grade tumor. patient 's sex, age (less or more than 40 years), histological origin (neuroepithelial versus non - neuroepithelial), level of tumor extension, and extend of tumor resection (total resection, subtotal resection or biopsy) were not associated with postoperative neurological outcome. in the past, some physicians recommended conservative surgery followed by irradiation for imscts because total removal of tumor may injure normal spinal cord around the tumor34). however, improving medical instruments such as high field mri, surgical tools including microscope with high - definition technology, ultrasonic aspirator and intraoperative monitoring, surgery of imsct has become much safer1). recently, a microsurgical resection is considered as gold standard in the treatment of imsct with the aim of complete tumor removal3). radiotherapy and chemotherapy should only be preserved for high grade lesions or some cases of low grade tumor with incomplete resection and clinical worsening28). although very limited number of cases was done, they suggested that stereotactic radiosurgery is an effective and safe alternative option to conventional radiotherapy. this study shows better outcomes than previous study (unpublished) of our institution on imscts. however, 10 (23.3 %) patients showed worsening of functional outcome whereas only 6 patients out of 69 (8.7%) showed neurological worsening in this study. even though improving medical techniques may result better clinical outcome various clinical factors were proposed as predictive factors of functional outcome after imscts surgery. in this study preoperative inability to walk, thoracic location and high - grade tumor were independently associated with poor post - operative functional outcome. patient 's sex, age, histological origin, level of tumor extension and extend of tumor resection were not significantly associated with postoperative functional outcomes in this study. this study also supports the findings of previous reports about predictors about imsct that preoperative ability to walk is the strongest predictive factor of postoperative outcome5,6,9,10,11,13,15,32,33). these results not only emphasize the importance of early recognition of symptoms and evaluations of imsct but also recommend early surgical treatment outcome10,11,13,20,24,25). the operation should be immediately performed not to wait the onset of clinical deterioration to maximize a chance of preserving an ability of walk. furthermore, in our study the tumors in thoracic lesion were associated with poor prognosis (fig. various studies also showed that tumors arising at thoracic cord were related to poor neurological prognosis1,13,24,33). these could be occurred irreversible damages caused by poor microcirculation and the narrow spinal canal than other spinal lesions during intraoperative maneuver. early surgical interventions should be important in cases with thoracic tumors, even if the neurologic defect is mild. because prolonged compression of imsct, amount of blood flow was increasingly reduced. additionally, thoracic spinal cord had been reported to be more liable to radiation damage18,27), which may be a cause of neurologic defects in survivors with malignancy tumors. on the other hands, tumors located in the thoracic spinal cord take longer time to invade respiratory center leading to respiratory failure (common cause of death among patients with intramedullary malignancy tumors) than tumors initially growing from the cervical spinal cord25). high grade tumor tends to infiltrate to normal spinal cord which results obscure surgical plane. removal of high grade tumor may also injured normal spinal cord resulting poor neurological outcome. additionally, high - grade tumors require pre- or post - operative radiotherapy, which could result poor functional outcome38,39). preoperative radiotherapy may cause radiation - induced myelopahty and/or myelitis19) and compromise the spinal cord microvasculature which lead to spinal cord ischemia. beside of neurological outcome there are some reports that the patients with glioblastoma had a higher mortality and poor prognosis in malignancy spinal cord tumors21,29,36). the rate of complete resection was approximately 90% for ependymoma and hemangioblastoma and 50 - 76% in low grade astrocytoma11). but adult patients with malignant intramedullary tumors treated with gross total resection had a significantly lower mortality and prognosis than patients treated with subtotal resection, biopsy, or non - surgical measures36). recently, advanced microsurgical skills and intraoperative neurophysiological monitoring have made more aggressive chances for total and nearly total resection of imsct8,17,30). in this study, but several authors reported a series with complete tumor removal and good postoperative functional outcome9,10,22). we did n't found statistical results between extent of tumor resection and neurological outcome, thought that total resection should be tried in low - grade tumors. only in high - grade tumors and the tumors where total removal had not been possible, we suggested partial resection or biopsy with radiotherapies and chemotherapies. neurosurgical advances such as improving neuroimaging, intraoperative neurophysiological monitoring, microsurgical technique, and operative instruments made it possible to achieve total resection of imscts. so neurosurgeons should be having a challenging attitude to total removal of low - grade tumors. the intraoperative neurophysiologic monitoring (ionm), combined recording of sseps and transcranial meps, has become a best material for intraopertative testing of spinal cord function in intramedullary spinal cord tumor surgery7). in many reports showed that imsct surgery with ionm was appeared aggressive removal of imsct and good state of neurologic performance16,23,31). these reports used a combined d - wave (epidural meps) and muscular meps (mmeps). hyun.14) reported that ionm combined sseps and mmeps was good method to prevent irreversible pyramidal tract damage. however, choi.4) reported that the groups performed operation under sseps and mmeps did n't significantly associate with total excision of tumor and postoperative well neurologic outcomes. in our study, only 7 patients ' operations were performed with ionm combined sseps and mmeps and one patient showed decreased mep during tumor removal. for this reason, it is hard to explain the relationship between the usage of ionm and the neurological outcome in this study. it was necessary that prospective and randomized controlled research to confirm an association between ionm(sseps and mmeps) and postoperative neurologic outcomes. the recent surgical result of patients with imscts was relatively good with minimal deterioration and complication rate. in addition, imscts in thoracic region showed relatively bad outcome and had a risk of postoperative morbidity compared to that in other regions.	objectiverecently, surgical outcomes of patients with intramedullary spinal cord tumors (imsct) have been improved due to advances of medicine. the purposes of our study were to evaluate the recent neurological outcomes after surgical treatment of imscts.methodswe retrospectively reviewed 69 patients who underwent surgical treatment for imsct in our hospital between 1998 and 2013. patient 's age, sex, histological origin and grade, tumor location, tumor extension, preoperative neurological state, initial presenting symptom, and extend of tumor resection were analyzed to evaluate predictive factors that affect postoperative functional outcome.resultsthe neurological states at last follow - up were improved in 16 patients (23.2%), unchanged in 47 (68.1%), aggravated in 6 (8.7%). in all patients, the functional outcomes were good in 52 patients (75.4%), fair in 10 (14.5%), poor in 7 (10.1%). preoperative good neurological state was the strongest positive predictor of good functional outcome (p<0.05). in tumor location, functional outcomes of thoracic tumors were poor than those in cervical and conus medullaris region (p=0.011). high - grade tumor shows poor outcome compare to low - grade tumor (p=0.03).conclusionthe most reliable predicting factor of surgical outcome was the preoperative neurological state. in addition, imscts in thoracic region and high - grade tumor showed relatively bad outcome and had a risk of postoperative morbidity.
periprosthetic joint infection (pji) is one of the most serious complications that could arise after total hip arthroplasty (tha)12). although the incidence of pji ranges from 0.2% to 1%34567), the pji is a significant (or major) cause of implant failure and requires multiple operations including revision arthroplasty89). microorganism for pji could be originated not only from exogenous sources such as intraoperative contamination, but also from endogenous or hematogenous sources including remote site skin infection, urinary tract infection (uti), and pneumonia in patient themselves1213141516). among the endogenous sources, upper uti, including acute pyelonephritis and intra- /peri - renal abscess, should be treated prior to tha, because the upper uti can develop to uro - sepsis, and result in impairment of renal function20). some authors reported that hip arthroplasty should be delayed until the treatment of uti, because patients with uti had a high risk of pji412171819). but, others demonstrated that preoperative uti was not associated with pji1319). to prevent an occurrence of pji, surgeons should identify the patients with high risk factors of pji2122), and correct the modifiable factors prior to tha323). however, unnecessary delays could result in dissatisfaction from patients who wait for tha2425). our purposes were (1) to evaluate the incidence of pji after elective primary tha at a single institute and (2) to determine whether the lower uti was associated with a risk of pji after elective primary tha. after approval of institutional review board, we retrospectively reviewed the medical records of 544 patients (635 hips), who underwent primary tha by two high volume surgeons at a single institute from may 2003 to october 2007. fifteen patients (15 hips) who were treated for hip fracture and 2 patients who were treated for pathologic fracture were excluded, because they were not elective. the remaining 527 patients (618 hips) were identified for elective primary tha. among 91 patients who underwent bilateral tha, 25 patients underwent simultaneous bilateral tha, and remaining 66 patients underwent staged bilateral thas. of the 593 procedures, uti group was defined as patients who underwent primary tha in status of having an uti, otherwise, they were defined as control group. during preoperative workups including complete blood count, electrolyte panel and urine analysis, uti was diagnosed if symptomatic patients have white blood cells greater than 110/ml or a bacterial count greater than 110/ml14). if a patient was diagnosed with lower uti, urine culture was performed before tha and prophylactic antibiotics were started at the operation day. it was administered until identification of microorganism. if the patients had fever or leukocytosis, we delayed tha until the control of uti (fig. the remaining 514 patients (580 procedures) were classified as control group. in uti group, there were 11 females and 2 males with a median age of 57 years (range, 25 - 74 years). the median of body mass index (bmi) was 26.7 kg / m (range, 19.0 - 33.6 kg / m). in control group, there were 247 females and 273 males with a mean age of 51.0 years (range, 19 - 83 years). their mean bmi was 24.2 kg / m (range, 14.6 - 37.7 kg / m) (table 1). during the study period, we performed all tha in operating rooms equipped with vertical laminar flow with the use of a hepa filter. each member of the surgical team put on a cap cover with string (string hood cap ; km healthcare co., guri, korea) and a surgical jacket (km healthcare co.) (fig. surgical scrub and skin preparation included the use of 2% chlorhexidine - alcohol and aqueous solutions of 10% povidone - iodine (povidin ; sung kwang pharm, bucheon, korea). after then, we used an incise adhesive drape (ioban ; 3 m corporation, st. we used prophylactic intravenous antibiotics within 1 hour of the tha and for 7 days postoperatively. we performed tha through a posterolateral approach in 289 procedures, modified anterolateral approach in 260 procedures, and combined approach in 31 procedures. closed suction drains were used in all cases, and removed at 2 or 3 days postoperatively. postoperative wound management included utilization of a sterile dressing that was applied to the incision in the operating room and usually changed at 2 days postoperatively. the wound was evaluated and the sterile gauze was changed twice a week for 1 week to 2 weeks in the ward. during hospitalization, thigh - length antiembolic stockings were applied and the ankle pump was encouraged without any pharmacological thromboprophylaxis. after discharge, patients were routinely followed up on their 6th week, 3rd, 6th, and 12th month and every 1-year thereafter. the diagnosis of pji was made by musculoskeletal infection society criteria, which is gold standard to diagnose pji (table 2)27). we also evaluated sex, age, bmi, type of anesthesia (regional or general), co - morbidity assessed by american society of anesthesiologists (asa) score, and operation time to determine the relationships between these variables and the development of pji. chi - square test was used for categorical variables. the spss version 16.0 (spss inc., two patients have deceased and 12 patients were lost during follow - up before the minimum 12 months postoperatively. we evaluated 513 patients (579 procedures), who could be followed up at the minimum of 12 months. among the 579 procedures, we evaluated the overall incidence of pji and the association of lower uti and pji after elective primary tha. the overall incidence of pji was 0% in our study, regardless of existence of lower uti. many of studies have reported the incidence of pji ranges from 0.2% to 1%34567). in our series universal aseptic technique and well - established facility might be a reason for the absence of pji in this study. in addition, we did not use routine chemical thromboprophylaxis, which has been considered as a risk of pji28). although several studies recommended the use of chemical thromboprophylaxis after tha, we did not do so as venous thromboembolism is uncommon in east asian patients after tha2930). impaired immune status, systematic disease such as rheumatoid arthritis, lupus, diabetes, and malignancy, advanced age, malnutrition, obesity, previous joint infection, longer operation time, and longer preoperative hospitalization have been reported to be associated with a risk of pji2122). in this study the presence of remote site infection might be associated with pji, and we agreed that it should be identified and controlled before surgery, especially in those with fever or leukocytosis. generally, fever and leukocytosis could be considered as surrogate parameter of systemic bacteremia, which might result in hematogenous pji20). however, a prospective randomized comparative study is barely possible in this topic, because of ethical issue. third, there might be a selection bias, because our hospital is a referral - only university - hospital with high quality equipment. our results showed that cautiously performed tha can have a very low incidence of pji, and lower uti was not associated with a risk of pji when tha was cautiously performed with universal aseptic technique.	purposeperiprosthetic joint infection (pji) after total hip arthroplasty (tha) is a grave complication. urinary tract infection (uti) as a source for pji is controversial. our purposes were, (1) to evaluate the incidence of pji after elective primary tha and (2) to determine whether uti was associated with a risk of pji after elective primary tha.materials and methodswe retrospectively reviewed the medical records of 527 patients who underwent elective primary tha by using universal aseptic technique from may 2003 to october 2007. uti group (13 patients) was defined as patients who underwent tha in status of having an uti, and the remaining patients were defined as control group (514 patients). we compared the incidence of pji in both groups.resultsduring the study period, the incidence of pji was 0%, regardless of existence (or presence) of uti.conclusionthere was no significant association between uti and pji, when cautiously performed tha.
the utah lions eye bank has harvested 159 eyes from strangulations or hangings since 2003. of these, 18 eyes (11%) were not suitable for surgery, with 13 eyes being excluded during the screening process. the reasons for exclusion included hepatitis b positivity, human t lymphotrophic virus positivity, high risk of creutzfeldt - jakob disease, low endothelial cell count, or time spent incarcerated. the final two eyes belonged to a 25-year - old male who was found hanging and unresponsive. his past medical history was significant for depression, previous suicide attempts, peritonitis, and an appendectomy, with an unremarkable past ocular history. following dissection of the corneoscleral buttons by the utah lions eye bank, total detachment of descemet s membrane was seen in both corneas (figure 1). the corneas were deemed unsuitable for transplant surgery, and the tissue was sent to another eye bank for use in keratolimbal allografts. the donor s corneoscleral buttons were retrieved by a skilled tissue recovery specialist with many years of experience. iatrogenic trauma during tissue retrieval can not be ruled out since corneas of intact globes are not routinely examined prior to removal. however, this is unlikely to cause such dramatic findings of complete descemet s membrane detachments bilaterally. to our knowledge, this is the first reported case of extensive descemet s membrane detachment following a hanging. dyer observed the hanging of a prisoner and reported a post mortem fracture through the crystalline lens and anterior lens capsule in the prisoner s right eye. he reproduced these findings by hanging three dogs, with two of six eyes showing similar damage following death.1 in 1869 he saw five more hangings, and found crystalline lens and anterior capsule fractures in three of 10 eyes.2 we speculate that a dramatic rise in intraocular pressure during hanging may stretch the cornea and cause rupture of descemet s membrane. tears in descemet s membrane may occur in congenital glaucoma when the intraocular pressure increases, causing an elongation in axial length and subsequent stretching of the cornea.3 recent research has examined how forces applied to the head influence intraocular pressure. bakaran measured intraocular pressure in yoga practitioners during sirsasana (the headstand posture) and found mean intraocular pressure increased two - fold in patients immediately after assuming this position.4 aykan found that the intraocular pressure increases significantly during valsalva movements.5 hanging may cause increased intraocular pressure secondary to increased venous pressure in the brain. he examined brain lesions caused by hanging via magnetic resonance imaging and found a venous distribution of brain damage.6 we suggest that further research on the effect of increased venous pressure and iop on corneal tissue is needed. furthermore, we would be interested in learning if other eye banks have observed similar corneal tissue changes following strangulation or hanging.	the eyes of a 25-year - old male were collected by the utah lions eye bank after his suicide by hanging. following dissection of the corneoscleral buttons from intact globes, bilateral detached descemet s membranes with subsequent scrolling in the periphery were observed. we believe these findings were caused by a large increase in intraocular pressure secondary to the hanging. lens and anterior capsule fractures after hanging have been reported, but corneal damage has never been discussed. we invite transplant surgeons and eye bank recovery specialists to share their experience of similar corneal changes in donated eyes following strangulation or hanging.
during march and april 2011, diagnoses of acute hev infection were made for diagnosed for 5 patients admitted to 3 hospitals in lazio : 2 in rome and 1 in latina. diagnosis was made on the basis of clinical and laboratory signs of acute hepatitis and detection of igg and igm against hev by immunoassay (radim s.p.a., rome, italy) ; other causes of acute liver injury, including drug toxicity, autoimmune hepatitis, and infection by hepatitis a, b, and c viruses, as well as by cytomegalovirus and epstein - barr virus, were excluded (table 1). hev, hepatitis e virus ; ab, antibody ; hbv, hepatitis b virus ; ebv, epstein barr virus ; ea, early antigen ; vca, viral capsid antigen. the 5 case - patients lived in the same area (maximum distance apart : 27 km) (figure 1). the cases were reported to the health authorities, who conducted a structured interview to determine travel history and other risk factors (table 2). none of the case - patients reported recent travel to disease - endemic areas, and relationships among them or exposure to a common source were not identified. it was not possible to conduct the complete interview for 1 patient (isolate no. b) residence location of 5 cases of hepatitis e virus (hev) subtype 4d infection involved in an autochthonous outbreak, march (map source : cartinegeografiche, catania, sicily, italy ; www.cartinegeografiche.eu). serum samples were tested for rna by reverse transcription - nested pcr by using primers designed within open reading frame (orf) 1 and orf2 (12,13). the sequence data from these genetic regions identified a monophyletic strain belonging to genotype 4, subgenotype d. orf1 nucleotide sequences (172 bp) from the outbreak showed high similarity among patients (99.2%) and 96% and 95% identity with hev4d swine hb-3 and human t1 isolates from china, respectively (genbank accession nos. sequences identified in the orf2 region (411 bp) in samples from the 5 case - patients showed 100% similarity. the sequences also were closely related to the strains kmsw-3 (nucleotide similarity 98.5% ; accession no. hq008863) isolated from swine and to the strain gs - nj-13 isolated from humans (97.8% ; accession no. phylogenetic trees based on partial open reading frame (orf) sequences of the hepatitis e virus monophyletic strain involved in an outbreak in lazio, italy, march sequences from the outbreak in italy could not be submitted to genbank, being 80 are shown. reference viral strains are identified by genbank accession number, source, country of origin, and respective genotype and subtype. the similarity of the strain from this outbreak to strains from recent autochthonous hev genotype 4 infections in europe was relatively low : 73.5% in the overlapping 37172-nt orf1 region of the isolate from germany (genbank accession no. eu879120) (6), 85.7% in the overlapping 31373-nt orf2 region of the isolate from northern france (accession no. gu982294) (7), and 84.7%84.8% in the 329-nt overlapping orf2 region of 5 isolates from southern france (9). consistent with these data, 85.1% and 85.3% identity was observed in the 537-nt orf2 region of 2 of the isolates from southern france (accession nos. jf900631 and jf900632) (8), (technical appendix). an outbreak of hev infection caused by genotype 4 in italy involved 5 case - patients who lived in the same area and did not travel to disease - endemic areas. the high genetic similarity in orf1 and orf2 among the 5 hev isolates supports a point - source outbreak and not sustained local circulation of this strain. epidemiologic information did not identify the transmission route ; available data ruled out direct transmission among patients, and parenteral transmission is unlikely because none of the patients had received blood transfusions, tattoos, or drug injections. three patients had igg against hepatitis a virus (hav) ; no information on hav vaccination status was available. however, the presence of these antibodies does not necessarily imply higher than average levels of exposure to enterically transmitted viruses ; a hav seroprevalence > 60% has been reported in persons in the birth cohorts of the 5 case - patients in central italy (14). consumption of contaminated food (i.e., pork or wild animal meat, bivalve mollusks, or shellfish) is considered the most likely source of infection with hev genotype 3 in europe (8,12). for genotype 4, uncooked deer meat was indicated as a source of human infection in japan (15), and undercooked pork meat was the probable source of infection in southern france (8,9). the isolates from this study had the highest genetic similarity to subgenotype 4d strains of swine origin from china (figure 2), which suggests a possible zoonotic origin through either direct (e.g., ingestion of raw or undercooked pork products) or indirect (e.g., by water contaminated with animal excreta) transmission. possible sources of infection with this hev strain that can not be ruled out include contaminated food from abroad and direct introduction through infected immigrants from china or other countries in asia. however, the proportion of immigrants from asia in this area of italy, 0.77%, is lower than the national average (1.26% ; www.comuni-italiani.it/statistiche/stranieri.html). available data do not support correlations between immigration from china and spread of this hev genotype in lazio. strong sequence similarity (> 96%) was observed between hev isolates from human cases in northern and southern france and the strain isolated from swine in belgium (5), classified as subgenotype 4b. human infection with hev genotype 4 reported in germany in 2008 (6) was attributed to a different subgenotype (4f). the strain involved in the outbreak in italy showed relatively poor genetic resemblance with any of these strains, which indicates that different hev genotype 4 strains have been recently introduced in europe. in summary, this outbreak of hev genotype 4 infection in italy was not linked to infection by imported foods or persons traveling from endemic areas, which suggests the possibility that newly imported strains might spread this virus to new areas. molecular characterization of hev outbreaks in europe is needed to implement epidemiologic mapping of infection with introduced strains of hev and subsequent circulation. methods used to generate partial open reading frame 2 sequences, including the 537-nt region of sequences from southern france described by colson. (1).	during 2011, 5 persons in the area of lazio, italy were infected with a monophyletic strain of hepatitis e virus that showed high sequence homology with isolates from swine in china. detection of this genotype in italy parallels findings in other countries in europe, signaling the possible spread of strains new to western countries.
study participants were recruited from the medical retina clinic at the eye ear nose & throat hospital of fudan university, shanghai, from july 2014 to april 2016. this study was approved by the institutional review board of eent hospital of fudan university and informed consent was obtained from all participants. all investigations followed the tenets of the declaration of helsinki. patients with a large area of hemorrhage on the fundus or cloudy media that could significantly reduce the intensity of the octa signal were excluded. in this study, all pcv patients enrolled underwent a multimodal retinal imaging examination, including fundus photography, fluorescein angiography (fa), icga, spectral - domain optical coherence tomography (sd - oct), and octa. two retinal specialists (wm, lw) who were experienced in the assessment and management of pcv made the diagnosis of pcv. simultaneous fa and icga was performed with the heidelberg spectralis hra+oct (heidelberg engineering, heidelberg, germany). optical coherence tomography angiography scans were performed using the rtvue - xr avanti (optovue, inc., fremont, ca, usa), a 70 khz spectral - domain oct system with a center wavelength of 840 nm. each angiography scan consisted of one horizontal priority (x - fast) and one vertical priority (y - fast) raster scan. for each volumetric raster scan, there were 304 a - scans per b - scan, and two consecutive b - scans at 304 locations. briefly, ssada assesses the oct reflectance variation on a voxel - by - voxel basis between two consecutive b - scans at each location via decorrelation to differentiate between flow (high decorrelation) and static tissue (low decorrelation). split - spectrum processing of the oct signal is used to improve the signal - to - noise ratio of flow detection. to correct for motion artifacts, the contained software registered and merged the x - fast and y - fast scans. maximum flow projection between the inner limiting membrane to inner (ipl) and outer (opl) plexiform layers to bruch 's membrane produced en face angiograms of the superficial and outer retina, respectively. to create composite en face angiograms of the inner retina and neovascular membrane, the inner retina angiogram (purple) composite cross - sectional b - scan images were created in a similar fashion by overlaying flow in the superficial and outer retina on structural oct images. two patient had multiple anti - vegf injections and one underwent photodynamic therapy (pdt). in each case the polypoidal lesion showed high flow signals in different patterns in 12 cases at the outer retina slab on octa. the polyps were located anatomically under the top of the ped. in one case, a polypoidal lesion was not detectable at the outer retinal slab. the patterns of the bvn on octa showed as seafan, medusa, and tangle. the bvn was located anatomically in the space between the rpe and bruch 's membrane on octa, and it showed clearer than on icga. patients ' information summary in a 59-year - old male patient (patient 11 in the table), the polypoidal lesions appeared as several orange - red lesions on fundus photography. indocyanine green angiography in the early phase identified 5 typical polypoidal choroidal dilations. on structural oct, the polypoidal lesion corresponded to the ped and the bvn corresponded to the double layer sign. on octa, the polypoidal lesions were detected as high flow signal spots in the left part of the bvn. bvn showed as a high signal (yellow). on composite b scan, the blood flow signal of the bvn was in the space between the rpe and bruch 's membrane, and showed at lines 1 and 2. both polyps and the bvn showed clearly on pseudo - color semiautomated segmentation. in a 58-year - old male patient (patient 10 in the table), a large orange - red lesion showed on fundus photography. indocyanine green angiography in the early phase identified a very obvious polypoidal choroidal lesion showing hyperfluorescence surrounded by a hypofluorescent pool and the bvn. indocyanine green angiography combined with sd - oct demonstrated that the polypoidal choroidal lesion and bvn on icga corresponded to the ped and double layer sign on sd - oct. the semiautomated segmentation showed that the blood flow signals of polyps were under the top of the ped and those of the bvn were between the rpe and bruch 's membrane. in a 59-year - old male patient (patient 11 in the table), the polypoidal lesions appeared as several orange - red lesions on fundus photography. indocyanine green angiography in the early phase identified 5 typical polypoidal choroidal dilations. on structural oct, the polypoidal lesion corresponded to the ped and the bvn corresponded to the double layer sign. on octa, the polypoidal lesions were detected as high flow signal spots in the left part of the bvn. the blood flow signal of the bvn was in the space between the rpe and bruch 's membrane, and showed at lines 1 and 2. both polyps and the bvn showed clearly on pseudo - color semiautomated segmentation. in a 58-year - old male patient (patient 10 in the table), a large orange - red lesion showed on fundus photography. indocyanine green angiography in the early phase identified a very obvious polypoidal choroidal lesion showing hyperfluorescence surrounded by a hypofluorescent pool and the bvn. indocyanine green angiography combined with sd - oct demonstrated that the polypoidal choroidal lesion and bvn on icga corresponded to the ped and double layer sign on sd - oct. the semiautomated segmentation showed that the blood flow signals of polyps were under the top of the ped and those of the bvn were between the rpe and bruch 's membrane. optical coherence tomography angiography is a noninvasive imaging technology for detecting the blood flow of pcv in a short testing time. we could obtain good quality octa images if the subject had relatively good fixation and clear media. we studied 13 cases of pcv with different manifestations on octa and icga. from the octa findings of these 13 cases, a polypoidal lesion could be detected in 12 (92.3%) cases with the outer retinal slab. kim. mentioned that only 50% polyps were hyperreflective lesions on the outer retinal reference on octa. inoue. scanned patients with pcv and polypoidal cnv (pcnv), and found that the polyps flow within a focal point in the polypoidal lumen and that polyps were not clearly discernible on en face octa with the reference plane in the choroid capillary level. srour. indicated that 75% eyes with pcv showed a hypoflow round structure in correspondence with the polypoidal lesion on octa with segmentation of the choriocapillaris layer. compared to the studies above, the polyp detection rate was higher in our study. this could miss some signals from the top of the ped, which is the location of the polyps. according to our experience, it would be better to choose the outer retina slab for observation of polyps on octa. the default en face display slab definitions of the outer retina are from 70 m below the ipl to 30 m below the rpe and the choroid capillary slab is from 30 to 60 m below the rpe in the angiovue software. actually, in eyes with a detached rpe, the outer retina slab included the flow signal from the top of the detached rpe to bruch 's membrane. among these 12 cases, polypoidal lesions showed as different patterns, such as nodular, ring, dot, and cluster (figs. 1, 2, 3). by comparing the en face octa of the polyps of patient 3 with the corresponding area on icga (figs. 3a1, 3a2), we found that the obvious hyperfluorescence polyps on icga only showed a high flow signal as a ring at the periphery of the polypoidal lesion. the polyps of patient 3 manifested as a slow icg filling by icga and this is not rare in pcv on icga, which probably is caused by the turbulence within the dilatation vessel. according to the principal of ssada, the slow velocity of the blood flow can not be captured in octa due to the low decorrelation value and the flow velocity at the periphery of this large polypoidal lesion could be faster than at the center, so octa captured the faster flow signals at the periphery and made them present as a ring. sometimes the polypoidal lesion on icga was so small that the blood flow signal on octa was difficult to detect (figs. this could cause shrinkage of the polyps and difficulty in detecting them with octa. to the best of our knowledge, this is the first study to show the distinct blood flow changes inside the polyps with octa. (a) the fundus photograph shows orange - red polypoidal lesions (red arrow). (c) spectral - domain oct scans corresponding to the interrupted white lines in (b) identify ped (red arrow, cross - section 1 on top) and bvn (white arrow, cross - section 2 on bottom). (d) 3 3 mm macular en face octa of the outer retina reference. the high flow signal spots (red arrow) indicates the location of the polyps. (e) enlarged icga (yellow rectangle in [b ]) shows polyps (red arrows) and bvn. (f) 3 3 mm en face octa of the macula shows bvn (white - dotted line enclosure). (g) color composite en face octa shows bvn flow (yellow outer retinal flow) in the context of normal retinal circulation (purple inner retinal flow). (h) color composite cross - sectional octa shows that the bvn was in the space between rpe and bruch 's membrane (white arrow, cross - section 1 on top) and the polyps were just below the top of the ped (red arrow, cross - section 2 on bottom). (i) en face octa of the slab from outer boundary of opl to bm shows polyps (red arrows) and bvn (white dotted line enclosure) clearly at the same time. (a) fundus photography shows a large orange - red lesion (red arrow) (b) early - phase icga shows the polyps as a hyper - fluorescent nodule surrounded by the hypo - fluorescent pool. (c) the cross - sectional oct crossing polyps (interrupted white line on [b ]) identifies dome - shaped ped (red arrow) and bvn (white arrow). (d) 3 3 mm en face octa with the outer retina slab identifies the polyps (red arrow). (e) the yellow rectangle in (b) was enlarged for better comparison with octa. (f) 3 3 mm en face octa with the choroid capillary reference plane shows the bvn (white - dotted line enclosure). (g) color composite en face octa identifies the polyps (red arrow) and bvn (white - dotted line enclosure). (h) color composite cross - sectional octa shows that the signals of polyps (red arrow) were just below the top of ped and the signals of the bvn (white arrow) were in the space between the rpe and bruch 's membrane. optical coherence tomography angiography (a1) and icga (a2) of patient 3. optical coherence tomography angiography with the outer retina slab shows the signals of the polyps as a circle (red dotted line enclosure). early - phase icga shows a large hyperfluorescent nodule (red dotted line enclosure). optical coherence tomography angiography (b1) and icga (b2) of patient 13. optical coherence tomography angiography with the outer retina slab shows the signals of the polyps as some spots (red dotted line enclosure). optical coherence tomography angiography (c1) and icga (c2) of patient 5. optical coherence tomography angiography with the outer retina slab shows polyps as a cluster of high flow signals (red dotted line enclosure). optical coherence tomography angiography (d1) and icga (d2) of patient 12. optical coherence tomography angiography with the outer retina slab can not detect the flow signal at the location the polyps should be (red dotted line enclosure). early - phase icga shows polyps as a hyperfluorescent spot (red dotted line enclosure). color composite cross - sectional octa identified the polypoidal lesion beneath the top of the ped (figs. summarized sd - oct findings in 17 eyes with pcv and khan. analyzed 18 pcv cases with icga and oct. they both demonstrated that the polyps were located in the sub - rpe space adherent to the posterior surface of the rpe. color composite cross - sectional octa of patients 2 (a) and 4 (b) show the signal of polyps (yellow signals). signals of the polyps are just below the top of the ped (red arrows). color composite cross - sectional octa of patients 12 (c) and 1 (d) show the signal of bvn (yellow signals). signals of the bvn are in the space between the rpe and bruch 's membrane (white arrows). among our 13 patients, octa was able to detect the bvn in each case (100%) and the bvn showed more clearly on octa than on icga. the patterns of the bvn on octa included seafan, medusa, and tangle (fig. 5). with color composite cross - sectional octa, the bvn flow signals were detected between the rpe and bruch 's membrane (figs. 1h line 1, 4c, 4d), not in the inner choroid as previous literature suggested. the patterns of the bvn on octa were very similar to type i cnv in wet amd. kuehlewein. described the different patterns of type i cnv on octa in wet amd as medusa and seafan, which were similar to the patterns of the bvn on octa in our study. optical coherence tomography angiography (a1) and icga (a2) from patient 6. optical coherence tomography angiography with the choroid capillary slab shows the bvn with seafan pattern more clearly than icga (yellow dotted line enclosure). optical coherence tomography angiography (b1) and icga (b2) from patient 3. optical coherence tomography angiography with the choroid capillary slab shows feeder vessel and draining vessel (yellow arrows). early - phase icga shows corresponding feeder vessel and draining vessel (yellow arrows). optical coherence tomography angiography (c1) and icga (c2) from patient 1. optical coherence tomography angiography with the choroid capillary slab shows the bvn with medusa pattern more clearly than icga (yellow dotted line enclosure). optical coherence tomography angiography (d1) and icga (d2) from case 8. optical coherence tomography angiography with the choroid capillary slab shows the bvn with a tangle pattern more clearly than icga (yellow dotted line enclosure. second, we only selected typical cases of pcv. third, the octa system did not allow us to customize the segmentation to follow the contour of the lesion in some cases. this could mislead interpretation of the octa image. in conclusion, octa is a noninvasive imaging tool for detecting the blood flow changes in pcv. the blood flow signal inside the polypoidal lesion showed a different pattern on octa because octa just does not image the entire polypoidal lesion as well as icga. the pattern of the flow signal of the polypoidal lesion and bvn on octa is helpful to better understand the pathology of pcv.	purposewe observed and analyzed the morphologic characteristics of polypoidal lesions and abnormal branching vascular network (bvn) in patients with polypoidal choroidal vasculopathy (pcv) by optical coherence tomography angiography (octa).methodsa retrospective observational case series was done of patients with pcv. all patients were scanned with a 70-khz spectral - domain oct system using the split - spectrum amplitude - decorrelation angiography (ssada) algorithm to distinguish blood flow from static tissue. the octa images of these patients were compared to those from indocyanine green angiography (icga). semiautomated segmentation was used to further analyze the polypoidal lesion and the bvn.resultswe studied 13 eyes of 13 patients 51 to 69 years old. a total of 11 patients were treatment - naive. two patients had multiple anti - vegf injections and one underwent photodynamic therapy (pdt). optical coherence tomography angiography was able to detect the bvn in all cases. using cross - sectional octa, bvn locations were shown to be in the space between the rpe and bruch 's membrane. using en face octa, the bvn vascular pattern could be shown more clearly than by icga. polypoidal lesions showed high flow signals in different patterns in 12 cases in the outer retina slab. using cross - sectional octa, the polyps were shown to be just below the top of the pigment epithelial detachment (ped). in one case, the polypoidal lesion was not detectable at the outer retina slab.conclusionsoptical coherence tomography angiography is a noninvasive imaging tool for detecting vascular changes in pcv. branching vascular networks showed more clearly on octa than on icga. polypoidal lesions had variable patterns on octa and were not always detected. the octa patterns of the polypoidal lesions and the bvn are helpful in understanding the pathology of pcv.
epidemiological studies have indicated that physical inactivity is associated with a variety of noncommunicable diseases (ncds), and overall 1.9 million deaths are attributable to physical inactivity. hypertension is one of the most common ncds affecting a quarter to about half of the adult population in many countries including kingdom of saudi arabia (ksa).[36 ] it is a complex disorder with many genetic and environmental causes. poor lifestyle such as physical inactivity is one of the risk factors predisposing to its occurrence and complications. it is claimed that virtually all individuals (hypertensives and normotensives) can benefit from regular physical activity, whether it is vigorous exercise or some moderate health - enhancing physical activity. prospective cohort studies have generally demonstrated that physical activity or physical fitness is associated with a reduced risk of hypertension.[810 ] all blood pressure measurements were greater in the least physically active persons including prepubertal children.[1113 ] in ksa, as in other gulf countries, a dramatic rise in the standard of living, increased mechanization, and the use of computer and telecommunication technology have resulted in low levels of physical activity and sedentary living.[1418 ] the current levels of physical inactivity are also partly due to little participation in physical activity during leisure time and an increase in sedentary behavior in work - related and domestic activities. likewise, an increase in the use of passive modes of transport has also been associated with the declining level of physical activity. increased urbanization resulting in several environmental factors such as violence, heavy traffic, pollution, and absence of parks, sidewalks, and sports and recreational facilities these changes may have contributed to the high prevalence of many chronic ncds such as hypertension. previous studies in ksa have reported the association of hypertension with physical inactivity in general, but the studies did not discuss the association of hypertension with the levels, patterns, or duration of physical activity. this study aims to compare patterns of physical activity of normotensive and hypertensive adults and identify significant risk factors of physical activity and predictors of hypertension. to the best of our knowledge, it is hoped that the results will be beneficial in designing programs for the prevention and control of hypertension. this is a cross - sectional community - based study done in 2005 covering the entire kingdom of saudi arabia. the who stepwise approach to surveillance (steps) of ncd risk factors was the basis for the conduct of the survey and for the collection of data. a multistage stratified cluster random sampling technique was used to recruit a representative sample of the total population, aged 1564 years, in saudi arabia. stratification was based on age (five 10-year - old groups) and gender (male / female, two groups). all health regions of the country (20 regions) were covered. based upon the proposed methodology of the who stepwise approach, a sample size of 196 a list of all primary health care centers (phccs) in each region was prepared and 10% of these phccs were randomly chosen, and allocated a regional sample that was proportionate to the size of their catchment population in the sampled phccs. to identify the households, a map of the health center coverage area data were collected using the who step wise approach which includes a questionnaire, physical measurements plus biochemical measurements including hypertension, other chronic diseases, and risk factors in addition to the pattern, intensity, and duration of physical activity. the questionnaire was translated into arabic by a team of physicians and translated back to ensure accuracy of translation. the arabic instrument was pretested and corrected before being tried on 51 eligible respondents to check the accuracy of the wording and comprehension of the questions. all required adjustments were then made in the instrument in the light of the pretest. data were collected by 54 males and 54 females working in teams of four persons each : a male data collector, a female data collector, a driver, and a female assistant. data collection teams were supervised by a hierarchy of a local supervisor, regional coordinators, and a national coordinator. all persons involved in data collection attended a comprehensive training workshop which explained the interview techniques, data collection tools, practical applications, and field guidelines. the respondent was advised to sit quietly and rest for 5 min with the legs uncrossed and the right arm free of any clothing. then, the right arm was placed on the table with the palm facing upward. taking of measurements involved the following : pushing the start button automatically inflated the cuff and displayed and recorded systolic blood pressure (sbp) and diastolic blood pressure (dbp) readings. a second reading was taken 5 min after the first and a third 5 min later. the subject was labeled hypertensive if the average of the three blood pressure measurements was 140 mmhg or above for systolic and/or 90 mmhg or above for diastolic blood pressure, or the subject was a known hypertensive previously diagnosed by a health professional. the physical activity measure used was the global physical activity questionnaire (gpaq) which comprised 19 questions about physical activity performed in a typical or normal week. the gpaq measure asked about the frequency (days) and the time (minutes / hours) spent doing moderate to vigorously intense physical activity in three domains : (i) work - related physical activity (paid and unpaid, including household chores) ; (ii) active commuting (walking and cycling) ; and (iii) discretionary leisure time (recreation) physical activity. gpaq is an instrument derived from the long and short forms of the international physical activity questionnaire (ipaq) which has been validated and widely used to assess physical activity patterns. the test retest reliability of gpaq (short - term assessment in a 3- to 7-day interval) produced good - to - excellent results (r = 0.670.81) and the concurrent validity against ipaq for total physical activity yielded a moderate - to - good correlation (r = 0.54) ; questions on sedentary activity generated a good correlation (r = 0.65). questionnaires collected from the field were reviewed by team leaders of each team before submission to the headquarters for data entry. double entry of the questionnaires was performed using epi - info 2000 software and epidata software. new variables were defined by adopting the standard steps variables (steps data management manual, draft version v1.5, october 2003). descriptive statistics, t - test, mann whitney test were used as appropriate after checking for normality. the protocol and the instrument of the surveillance were approved by the ministry of health, center of biomedical ethics, and the concerned authorities in ksa. confidentiality of data was assured indicating that data would only be used for the stated purpose of the survey. a multistage stratified cluster random sampling technique was used to recruit a representative sample of the total population, aged 1564 years, in saudi arabia. stratification was based on age (five 10-year - old groups) and gender (male / female, two groups). all health regions of the country (20 regions) were covered. based upon the proposed methodology of the who stepwise approach, a sample size of 196 a list of all primary health care centers (phccs) in each region was prepared and 10% of these phccs were randomly chosen, and allocated a regional sample that was proportionate to the size of their catchment population in the sampled phccs. to identify the households, a map of the health center coverage area data were collected using the who step wise approach which includes a questionnaire, physical measurements plus biochemical measurements including hypertension, other chronic diseases, and risk factors in addition to the pattern, intensity, and duration of physical activity. the questionnaire was translated into arabic by a team of physicians and translated back to ensure accuracy of translation. the arabic instrument was pretested and corrected before being tried on 51 eligible respondents to check the accuracy of the wording and comprehension of the questions. all required adjustments were then made in the instrument in the light of the pretest. data were collected by 54 males and 54 females working in teams of four persons each : a male data collector, a female data collector, a driver, and a female assistant. data collection teams were supervised by a hierarchy of a local supervisor, regional coordinators, and a national coordinator. all persons involved in data collection attended a comprehensive training workshop which explained the interview techniques, data collection tools, practical applications, and field guidelines. the respondent was advised to sit quietly and rest for 5 min with the legs uncrossed and the right arm free of any clothing. then, the right arm was placed on the table with the palm facing upward. taking of measurements involved the following : pushing the start button automatically inflated the cuff and displayed and recorded systolic blood pressure (sbp) and diastolic blood pressure (dbp) readings. a second reading was taken 5 min after the first and a third 5 min later. the subject was labeled hypertensive if the average of the three blood pressure measurements was 140 mmhg or above for systolic and/or 90 mmhg or above for diastolic blood pressure, or the subject was a known hypertensive previously diagnosed by a health professional. the physical activity measure used was the global physical activity questionnaire (gpaq) which comprised 19 questions about physical activity performed in a typical or normal week. the gpaq measure asked about the frequency (days) and the time (minutes / hours) spent doing moderate to vigorously intense physical activity in three domains : (i) work - related physical activity (paid and unpaid, including household chores) ; (ii) active commuting (walking and cycling) ; and (iii) discretionary leisure time (recreation) physical activity. gpaq is an instrument derived from the long and short forms of the international physical activity questionnaire (ipaq) which has been validated and widely used to assess physical activity patterns. the test retest reliability of gpaq (short - term assessment in a 3- to 7-day interval) produced good - to - excellent results (r = 0.670.81) and the concurrent validity against ipaq for total physical activity yielded a moderate - to - good correlation (r = 0.54) ; questions on sedentary activity generated a good correlation (r = 0.65). questionnaires collected from the field were reviewed by team leaders of each team before submission to the headquarters for data entry. double entry of the questionnaires was performed using epi - info 2000 software and epidata software. new variables were defined by adopting the standard steps variables (steps data management manual, draft version v1.5, october 2003). descriptive statistics, t - test, mann whitney test were used as appropriate after checking for normality. the protocol and the instrument of the surveillance were approved by the ministry of health, center of biomedical ethics, and the concerned authorities in ksa. confidentiality of data was assured indicating that data would only be used for the stated purpose of the survey. of the total 4758 subjects who participated in the study, about 51% were females. a total of 1213 were hypertensives giving a prevalence of 25.5% (27.1% males and 23.9% females). of the subjects, only 12.1%, 20.2%, and 46.1% were physically active in recreational, work- and transport - related activities, respectively. physical inactivity in general was significantly associated with females, the elderly, retired, and persons living in the central region. pearson 's correlation coefficient was calculated for systolic and diastolic blood pressures separately and the total duration of physical activity in minutes per day for all subjects (hypertensives and normotensives). there was a significant negative correlation indicating that as the total duration of physical activity increased, both systolic and diastolic pressures decreased (r = -0.048, p = 0.001, for systolic pressure ; r = -0.037, p = 0.013, for diastolic blood pressure). hypertension was significantly negatively associated with the total level of physical activity as shown in table 1. in table 2, the comparison between physical activity patterns in hypertensive and normotensive subjects using odds ratios and 95% confidence intervals. all lower levels of physical activity in leisure, transport, and work were significantly associated with an increased risk of hypertension. table 3 compares the mean duration of the different patterns of the physical activity of the hypertensives and normotensives. the table shows that for hypertensives durations of all patterns of physical activity were significantly shorter. all significant risk factors in tables 2 and 3 were included in the logistic regression model. these were : work involving moderate physical activity for 10 min, walking / cycling for 10 min continuously, and vigorous activity in leisure time. overall level of physical activity among hypertensives and nonhypertensives association of physical activity patterns and hypertension status duration of physical activity patterns and blood pressure status logistic regression model to predict hypertension from some physical activity patterns and levels after adjusting for age this study as well as previous studies has shown that physical inactivity in ksa was high indicating the sedentary nature of the saudi population.[341417 ] the overwhelming majority of men and women did not reach the recommended physical activity levels required for the promotion of health and the prevention of disease. the knowledge of students in ksa of the role of physical activity in the prevention of hypertension is rather weak. consequently, it is not surprising that the study revealed that hypertension affected more than a quarter of the population, physical inactivity being a significant contributor. significant risk factors for hypertension included low levels of physical activity for all patterns of activities (leisure-, work-, and transport - related and recreational activities). these were work related involving a moderate physical activity for 10 min, walking / cycling for 10 min continuously, and vigorous activity in leisure time. physical inactivity was significantly negatively correlated with both systolic and diastolic blood pressures in this study, and in previous studies in ksa.total physical inactivity was significantly associated with hypertension. studies have reported that both fitness and activity were inversely associated with the development of hypertension and that physical activity of a moderate intensity lowered systolic and diastolic pressures in both normotensive and hypertensive subjects by 27 mmhg. walking during leisure or working in many communities physical inactivity was definitely and strongly associated with hypertension in this and other studies, but the pattern of the levels and intensity of physical activity were not consistent. meta - analyses of randomized control trials assessing the effectiveness of exercise training in lowering blood pressure revealed significant heterogeneity, reflecting the varied subject demography and exercise schedules used in different studies. the available data suggest that aerobic exercise of a moderate intensity is associated with a significant reduction in blood pressure in hypertensive and normotensive participants and in overweight as well as normal weight participants. increasing the exercise intensity above recommended levels did not have any additional impact on the reduction of blood pressure. even though it is well established that moderate exercise lowers blood pressure in most hypertensives, in some studies about a quarter did not respond to exercise training. this study detailed the association of physical activity and hypertension in terms of intensity, duration, and patterns. although the mechanism of how physical activity reduces the risk of hypertension remains unclear, the finding that an increase in activities in daily life is associated with a lower risk of hypertension as reported in this study, and others should encourage all of us to advocate physical activity in all age groups. aerobic activity is the preferred type of activity as a means of lowering blood pressure, but all other patterns are useful. as found in a recent study in ksa, the enhanced quality and a broadened scope of health education if given to patients who go to easily accessible phccs all over the country would improve the awareness and practice of healthy behaviors such as the inclination to become more physically active. regular physical activity, fitness, and exercise are critically important for the health and well being of people of all ages. this study and others have demonstrated that virtually all individuals (hypertensives and normotensives) can benefit from regular health - enhancing physical activity. specifically tailored and culturally sensitive interventions including physical activity that address multiple health behaviors are necessary for the prevention, and control of hypertension and other chronic morbidities.	background : to formulate all intervention strategies for hypertension in the community, it is essential to quantify the magnitude of the disease and its risk factors. the patterns of physical activity have not been studied in terms of their being a risk factor or a predictor of hypertension in saudi arabia.materials and methods : this was a community - based cross - sectional study using the step - wise approach of adults and a multistage, stratified, cluster random sample. data were collected using a questionnaire which included sociodemographics, blood pressure, patterns, levels and duration of physical activity.results:of a total of 4758, 1213 (25.5%) were hypertensives. hypertension was significantly negatively associated with total levels and duration of physical activity in leisure, transport, and work. significant predictors of hypertension included lower levels of work involving a moderate physical activity for 10 min, walking / cycling for 10 min continuously, and vigorous activity during leisure time.conclusions:hypertension is prevalent among adults ; physical inactivity is a significant risk factor and predictor. targeting this modifiable risk factor can help in prevention, early diagnosis, and control.
there is strong epidemiological evidence of excess mortality in association with the diabetic foot syndrome. there is a greater than twofold increase of mortality in diabetic foot ulcer (dfu) patients compared to nonulcerated diabetic patients, regardless of age, type and duration of diabetes, treatment of diabetes, glycated haemoglobin concentration, history of lower extremity amputation, and cumulative pack years of cigarette use. diabetic patients with leg and foot ulcers have a lower 5-year survival (43%) than nondiabetic ulcerated subjects (56%) and general population controls (68%). reported mortality rates for diabetic foot ulcer (dfu) patients range from 10% on a median follow - up of 16 months to 24% after 5 years. a study from a liverpool foot clinic indicated a 5-year mortality rate as high as 44% in patients presenting with new dfus. a large community based norwegian study over a 10-year follow - up period reported an increased mortality of 49% in diabetic patients with a history of dfu compared with 35.2% of diabetic patients without a history of foot ulcers and 10.5% of nondiabetic individuals. we previously reported preliminary results on our patients but only had information on 112 of 243 death certificates and 41 out of 80 postmortem examinations that were carried out. the present paper has information on the full number of 243 death certificates and 80 postmortem examinations. the aim of this study was to establish the precise cause(s) of death in dfu patients and to examine the relationship between cause of death and ulcer type. to delineate causes of death in dfu patients, we conducted an 11-year retrospective (death search) audit of all deceased subjects who attended the diabetic foot clinic at king 's college hospital and whose records were available in this institution. information provided for each patient included full name, address, date of birth, date of death, age at death, and place of death. causes of death were established for deaths occurring between april 1989 and january 2000 from the following : death certificates obtained from the central register for england and wales held in southport through the family record centre in london. in cases where death certificates could not be retrieved from the central register, individual registry offices of various boroughs of london were approached for those certificates;postmortem examination results as stated in hospital records and coroners ' reports on death certificates.study subjects included type 1 and type 2 diabetes patients as per who classification (1997) (table 1). neuropathy was determined by the presence of a stocking distribution of sensory loss to light touch (cotton wool). patients with signs of neuropathy and palpable pulses were said to have neuropathic ulcers and patients with absent foot pulses with or without neuropathy were deemed to have ischaemic ulcers. death certificates obtained from the central register for england and wales held in southport through the family record centre in london. in cases where death certificates could not be retrieved from the central register, individual registry offices of various boroughs of london were approached for those certificates ; postmortem examination results as stated in hospital records and coroners ' reports on death certificates. we sought a control group of diabetic patients who attended the diabetic clinic at king 's college hospital and died over a 10-year period from march 1990 to march 2000. they were stratified to match the dfu group in age and type of diabetes. in the uk, the major immediate cause of death death from ischaemic heart disease (ihd) was defined as death due to coronary artery disease or fatal myocardial infarction from occlusive coronary thrombosis or secondary fatal dysrhythmias. mean age at death was calculated, including its standard deviation, and significance was tested at 95% confidence interval. a p value of < 0.05 was considered statistically significant. the significance of the risk of death from ihd in diabetic patients with neuropathic ulcers compared to those with ischaemic ulcers was also assessed using the chi - squared and fischer exact tests (software used was gb stat v6.5). the odds ratio was also determined with confidence intervals calculated at 95% level of certainty. also, levels of significance of causes of death between dfu patients and control patients and between neuropathic and ischaemic ulcer patients on both death certification and postmortem results were calculated and expressed as p values in tables 2, 3, and 4. we identified 268 patients who no longer attended the diabetic foot clinic between april 1989 and january 2000. there were 243 confirmed deaths in dfu patients who attended the clinic during the above period. the remaining 25 subjects with dfu whose deaths could not be confirmed or information on cause of death was unobtainable were excluded from the study. the dfu group consisted of 147 (60.5%) male patients and 96 (39.5%) female patients with an age range of 30 to 95 years. of these, 187 had ischaemic ulcers and 56 had neuropathic ulcers as defined in materials and methods. a control group of 121 deceased diabetic patients without foot ulceration was identified from the general diabetic clinic at king 's college hospital. of the 139 patients who no longer attended the diabetic clinic, 121 patients had confirmed deaths and causes of deaths were obtained as for the ulcerated group. in 12.9% the control group comprised 54 (44.6%) male patients and 67 (55.4%) female patients with an age range of 44 to 92 years. the majority of the control patients, 103 (85%), died in hospital, which was comparable to the dfu group.. the characteristics of both dfu and control group patients are summarised in table 1. there was no significant difference apart from the proportion of male patients which was significantly higher in the dfu group compared with the controls. in addition to death certification, 80 (33%) dfu subjects and 30 (25%) control patients had postmortem examination. there were 25 dfu patients and 18 control patients whose cause of death could not be acquired. these patients may have moved from one region to another within the uk or possibly emigrated abroad. there are no reliable procedures for confirmation of death in this group, as deaths have not been notified to the central register in england and wales. it was not possible to obtain the death certificate (or postmortem results) for these subjects as there was no trace at the family records centre or the local registry office. the dfu group consisted of 12 (48%) male patients and 13 (52%) female patients. there was no significant difference between patients whose cause of death was ascertained and those whose cause of death was not obtained for both age (p = 0.174) and proportion of males to females (p = 0.230). the control group consisted of 9 (50%) male and 9 (50%) female patients. there was no significant difference between patients whose cause of death was ascertained and those whose cause of death was not obtained for both age (p = 0.107) and proportion of males to females (p = 0.670). overall, ihd was the major immediate cause of death (stated as cause 1a, 1b, or 1c on the death certificate) in 111 (45.7%) dfu patients (table 2). the ihd category comprised coronary atheroma and coronary artery disease which were reported as cause of death in 89/111, myocardial infarction (mi) or coronary thrombosis and occlusion in 47/111, and cardiac arrest in 5/111 death certificates. each one of ihd subcategories may have been reported alone or in combination with one or more of the other subcategories. category comprised deaths due to hypertensive heart failure, myocardial degeneration and fibrosis, and haemopericardium. the results were similar in the control group with ihd accounting for 55 (45.5%) of all deaths. in the ihd category, coronary atheroma and coronary artery disease were stated in 45/55, mi or coronary thrombosis and occlusion in 32/55, and cardiac arrest in 1/55 on death certificates. there was no significant difference in the other causes of death between dfu patients and controls apart from a significantly higher number of deaths in the control group from cancer (19/121) compared with the dfu group (20/243) (odds ratio 2.077, 95% confidence interval 1.0624.060, and p = 0.032) (table 2). the results confirmed that ihd was the commonest immediate cause of death and the proportion had risen to 62.5% of all dfu deaths compared with 45.7% from death certification (table 2). the postmortem results in 30 control group patients showed a slightly higher proportion of ihd deaths, 70.0% (21) compared to 62.5% (50) for the dfu group. this difference, however, did not reach statistical significance (p = 0.465) (table 2). on postmortem examination, there was no significant difference in other causes of death including cancer between the dfu and control groups. to examine the relationship between cause of death and ulcer type of the 80 dfu patients who had postmortem examination, 24 were neuropathic and 56 were ischaemic dfus. the postmortem data demonstrated that mortality from ihd was significantly higher in the neuropathic group compared to the ischaemic group (79.2% versus 55.4%, resp.) (odds ratio 3.064, 95% confidence interval 1.0039.366, and p = 0.049) (table 3). specifically, there was a significantly increased mortality from myocardial infarction / coronary thrombosis in the neuropathic group (13/24) compared with ischaemic group (13/56) (odds ratio 3.909, 95% confidence interval 1.41719.783, and p = 0.009) and also a trend to increased mortality from cad / atherosclerosis in the neuropathic group (16/24) compared with ischaemic group (24/56) (odds ratio 2.667, 95% confidence interval 0.9817.250, and p = 0.055) (table 4). there was no other significant difference in the cause of death between patients with neuropathic and ischaemic ulcers. regarding patients whose death certificate was available, 56 had neuropathic and 187 had ischaemic ulcers. death certificate data showed that mortality from myocardial infarction / coronary thrombosis was significantly increased in the neuropathic group (17/56) compared with the ischaemic group (30/187) (odds ratio 2.281, 95% confidence interval 1.4344.551, and p = 0.0193) (table 4). there was no other significant difference in the cause of death between patients with neuropathic and ischaemic ulcers. we further determined the mean age at death in patients who had a postmortem examination. the mean age at death from ihd in the dfu group was five years lower than that in the control group (68.2 8.7 years versus 73.1 7.96 years, p = 0.015). in the neuropathic ulcer group, the mean age at death from ihd on postmortem was 67.9 8.5 years compared with a mean age at death for the ischaemic ulcer patients of 68.5 8.9 years (p = 0.407). the mean age at death for all causes combined (on death certificates and postmortem examinations) showed no significant difference between the dfu group and the control patients, 71.2 11.1 years versus 72.8 10.1 years (p = 0.091). the mean age at death from ihd (on death certificates and postmortems combined) in the dfu group was 69.5 9.5 versus 72.6 8.3 years for the control group (p = 0.051). in particular, death from myocardial infarction was significantly higher in the neuropathic group compared with the ischaemic group both on death certification and postmortem findings. our results concur with other studies which showed the increased mortality risk to be ascribed to cardiovascular disease in particular ihd [10, 11 ]. in addition, our study established that the risk of premature mortality from ihd is greater in patients who develop neuropathic ulceration. we have earlier addressed the role of ihd in the increased mortality rate associated with dfu through the application of a proportionate model of the dfu population. we used the model to show that a 25% reduction in the number of neuropathic dfu patients dying earlier than nonulcerated subjects eliminated increased mortality. the link between neuropathic foot ulceration and the excess mortality from ihd demands further clarification. latest evidence shows that dfu has a major independent influence on lower extremity amputation and mortality risk which is quite apart from other baseline complications. our postmortem data indicated a significantly high frequency of ihd in neuropathic ulcer patients (79.2%) compared to the ischaemic group (55.4%). this is in keeping with the study of boyko. stating that, amongst patients with diabetic foot ulcers who died, 64% of these ulcers were judged to be due to neuropathy, and mortality was independent of macrovascular disease as measured by ankle - arm index. more recent studies have confirmed that mortality in patients presenting with neuropathic ulcers was unexpectedly high with an average 14-year reduction in life expectancy related to neuropathy whether an ulcerated neuropathic or a charcot foot. large nerve fibre dysfunction related to diabetes, as measured by vibration perception threshold, is strongly linked with a high risk of foot ulceration. it also predicts amputation and mortality even in young type 1 diabetes patients and is associated with increased cardiovascular risk. because of the established strong association between lower extremity neuropathy and diabetic foot lesions, death related to diabetic foot problems (including ulceration) has been used as an estimate of mortality associated with peripheral neuropathy. in a 14-year observational study, the main microvascular complications of diabetes (peripheral sensory neuropathy and nephropathy) were associated with increased mortality in diabetic patients although abnormal vibration threshold was more strongly associated with increased mortality than other microvascular complications. patients with large fibre neuropathy also have evidence of small fibre neuropathy including autonomic neuropathy, which is associated with increased mortality from cardiovascular disease, particularly sudden cardiac death. peripheral autonomic neuropathy (small fibre) is associated with the development of foot ulceration in diabetic subjects. measures of peripheral autonomic neuropathy in terms of peripheral vascular, cardiovascular, and neurophysiological measurements are worse in neuropathic ulcer patients when compared with nonulcerated patients. diabetic patients with autonomic dysfunction affecting cardiac efferent sympathetic signals have impaired sympathetically mediated dilation of coronary resistance vessels and the severity is related to the degree of sympathetic dysfunction. impaired dilation of these vessels can lead to myocardial ischaemia and left ventricular dysfunction, even in the absence of overt atherosclerosis [20, 21 ]. silent ischaemia is significantly more common in diabetic men with autonomic neuropathy than in those without as it prevents the development of angina pain. evidence of fresh infarction may not always be found at postmortem in sudden deaths in patients with autonomic neuropathy. neuropathy is also closely linked to calcification of vascular smooth muscle, a process thought to be mediated by receptor activator of nuclear factor kappa b ligand (rank - l)/osteoprotegerin signalling pathway implicated in coronary and peripheral vascular disease. vascular calcification in diabetic neuropathy may be a significant factor in increased cardiovascular risk in neuropathic ulcerated patients independent of autonomic neuropathy and cardiac denervation. therapeutic options targeting these emerging pathways may help modulate macrovascular complications and have a beneficial effect on cardiovascular outcomes in this population of diabetes patients. neuropathy may also be a marker of associated nephropathy which is a well - established risk factor for cardiovascular death. microalbuminuria, an independent predictor of progressive nephropathy, is associated with endothelial damage and reflects atherosclerotic disease and vascular dysfunction and has also been shown to be strongly associated with the development of diabetic foot ulcers in type 2 diabetic patients. patients with diabetic nephropathy have a high frequency of autonomic neuropathy and both factors are associated with and contribute independently to the risk of silent ischaemia. cardiac autonomic neuropathy is also an independent risk factor for cardiovascular morbidity and mortality in type 1 diabetic patients with nephropathy. moreover, survival after amputation is lower in diabetic patients with chronic kidney disease and those on dialysis and this may be related to the severity of neuropathy amongst other comorbidities in these patients. autonomic neuropathy, however, is difficult to diagnose on postmortem studies, ascertain the degree of severity of autonomic dysfunction. some of the excess mortality has also been thought to be due to uncontrolled sepsis. we have shown that foot infection with staphylococcus aureus, which is a very common offender in dfus, increases the mortality rate 2.6 times compared to those without staphylococcus infection. it is postulated that staphylococcus aureus could increase the risk of mortality through a cytokine response, which might cause plaque rupture and subsequent death from myocardial infarction. strong evidence exists for the importance of a vagus nerve - mediated pathway in controlling cytokine production essential for preventing pathological inflammation. the activation of the efferent vagus nerve stimulates the release of acetylcholine which inhibits the release of tumor necrosis factor (tnf), high mobility group box-1 (hmgb1), and other proinflammatory cytokines from resident tissue macrophages (the cholinergic anti - inflammatory pathway) without affecting the production of anti - inflammatory cytokines. this inhibits excessive systemic inflammation and protects against endotoxaemia and ischaemia reperfusion injury [31, 32 ]. this process is attenuated in autonomic dysfunction resulting in decreased vagus nerve anti - inflammatory output, which might be associated with loss of tonic suppression of inflammatory processes. thus neuropathic patients may be at increased risk of more flagrant inflammation and more extensive endothelial dysfunction of their coronary arteries. potential confounding factors of the relationship between cause of death and ulcer type include ageing. the increased mean age of this sample increases the possibility of ihd being a late diabetic complication. on the other hand, published data show that diabetes per se is associated with excess mortality, even in an area with high background death rates from cardiovascular disease. more recently, studies have implicated qtc prolongation in type 2 diabetes patients with foot ulcers in the excess mortality observed in these patients. the use of our local nonulcerated diabetic population, as opposed to the general population, as a control group minimised bias in the estimates of relative mortality rates. the classification of causes of death facilitated comprehensive comparisons of all causes of mortality between the two groups with and without foot ulcers. notwithstanding the loss to follow - up rate of 9.3% in acquiring the cause of death in dfu patients, the data collection method allowed for representation of most of the population in the south east of england referred with foot ulceration to this centre with no specific groups excluded. it was not possible to acquire death certificates in 25 of the dfu group and 18 of the controls. however, there was no significant difference between patients whose cause of death was ascertained and those whose cause of death was not obtained in both dfu and control patients for both age and proportion of males to females. it was not feasible to look at lifestyle factors or analyse the events preceding their deaths. it was also not possible to adjust for independent risk factors for ihd in the neuropathic group, for example, lipid profile and smoking habits. it is accepted that the deaths took place from 1989 up to 2000. since then, newer diabetes treatments may have improved cardiovascular outcome. however, despite modern treatments, mortality of diabetic foot patients is still high. controlling for dfu patients also poses a significant problem as the general population does not provide specific controls, which in the case of dfu patients is difficult to determine. although the dfu group had a higher proportion of males compared with the control group, the frequency of the causes of death was similar. while death certification showed a significantly higher number of deaths in the control group from cancer compared with the dfu group, this data was not available for the presence of ischaemia or neuropathy in the control group. this emphasises the importance of a prospective study to account more accurately for levels of ischaemia and neuropathy and possibly the rate of change of these baseline categorisations. this study provides strong evidence to reiterate its importance as a factor, placing neuropathic diabetic foot ulcer patients at a considerable risk of premature death. we have attempted to elucidate the mechanisms implicating ihd in the excess premature mortality in neuropathic dfu patients. the survival benefits of introducing an aggressive cardiovascular risk management programme in dfu clinics have also been proven and this can direct future implementation of national programmes.	diabetic foot ulcer (dfu) patients have a greater than twofold increase in mortality compared with nonulcerated diabetic patients. we investigated (a) cause of death in dfu patients, (b) age at death, and (c) relationship between cause of death and ulcer type. this was an eleven - year retrospective study on dfu patients who attended king 's college hospital foot clinic and subsequently died. a control group of nonulcerated diabetic patients was matched for age and type of diabetes mellitus. the cause of death was identified from death certificates (dc) and postmortem (pm) examinations. there were 243 dfu patient deaths during this period. ischaemic heart disease (ihd) was the major cause of death in 62.5% on pm compared to 45.7% on dc. mean age at death from ihd on pm was 5 years lower in dfu patients compared to controls (68.2 8.7 years versus 73.1 8.0 years, p = 0.015). ihd as a cause of death at pm was significantly linked to neuropathic foot ulcers (or 3.064, 95% ci 1.0039.366, and p = 0.049). conclusions. ihd is the major cause of premature mortality in dfu patients with the neuropathic foot ulcer patients being at a greater risk.
endotoxin - induced uveitis (eiu) is an animal model of acute ocular inflammation. usually, this experimental pathology lasts up to 72 hours and has similarities with the human pathology. cytokines, chemokines, nitric oxide (no), and impairment of stress - sensitive enzymes such as heme oxygenase-1 increase the inflammatory response to eiu. augmentation of local recruitment of pmn promotes the inflammation and cell injury of the ciliary bodies in this pathology. thus, prospectively pharmacological agents that promote endogenous defensive responses, reduce the burden of inflammatory mediators released within the eye structures or reinforce the resolution of the inflammation that may exert cytoprotection and good outcome for uveitis. a growing body of evidence indicates that a good resolution of the inflammation could be achieved by means of resolvins. these are a family of potent lipid mediators derived that promote the resolution of the inflammatory response back to a noninflamed state. resolvin d1 (rvd1) is the major actor of the resolvins family ; it is produced physiologically from the sequential oxygenation of dha by 15- and 5-lipoxygenase and it has effects on important components of the inflammation. resolvin d1 reduces human polymorphonuclear leukocyte (pmn) transendothelial migration, the earliest event in acute inflammation, and exhibits a dose - dependent reduction in leukocyte infiltration in a murine model of peritonitis with a maximal inhibition of ~35% at a 10100 ng dose. rvd1 also acts as a scavenger of cytokines and chemokines from the inflamed site and inhibits the production of pmn - derived free radicals [58 ]. other biological actions have been reported, with a therapeutical potential such as a reduction in inflammatory pain. on this base, we have investigated whether the stimulation of the resolutive phase of the inflammation through the use of the resolvin d1 ameliorates the immunoinflammatory profile of the rat eye following experimental uveitis. male sprague - dawley rats (180220 g) were injected in one footpad with 200 g of lipopolysaccharide (lps, salmonella minnesota, sigma, st louis, mo, usa) in 0.1 ml of sterile pyrogen - free saline for the induction of eiu. the rats were treated with vehicle, lps or lps + resolvin d1 (rvd1) (n = 6 for each) and killed 24 h after treatment. the doses of rvd1 (101001000 ng / kg) were chosen in the range of those used in murine models of inflammation ; they were injected by intravenous bolus (in 200 l of sterile saline) via the tail vein 1 h following lps injection. clinical manifestations of eiu were graded from 0 to 4 in a blinded fashion according to the previously reported scoring system [10, 11 ] : 0 = no inflammatory reaction ; 1 = discrete dilation of iris and conjunctival vessels ; 2 = moderate dilation of iris and conjunctival vessels with moderate flare in the anterior chamber ; 3 = intense iridal hyperemia with intense flare in the anterior chamber ; and 4 = same clinical signs as 3 with presence of fibrinoid exudation in the pupillary area and miosis. no signs of uveitis were observed in the animals at the beginning of each experiment. eiu clinical data shown were representative of 3 sets of experiments and presented as mean sem of 6 observations. immediately after the biomicroscope examination, the animals were killed with an overdose of anesthesia. aqueous humor (aqh) was collected immediately from both eyes by an anterior chamber puncture (3040 l / rat), using a 30-gauge needle under a surgical microscope and stored in ice water until testing. one unit of mpo activity has been reported to be equivalent to approximately 2 10 pmn [12, 13 ]. after 24 h of eiu, the eyes were harvested and cut in two halves. one half of each eye was immediately fixed by immersion in 10% buffered formalin and paraffin - embedded for immunohistochemistry. sections were serially cut at 5 m, placed on lysine - coated slides, and stained with hematoxylin and eosin and with the trichrome method. the other half of each eye was immediately frozen in liquid nitrogen and stored at 80c for the later biochemical assays described below. subsequently the frozen tissues were homogenized in a solution containing 0.5% hexa - decyl - trimethyl - ammonium bromide dissolved in 10 mm potassium phosphate buffer (ph 7) and centrifuged for 30 min at 4,000 g at 4c. paraffin - embebbed eye samples were treated with an xylene substitute (hemo - de ; fisher scientific) in order to remove the paraffin, and tissue sections were rehydrated with ethanol gradient washes. tissue sections were quenched sequentially in 3% hydrogen peroxide aqueous solution and blocked with pbs 6% nonfat dry milk (biorad, milan, italy) for 1 h at room temperature. sections were then incubated with specific antibodies anti - cd11b, anti - cd4, and anti - cd8 (santa cruz biotec, usa). specific labelling was detected with a biotin - conjugated goat anti - rabbit igg and avidin - biotin peroxidase complex (dba, milan, italy). the specimens were analyzed by an expert pathologist (intraobserver variability 6%) blinded to the experimental protocol. six distinct preparations for each group of animals were done and 20 microscopic fields were analyzed in each preparation at 400x magnification. tnf - alpha levels in tissue homogenates (50 l) were determined using a commercially available elisa specific for the rats cytokine, purchased from r&d systems (abingdon, uk). briefly, tissue supernatant aliquots (50 l) were assayed for tnf - alpha and compared to a standard curve constructed with 0 - 1 ng / ml of the standard cytokine. the elisa showed negligible (< 1%) cross - reactivity with several murine cytokines and chemokines (data as furnished by manufacturer). a similar procedure was followed for determination of the chemokines cxcl8 and rantes by elisa (r&d systems, uk) and used according to the manufacturer instructions. all values are expressed as mean sem of number (n) of rats for the in vivo experiments. statistical analysis was assessed either by student 's t - test (when only two groups were compared) or one - way anova followed by dunnett 's test (more than two experimental groups). a probability p value less than 0.05 was considered significant to reject the null hypothesis. 24 h after the administration of 200 g lps into the footpad of sprague - dawley rats, severe changes of the structure of the eye occurred with a clinical score of 3.90 0.3 attributed (figure 1). rvd1 (101001000 ng / kg) dose - dependently attenuated the development of the ocular inflammation caused by lps and improved the clinical score attributed in a dose - dependent manner (figure 1). particularly, the insurgence of eiu was effectively reduced (22.6 1.4% less) by the intermediate dose of rvd1, while the highest dose protected the eye until the 74 3% (figure 1). the uveitis clinical scores for these two doses were significantly reduced to 1.02 0.2 (p < 0.05) and 2.98 0.3 (p < 0.01) compared with vehicle - treated group. concomitantly, a severe inflammatory and immune response rose within the eye of the rats. the immunohistochemistry performed on the eye revealed tissues were largely oedematous and telangiectatic with an oblong profile of the blood vessels (figure 2), caused by the development of the oedema that tissues were largely oedematous and telangiectatic with an oblong profile of the blood vessels (figure 2). particularly, both the external fibrous (sclera) and vascular median tunics (choroid) were markedly infiltrated of flogistic elements (figure 2). these were composed predominantly of pmn leukocytes cd11b and t - lymphocytes cd4 and cd8. in fact, figure 3 showed that rvd1 reduced the number of infiltrated cd4 and cd8 particles within the perivascular tissue. at 24 h, the number of cd4 particles for lps + rvd1 treated animals were significantly lower than those for the lps treated animals (p < 0.001) (figures 3 and 4). lps + rvd1 animals also had the lowest number of c8 particles compared to the lps group (figures 3 and 4). the immunohistochemistry also showed that rvd1 reduced the infiltration of pmn leukocytes marked within the uvea. cd11b positive particles were mainly localized inside the blood vessels with no infiltration within the adjacent tissues ; figure 3 shows the effects of rvd1 100 ng / kg. the actions of rvd1 were evident for the doses of 100 and 1000 ng / kg, while 10 ng / kg gave no significant results in term of reduction of number of inflammatory components within the uvea (figures 3 and 4). the development of eiu was paralleled by increase in mpo activity (figure 5). treatments of eiu rats with rvd1 1 hour after lps caused a dose - dependent decrease of mpo activity in the aqh. the decreases significantly started from the dose of 100 ng / kg (p < 0.05 versus vehicle) and reached the maximum with the highest dose of 1000 ng / kg (p < 0.01 versus vehicle) (figure 5). tissue homogenates from the eyes of vehicle - treated rats had slightly appreciable levels of tnf - alpha, cxcl8, and rantes (figure 6). in contrast, tissue homogenates from eiu lps - induced rats showed high levels of the tnf - alpha, cxcl8, and rantes as 280 20 pg / mg, 560 38 pg / mg, and 373 27 pg / mg, respectively (figure 6). resolvin d1 treatment, 1 hour after lps, dose - dependently produced a significant reduction in either the cytokine and the chemokine levels within eye tissues (figure 6). here we report that stimulation of resolvin d1 pathway in rats undergoing experimental uveitis ameliorates the immuno - inflammatory profile of the external and median tunics of the eye, accounting for eye protection. inflamed eye is the result of the altered functions of endothelial cells, leukocytes, retinal pigment epithelium, retinal neurons, glial cells, and other types of cells locally present. these cells are targets of signalling molecules such as lipid mediators and cytokines that favour the shifting of the tissue from a physiological shape to pathological one. targeting one, or more than one, of these mediators with specific agents prevents inflammation or promotes resolution of it. this latter response is limited at the site of the noxious stimulus, in order to restore the right homeostasis through specialized proresolving mediators with tissue - protective and resolution - stimulating functions. they are biosynthesized from eicosapentaenoic acid and from docosahexaenoic acid and so are denoted as e series and d series, respectively. the actions of the resolvins include the scavenging of cytokines and chemokines from the inflamed site, the inhibition of the de novo production of cytokines and chemokines, the inhibition of the leukocytes trafficking / infiltration to inflamed tissue, and the inhibition of the production of pmn - derived free radicals [58 ]. however, an action on the recruitment of nonphlogistic monocytes and phagocytosis is also accredited. from the molecular point of view, the proresolving properties of resolvins are exerted through the share of g - protein - coupled seven - transmembrane receptors located on human leukocytes with the anti - inflammatory peptide annexin 1 and chemerin. receptors are called alx / fpr2 (lxa4 receptor) and gpr32 (g - protein - coupled receptor) [1518 ] and are aimed to translate the rvd1 signal into leukocytes activation and movement impediment. according to this evidence here we report that the pro - resolving properties of resolvin d1 is exerted on the typical actors leading experimental uveitis, the white blood cells recruited into the eye specimens. mpo, a sensible marker of leucocytes infiltration, and tissue immunoreactivity for the cd11b were remarkably reduced by the rvd1. pmn leukocytes activation and infiltration are the key events of inflamed eye, because pmn leukocytes adhere to, roll along, infiltrate the endothelial wall of blood vessels and release reactive oxygen species to the site attacked causing inflammation. furthermore, this treatment also reduced the local generation of cytokines and chemokines which are known to promote leukocyte - endothelium interaction and finally eye damage. indeed, a cytokine able to increase the adhesive properties of the endothelial wall [21, 22 ], and that it is implicated in the pathology associated with experimental uveitis, the tnf - alpha was drastically reduced following the treatment of the rats with rvd1 as well as the chemokine cxcl8, a chemokine able to recruit neutrophils in rodent species during experimental inflammation [21, 24 ]. as chemoattractants, cxcl8 stimulates directional leukocyte migration and activates the expression of integrin on leukocytes such as cd11b, which increases leukocyte binding to the ligands icam-1 and vcam-1 on the endothelium. taken together, the present results show that the persistent inflammation of the eye and tissue damage following uveitis could be controlled by means of rvd1. an intriguing hypothesis would be that the rvd1, acts through its receptor alx / fpr2, ubiquitously present within the eye structures. consistent with this contention are the recent studies by odusanwo., in other settings. the resolution of the inflammation seems not be the only target to reach during uveitis ; unfortunately, the immune response has a major responsibility in it and thus needs to be suppressed. much experience on the immune - mediated damage has arisen from clinical and experimental models of uveitis [2729 ]. however, the interplay between innate and adaptive immunity and the crossover between autoinflammatory and autoimmune conditions need relative exploration. here we show that although the predominant infiltrating cell type in eiu is the pmn neutrophil, cd4 and cd8 t cells are also partners of this company. these cells have been found markedly infiltrated within the uveal structures together with pmn leukocytes. over the last two decades, the understanding of immunopathogenetic mechanisms associated with eiu is increased and supports a fundamental role of t cells in it, especially cd4 t cells. this is in accordance with a number of studies done through the last two decades supporting the fundamental role for t cells, especially cd4 t cells. other studies showed that that anti - cd4 antibody significantly reduced the severity of eiu in endotoxin - responsive strains of mice, while an anti - cd8 antibody had no influence on the disease. in 2002, avunduk. also showed cd4 and cd8 cells infiltration in the anterior uveal tract, paving the way to the later concept that activated lymphocytes can invade vascular endothelium by degrading subendothelial matrix, and so memory t lymphocytes may be the predominant introducers of the extra vascular tissues. activated t lymphocytes selectively cross the blood - aqueous barrier, accumulate in the uveal tissue of eiu animals, and secrete a number of damaging elements. rvd1 quantitatively reduced the number of infiltrated cd4 and cd8 elements within the perivascular uveal tissue. also, rvd1 associated a reduction of the t - chemokine rantes (regulated on activation, normal t expressed and secreted) within the uveal tissues. rantes, also known as ccl5, is an 8 kda protein member of the interleukin-8 superfamily of cytokines. firstly identified after t - cells activation, it was subsequently determined to be a cc chemokine and expressed in more than 100 human diseases. rantes expression in t lymphocytes is regulated by kruppel like factor 13 (klf13) [3537 ] and is chemotactic for t cells, eosinophils, basophils, neutrophils, and macrophages [3840 ], and through its receptor ccr5 plays an active role in recruiting leukocytes into inflammatory sites. rantes, along with the related chemokines mip-1alpha and mip-1beta, has been identified as a natural factor secreted by activated cd8 t cells and other immune cells. with the help of particular cytokines (i.e., il-2 and ifn-) that are released by t cells, ccl5 also induces the proliferation and activation of certain natural - killer (nk) cells to form chak (cc - chemokine - activated killer) cells. therefore, limiting the infiltration of immune cells into inflamed sites or reducing their products could be mandatory for return to eye homeostasis. in conclusion, our study shows that stimulation of the resolving phase of eiu through resolvin d1 pathway ameliorates the immuno - inflammatory profile of the rat eye.	this study investigated whether the administration of resolvin d1 to rats with endotoxininduced uveitis (eiu) ameliorates the immuno - inflammatory profile of the eye. 24 h after the administration of 200 g lps into the footpad of sprague - dawley rats, severe changes of the structure of the eye occurred concomitantly with a severe inflammatory and immune response. these latter included strong infiltration of pmn leukocytes cd11b+ t - lymphocytes cd4 + and cd8 + within the eye and a significant release of the cytokines / chemokines tnf - alpha, cxcl8, and rantes too. bolus of resolvin d1 (rvd1 ; 101001000 ng / kg in 200 l of sterile saline via the tail vein) significantly and dose - dependently (i) reduced the development of the ocular derangement caused by lps ; (ii) reduced the clinical score attributed to eiu ; (iii) reduced the protein concentration and myeloperoxidase activity (mpo) in aqueous humor (aqh) ; and (iv) reduced neutrophils, t - lymphocytes, and cytokines within the eye.
adrenal disorders are multifaceted involving genetics, autoimmunity, infection and environmental endocrine influences as the etiological trigger. it includes various disorders in the form of adrenal insufficiency, cushing syndrome (cs), secondary hypertension (pheochromocytoma and conn 's syndrome), and congenital adrenal hyperplasia (cah) which are treatable and have avoidable morbidity and mortality. in india, posttubercular adrenalitis leading to insufficiency has been noticed in 46%56% patients and when further investigated the number rose to 70%75%. increase in the incidence of hiv / aids has also contributed in increasing the number of cases presenting as adrenal insufficiency. adrenal insufficiency is one of the most common causes of cardiovascular instability leading to shock and death in india. cortisol deficiency leading to hypotension, shock, and nonreviability of patient is commonly encountered. the diagnosis and management of primary adrenal insufficiency needs a high degree of suspicion, acumen, knowledge about adrenal disorders, and a protocol - based approach. cah, an autosomal recessive disorder of defective steroid genesis, is as common as 1/10,0001/20,000 for salt wasting / simple virilizing. cah and for the milder varieties such as nonclassical cah (nccah), the incidence is 1/1000 cases. infertility, delayed puberty, and metabolic syndrome are the critical challenges which are associated with cah. india is at the epoch of socioeconomic transition and diseases such as obesity and polycystic ovarian disease are nowadays very common among young females. the burden of such adrenal disorders is very high in clinical practice, but the level of awareness is not that high among medical graduates and postgraduates students. very few studies have been conducted to test the awareness of adrenal disorders among interns and postgraduate students (general medicine). hence, the present study was conducted with the objective to assess the knowledge and preparedness of the interns and postgraduate students to tackle the adrenal disease. the present cross - sectional questionnaire - based study was performed on 56 participants of 1, 2, and 3 year postgraduate residents of general medicine (n = 14 3) and interns (n = 14) in the department of medicine, gandhi medical college, bhopal, madhya pradesh, among the postgraduate students and interns posted in the department of medicine. confidentiality of the participants and the data collected were maintained at every step and the data collected were used for study purpose only. after explaining the objective of the study, the questionnaire comprising different multiple choice questions were distributed. the questionnaire covered a range of questions (12 questions in total) that would test basic knowledge about of the participants on topic such as adrenal insufficiency, adrenal adenoma, cah, nccah, pheochromocytoma, and conn 's syndrome. no data on age and gender were collected to further protect the individual identity of the participants. the questionnaire was given to every participant, who answered the questions anonymously and the answers were kept confidential. the data were analyzed descriptively (frequency analysis) by disease type (primary adrenal insufficiency, cah, nccah, cs, pheochromocytoma, and conn 's syndrome), treatment, and dosing regimen. among the study participants, 14 (25%) participants scored < 5 marks, 33 (58.9%) scored between 6 and 9, and 9 (16.1%) scored between 10 and 12 [table 1 ]. the mean score among the participants was 6.38 2.505 with a range from 2 to 11 marks [table 2 ]. total score distribution among responders response to the question in numbers (12 question 14 participants in each group=168 responses) the number of correct answers by postgraduates residents of 1 year was 101, 2 year was 95, and 3 year was 93 and interns scored 68 out of total 168 questions in each group [table 2 ]. mean awareness score for residents of 1, 2, and 3 year participants and interns was 7.21 2.806, 6.79 2.119, and 6.64 2.818 and 6.63 2.505, respectively [table 3 ]. mean scores of the participants included in the study the residents who answered correctly for adrenal disease - related disorders were 45.5%, (maximum by 1 year : 52.45% and minimum by interns : 36.9%), related to diagnosis were 57.7% (maximum by 2 year : 69% and minimum by interns : 42.9%), and related to treatment were 64.3% (maximum by 1 and 3 year : 71.4% and minimum by interns 50%) [table 4 ]. percentage awareness among different doctor groups answers to a question regarding how commonly is adrenal insufficiency diagnosed in medical intensive care unit, none of the individuals responded correctly. awareness score regarding adrenal insufficiency was 47.6% (maximum by 2 year : 58.3% and minimum by interns : 28.6%), treatment for adrenal insufficiency was 40.5% (maximum by 2 year and minimum by interns : 33.3%), cah was 63.4% (maximum by 1 year : 78.5%), pheochromocytoma was 50.9% (maximum by 1 year : 57.1%), conn 's disease was 58.9% (maximum by 3 year : 78.6%), and overall correct response was maximum for cah (63.4%) and minimum for adrenal insufficiency (47.6%). endocrine disorders in several million children and adults in india remain undetected and untreated because of inadequate professional expertise and a lack of reliable diagnostic services. general practitioners and some specialists often fail to suspect endocrine disorders, even in likely cases. studies have shown that in india, one in every four patients with tuberculosis has adrenal involvement, yet few are investigated for this. endocrine training in india consists of a brief nonformal exposure during postgraduate training in the form of patients admitted in wards being treated for additional endocrine illnesses, the curriculum of examinations usually does not have cases of diabetes, thyroid, and other endocrine illnesses. neurology, cardiology, pulmonology, and gastroenterology form the major chunk of postgraduate curriculum in general medicine, where endocrine training is the less sort after. hence, the responsibility of endocrine training in india is at the superspecialty courses such as dm in endocrinology, dnb training in endocrinology, and post doctoral certificate courses. there are somewhere around seventy endocrine superspecialty postgraduates who are completing the training every year, i.e., we are having one newly trained endocrinologist every year to cater a population of roughly 2 million. the mean score obtained among the study participants was 6.38 2.505, score ranged from 2 to 11 marks. this is a clear reflection of the lack of priority, acumen, and awareness of adrenal disorders in our medical teaching curriculum which is actually followed. when we saw the breakup of correct responses, it was maximum by the 1 year resident, followed by 2 year and 3 year. this can be explained by the preparation during the postgraduate entrance examinations, which has improved the knowledge among the 1 year residents as compared to interns and later during the 2 and 3 year of residency, the knowledge has declined because of less discussion in clinical rounds, case presentation, and awareness of adrenal disorders during training. the trend of awareness, diagnosis, and management of adrenal disorders followed the same pattern as the responses given above. awareness score regarding adrenal insufficiency was 47.6% (maximum by 2 year : 58.3% and minimum by interns : 28.6%), treatment for adrenal insufficiency was 40.5% (maximum by 2 year and minimum by interns : 33.3%), cah was 63.4% (maximum by 1 year : 78.5%), pheochromocytoma was 50.9% (maximum by 1 year : 57.1%), conn 's disease was 58.9% (maximum by 3 year : 78.6%), and overall correct response was maximum for cah (63.4%) and minimum for adrenal insufficiency (47.6%). there is a huge responsibility on the medical teaching fraternity for the diagnosis, management, and treatment of adrenal disorders in central india. seeing to commonality of adrenal - related illnesses and a large number of cases, there should be awareness among the postgraduate students in medicine regarding adrenal - related illnesses. there are fewer opportunities in terms of number for endocrine - related post - md training in india. we need to make our postgraduates in medicine well versed with the knowledge of adrenal disorders. there is a lack of awareness regarding diagnosis, management, and treatment of adrenal disorders in central india. we need to prioritize training related to these illnesses in our postgraduate teaching curriculum in practice.	introduction : adrenal disorders could be a life - threatening emergency, hence requires immediate therapeutic management. for this awareness regarding its diagnosis, management, and treatment is prime important.aims and objective : to study the awareness of adrenal disorders among interns and postgraduates students of hamidia hospital, bhopal.materials and methods : a cross - sectional questionnaire - based study was performed. fifty - six participants, i.e., 1st, 2nd, and 3rd years postgraduate residents of general medicine (n = 14 3) and interns (n = 14) were included in the study. there were 12 questions on adrenal insufficiency, adrenal adenoma, congenital adrenal hyperplasia (cah), nonclassical cah (nccah), pheochromocytoma, and conn 's syndrome. one mark was awarded for each correct response.results:in the present study, 14 (25%) participants scored < 5 marks, 33 (58.9%) scored between 6 and 9, and 9 (16.1%) scored between 10 and 12. the mean score among the participants was 6.38 2.505, with a range from 2 to 11 marks. the number of correct answers by postgraduates residents of 1st year was 101, 2nd year was 95, and 3rd year was 93 and interns scored 68 out of total 168 questions in each group. mean awareness score for residents of 1st, 2nd, 3rd years participants and interns was 7.21 2.806, 6.79 2.119, and 6.64 2.818 and 6.63 2.505, respectively. most of the participants recorded correct responses related to diagnosis (57.7%) followed by responses related to treatment (64.3%). answers to a question regarding how commonly is adrenal insufficiency diagnosed in medical intensive care unit, none of the individuals responded correctly.conclusion:there was a lack of awareness regarding diagnosis, management, and treatment of adrenal disorders in central india. we need to prioritize training related to these illnesses in our postgraduate teaching curriculum in practice.
self - determination theory is a well - known psychological framework to study people 's behavior, based on the assumption that humans are innately motivated toward growth and health, a process which can be nurtured or thwarted by the social environment [1, 2 ]. much research on self - determination theory, particularly in the health domain, has focused on the study of the characteristics of motivation and associated regulatory processes. for instance, the degree to which people feel autonomous (i.e., self - determined) versus controlled in their motivated pursuits, and how this relates to behavioral persistence (e.g., [3, 4 ]). intrinsic motivation, the doing of an activity for its inherent satisfactions, is highly autonomous and represents the prototypic instance of self - determination, while extrinsically motivated behaviors, by contrast, cover the continuum between amotivation and intrinsic motivation, varying in the extent to which their regulation is autonomous. within extrinsic regulations, autonomous regulations (identified and integrated) reflect a sense of personal volition and recognition of the importance of the target behavior and its consequences. in contrast, in controlled regulations (external and introjected) people feel forced to comply with outside demands or feel guilty or ashamed if they do not perform the target behavior [1, 2 ]. it states that people have innate psychological needs that when fulfilled have an effect on personal growth, psychosocial adjustment, feelings of integrity, and well - being. additionally, it clarifies the relationships between the satisfaction of the needs for competence, autonomy, and relatedness and psychological functioning and well - being. it should be noted that, from a self - determination theory perspective, well - being is not concerned exclusively with hedonic or subjective well - being in the tradition of positive psychology, namely, the experience of happiness, usually characterized by high positive affect, reduced negative affect, and life satisfaction. self - determination theory favors a eudaimonic view of well - being, focused on feeling fully functioning, self - coherent, and with a deep sense of wellness, and vitality, rooted on the idea of fulfilling or realizing one 's daimon or true nature. as a consequence of this broader notion of well - being, which includes happiness and emotional well - being but also meaning and personal growth, psychological well - being has been assessed in self - determination theory studies with indicators of positive affect and mental health. some examples are self - esteem, vitality, life satisfaction, and also (low levels of) depression and anxiety [10, 11 ]. previous studies in exercise contexts which examined perceived choice, a marker of autonomy, in relation to well - being found that it was associated with reduced negative affect and positive well - being. more recent studies in the exercise domain showed that the satisfaction of basic psychological needs for competence, autonomy, and relatedness enhanced psychological well - being in the form of physical self - perception, subjective vitality and positive affect [10, 11 ], enjoyment and intrinsic motivation for exercise, and satisfaction with life. numerous studies have demonstrated that obese individuals experience significant impairments in health - related quality of life (hrqol) as a result of their weight, with greater impairments being associated with greater degrees of obesity [1618 ]. conversely, weight loss has been shown to improve quality of life in obese persons undergoing a variety of treatments. assessing quality of life is especially important to help determine the comparative efficacy of different treatments and to assess the impact of treatment on how patients feel and function in their everyday life [19, 20 ]. the use of both general and specific quality of life instruments is a methodological recommendation from previous obesity hrqol research. hrqol, as measured by the sf-36, improves after small to moderate amounts of weight loss with nonsurgical methods. not only weight loss but also weight maintenance is considered to be beneficial for physical hrqol. using obesity - specific measures, such as the iwqol and iwqol - lite, quality of life improvements were associated with decrease in body weight in different studies [22, 2426 ]. in a recent meta - analysis, weight loss treatment was associated with lowered depression and increased self - esteem ; only treatments that produced actual weight loss predicted increased self - esteem, whereas improvements in depression were independent of weight loss. in portugal, the prevalence of overweight and obesity is 53.6% in adult women ; of these 13.4% are obese and 34.4% are overweight. however, there are very few studies that have analyzed markers of quality of life and well - being among the portuguese population, particularly in overweight or obese individuals. one study showed that body image and physical dimensions of obesity - specific quality of life improved significantly during the course of treatment. another study indicated that changes in weight and body image may reciprocally affect each other during the course of behavioral obesity treatment, and that weight loss partially mediated the effect of treatment on quality of life and on self - esteem. in the present study, our goal was to assess the association of perceived need support, general self - determination (as measured by perceived choice), and treatment and exercise motivation (autonomous versus controlled regulation) with variables reflecting psychological well - being and quality of life, during a behavioral obesity treatment program lasting 1 year. based on self - determination theory, we predicted that higher perceived need support, higher self - determination, and more autonomous treatment and exercise self - regulation would be associated with higher hrqol and improved psychological well - being. to our knowledge, only a few studies have examined predictors of psychological well - being in overweight / obese persons during behavioral treatment [29, 30 ] and none have tested self - determination theory variables as putative predictors. the study was conducted within a randomized controlled trial in overweight and moderately obese women, primarily focused on increasing exercise self - motivation and exercise adherence, aiming at long - term weight control. the program 's principles and intervention style were based on self - determination theory, while the control group received a general health education program. the intervention and its theoretical rationale have been described in detail elsewhere [31, 32 ]. participants (n = 239, 37.6 7.1 years old ; bmi = 31.5 4.1 kg / m) were recruited from the community at large through media advertisements. about 67% of the study participants had at least some college education, 23% had between 10 and 12 years of school and 10% had 9 years or less of school. at baseline, women in the intervention group did not differ from those in the control group in terms of bmi, age, education, or marital status. there were also no differences between the 208 women who completed the 12-month intervention and the 31 who withdrew from the program, for any demographic or baseline psychosocial variable, with the exception of age ; women who stayed in the program were on average four years older (p =.01). data was collected at baseline, corresponding to the pretreatment scores, and at 12 months, corresponding to the end of the treatment. the instruments were validated portuguese versions of some of the most commonly used psychosocial instruments in obesity research and are described in detail below. self - determination was assessed with the perceived choice subscale from the self - determination scale, an instrument designed to evaluate individual differences in the extent to which people function in a self - determined way (e.g., i do what i do because i have to, what i do is often not what i 'd choose to do). i feel that the staff has provided me choices and options), involvement (e.g., the staff handles peoples ' emotions very well), and the provision of structure (e.g., the staff has made sure i really understand my condition and what i need to do). total score is calculated by summing response items, higher scores indicating higher perceptions of need support climate (cronbach 's = 0.96). self - regulation for treatment was measured with the treatment self - regulation questionnaire assessing the degree to which a person 's motivation for participating in treatment is autonomous versus controlled. items are summed into two subscales, one measuring autonomous (cronbach 's = 0.86), the other controlled (cronbach 's = 0.80) regulation. exercise regulations were assessed by the self - regulation questionnaire for exercise measuring exercise regulatory motives. the scale can also be summarized into two subscales, autonomous (cronbach 's = 0.91) and controlled exercise regulation (cronbach 's = 0.73). general quality of life was measured with the sf-36 [35, 36 ], composed of two scales and a total of 36 items, reflecting physical (physical component summary, pcs) and psychological (mental component summary, mcs) composite values (cronbach 's between 0.66 to 0.87), in which higher results represent greater quality of life perception. the weight - related aspects of health - related quality of life was assessed using the impact of weight on quality of life lite scale (iwqol - l) [37, 38 ], a 31-item questionnaire (cronbach 's = 0.93). self - esteem was assessed with the rosenberg self - concept / self - esteem scale [39, 40 ] with higher scores of the rses representing greater self - esteem (cronbach 's = 0.88). depression was evaluated with the beck depression inventory [41, 42 ], where higher scores represent greater levels of depressive symptoms (cronbach 's = 0.87). state anxiety was assessed with the state - trait anxiety inventory, where higher scores represent greater levels of anxiety (cronbach 's = 0.92). body weight was measured twice with a standardized procedure (average was used) at baseline and at the end of the treatment (12 months) using an electronic scale (seca model 770, hamburg, germany). height was measured with a balance - mounted stadiometer to the nearest 0.1 cm. for quality of life and psychological well - being variables, 12-month standardized residuals were used, calculated by regressing the 12-month value onto the baseline value, producing an outcome variable which is entirely orthogonal to (i.e., adjusted for) the baseline value. unadjusted 12-month scores were used for treatment and exercise motivation, perceived need support, and self - determination. this option was based on the fact that most participants did not engage in regular exercise at the beginning of the intervention, which yielded exercise self - regulation measures less valid (e.g., i exercise because i). also, treatment self - regulation (i.e., reasons to stay in treatment) and perceived need support from the intervention team could only be evaluated after participants started the intervention. for consistency, statistical analyses were completed using the statistical package for the social sciences (pasw statistics 18). pearson correlations and partial correlations were used to test associations among all variables in the study, and also to examine associations between the independent variables and a global well - being z - score, computed as mean value of all hrqol and psychological well - being z - scores, before and after adjusting for group. stepwise linear regressions were used to analyze the independent effect of self - determination theory 's factors on hrqol and psychological well - being. we ran the same analysis controlling separately for a potential intervention effect and for an effect of weight change on outcomes. tertiles were computed based on the mean z - score value to define success groups. mean differences in hrqol and psychological well - being between women in the low hrqol and psychological well - being and high hrqol and psychological well - being tertile - formed groups were tested using independent t - tests. type i error was set at = 0.05 (two - tailed) for all tests. at the end of the 12-month intervention, there was an overall 87% retention rate. correlations among independent variables and measures of well - being are presented in table 1. self - determination (p =.001), perceived need support (p <.001), treatment autonomous self - regulation (p <.001), and exercise autonomous self - regulation (p <.001) were positively associated with the global well - being z - score, while treatment controlled self - regulation was negatively correlated with well - being (p =.001). more specifically, self - determination correlated positively with hrqol and self - esteem, and negatively with depression and anxiety. regarding self - determination theory treatment variables, perceived need support was positively correlated with physical, mental, and weight - related hrqol, and with self - esteem ; in turn, correlations were negative with depression and anxiety. treatment autonomous self - regulation was positively associated with physical and weight - related hrqol, negatively with depression and anxiety, and not related to mental hrqol. conversely, treatment controlled self - regulation correlated positively with anxiety and negatively with self - esteem, physical, and weight - related hrqol. for exercise variables, results indicated that autonomous self - regulation was positively associated with hrqol and self - esteem, but negatively correlated with anxiety. next, we ran multiple regression models, using self - determination, perceived need support, treatment self - regulation, and exercise self - regulation as independent variables and global well - being as the dependent variable. self - determination, perceived need support, controlled treatment self - regulation (negative association), and exercise autonomous self - regulation were independent predictors of global psychological well - being and explained 29% of the variance. to test whether these relations held when adjusting for group (i.e., controlling for the intervention effect), the same regression model was run, this time with group assignment forced into the model. results were comparable to the unadjusted model (28% variance explained, p <.001) ; however, only self - determination, controlled treatment self - regulation (negatively), and perceived need support were found to predict psychological well - being (see model 2 in table 2). to test if weight change was a confounding factor in these relations, the same regression model was run with weight change also in the model (see model 3 in table 2). results were comparable to model 2 (33% variance explained ; p <.001). a final analysis was conducted, comparing z - scored self - determination theory variables at the highest and lowest of tertiles of global well - being (z - scored) (see figure 1). participants in the higher psychological well - being group had higher self - determination (p <.001), perceived need support (p <.001), lower treatment controlled self - regulation (p =.001), higher treatment autonomous self - regulation (p <.001), and higher exercise autonomous self - regulation (p <.001). the primary goal of this study was to examine the association of general, contextual (obesity treatment), and situation - specific (exercise - related) measures of self - determination with psychological well - being and hrqol. to briefly summarize our findings, higher self - determination and perceived need support, lower treatment controlled self - regulation, and higher exercise autonomous self - regulation were significant predictors of well - being in the course of a 1-year behavioral treatment for obesity, before and after adjustment for weight change. according to self - determination theory, more self - determined behavior, which is partially derived from need - supportive interactions with one 's environment, this causal path is supported by the present results, the first of this kind to be conducted in the context of behavioral obesity treatment. additionally, participants in this study who indicated more autonomous reasons to participate in treatment also reported higher scores on most markers of psychological well - being. finally, women with higher hrqol and enhanced psychological well - being were those for whom exercise behavior was associated with valued outcomes and/or for whom being physically active was enjoyable and optimally challenging (autonomous regulation). we have previously detailed the mechanisms by which interventions can promote autonomous self - regulation for treatment and specifically for exercise [31, 32 ]. in an exercise - specific context, a study by wilson. showed that if the needs for competence, autonomy, and relatedness in an exercise setting are satisfied, subjective vitality and the degree of positive affect typically experienced within one 's exercise session are enhanced. in overweight sedentary women, externally imposed exercise intensity led to a significant decline in ratings of pleasure during exercise session, compared to self - selected intensity. these findings have bearing on the importance of perceived choice in exercising, which could exert positive effects on autonomous regulation, intrinsic motivation, and adherence. another study with overweight and obese individuals, using positive and negative affect, subjective vitality, and satisfaction with life as measures of well - being, showed that exercise - related autonomy positively predicted satisfaction with life and that intrinsic motivation was a positive predictor of positive affect, while introjected regulation was found to be a negative predictor of subjective vitality. in fact, research generally indicates that more autonomous regulations enhance not only behavioral persistence but also psychological well - being [3, 46, 47 ]. in contrast, controlled regulations are typically associated with diminished psychological well - being, also reflected in lower self - esteem. results of the present work are consistent with these findings in showing that treatment and exercise autonomous self - regulation also predict better psychological well - being in overweight or obese women undergoing treatment. in the present study, as predicted, controlled however, controlled motivation towards exercise was generally not related to psychological outcomes. as assessed in this study, i exercise because i want others to see me as physically fit) and reasons which have been introjected or partially internalized (e.g., i exercise because i 'd be afraid of falling too far out of shape, i exercise because i would feel bad about myself if i did not do it). one interpretation for the present findings is that external demands and pressures to exercise in this study were perceived by participants as coming mostly from the intervention team. to the extent that this occurred, external contingencies and incentives, which normally could have been felt as controlling and a potential cause of anxiety, may have been perceived as normal, expected, and even positive by some participants. this was likely the case in the main intervention group, for whom introjected, integrated, and intrinsic exercise motivation were found to increase (compared to controls) at the end of an intervention which was overwhelmingly perceived as supporting participants ' autonomy. in fact, group differences in the psychological impact of introjected and/or external regulations may partially explain the generally nonsignificant results. according to self - determination theory, satisfaction of competence, autonomy, and relatedness, the three basic psychological needs, provide a basis for predicting when the pursuit and attainment of goals will be associated with more positive versus more negative well - being outcomes. in contrast, thwarted satisfaction of these needs results in negative functional consequences for mental health. persistent deprivation of any need has costs for health and well - being, leading to the development of compensatory processes, such as substitute motives and non - autonomous regulatory styles, which are expected to result in worse well - being. thus, need thwarting conditions lead to specifiable patterns of behaviors, regulations, goals, and affects that do not represent optimal development and well - being, which would occur in supportive environments. our findings suggest that, in the context of weight loss treatment, perceived need support and autonomous self - regulation will also lead to increased hrqol and improved psychological well - being. several studies showed that weight loss is important in overweight and obese populations in part due to beneficial effects on hrqol and psychological well - being. for example, kolotkin and colleagues found that obesity - specific hrqol changes were strongly related to weight reduction among 161 participants (88% women). additionally, improved mood, affect, and psychological well - being may also facilitate the adoption and maintenance of behavior needed to regulate one 's weight. thus, improving psychological well - being is not only ethically appropriate but could also be good clinical practice, to the extent that it contributes to enhanced treatment outcomes. unfortunately, investigating predictors of psychological well - being in obesity treatment has not been the focus of much prior research. interestingly, an increasing body of research shows that promoting autonomy is also advisable from a treatment efficacy viewpoint. for example, in weight loss and weight maintenance, more autonomous and intrinsic motivation have been shown to significantly predict more successful weight and exercise - related outcomes [3, 5153 ]. in conclusion, this work supports predictions based on self - determination theory in relation to correlates of quality of live and psychological functioning. for obesity treatment, this study furthers our understanding of mechanisms associated with enhanced psychological outcomes, which is of direct relevance for health care providers in this field. promoting self - determined motivation for health and health behaviors, particularly exercise, importantly, during weight control, these associations appear to hold independently of actual weight changes.	this study examined the association of autonomy - related variables, including exercise motivation, with psychological well - being and quality of life, during obesity treatment. middle - aged overweight / obese women (n = 239) participated in a 1-year behavioral program and completed questionnaires measuring need support, general self - determination, and exercise and treatment motivation. general and obesity - specific health - related quality of life (hrqol), self - esteem, depression, and anxiety were also assessed. results showed positive correlations of self - determination and perceived need support with hrqol and self - esteem, and negative associations with depression and anxiety (p <.001). treatment autonomous motivation correlated positively with physical (p =.004) and weight - related hrqol (p <.001), and negatively with depression (p =.025) and anxiety (p =.001). exercise autonomous motivation was positively correlated with physical hrqol (p <.001), mental hrqol (p =.003), weight - related hrqol (p <.001), and self - esteem (p =.003), and negatively with anxiety (p =.016). findings confirm that self - determination theory 's predictions apply to this population and setting, showing that self - determination, perceived need support, and autonomous self - regulation positively predict hrqol and psychological well - being.
thymosin alpha 1 (t1), a 28-amino - acid peptide with molecular weight (mw) of 3108 da, was first isolated from calf thymus in 1977 by goldstein. [1, 2 ]. t1 is mainly produced in vivo by thymic epithelial cells and is stable at 8090c. t1 has no aromatic amino acids (see figure 1), and n - acetylation is important for protecting towards proteolysis. t1 plays a role against viral infection, against tumors, and in immunomodulation. upon stimulation of t1, t cells become mature in vitro and express il-2, il-2r, and cd2 [4, 5 ]. t1 inhibits viral replication at least in hepatitis virus b - transfected hepg2 tumor cells [6, 7 ]. in addition, t1 antagonizes t cell apoptosis induced by dexamethasone, cd3 monoclonal antibody treatment, or addition of sera from tumor - bearing mice. it also downregulates tumor weight in mice, enhances major histocompatibility complex (mhc) class i antigen expression in tumor cells and antigen - presenting cells [1113 ], and partly restores cellular immunity. t1 is used in combination with ifn- for patients with chronic hepatitis virus b or c infection [15, 16 ]. recently, t1 has even been used as an adjuvant for vaccines against influenza and hepatitis b. in the past two decades, although the successful expression of the recombinant t1 (r t1) and prothymosin (thymosin comprises the n - terminus of prothymosin) in escherichia coli has been reported [19, 20 ]. however, t1 was expressed with a fusion protein or in a concatemer form [21, 22 ]. the expression of the t1 monomer peptide has not been reported, because it is difficult to extract the micromolecular peptide from the fermentation broth. the fusion protein all of these issues made it difficult to prepare active recombinant t1. in this study, we have expressed t1 concatemer protein and cleaved the concatemer protein with hydroxylamine to obtain active t1 monomer peptide. the pet-22b (+) vector, e. coli bl21 (de3) (hsds gal [cits857 ind1 sam7 nin5 lacuv5-t7 ]), the hepg2 liver carcinoma cell line, and the spc - a-1 lung carcinoma cell line were kindly provided by institute of biochemistry and cell biology (shanghai, cas, china). hepg2 and spc - a-1 cells were cultured in rmpi 1640 medium with 10% fetal bovine serum in humidified air at 37c with 5% co2. lymphocytes were isolated from adult mouse spleens (bal / c) and cultured in rmpi 1640 medium with 10% fetal bovine serum in humidified air at 37c with 5% co2. synthesized t1 (sinopep pharmaceutical, inc., hangzhou, china.) the synthesized t1 and the cleaved t1 were dissolved in phosphate - buffered solution (pbs ph 7.2) at 1 mg / ml (approximately 3 10 two single strand dna oligomers encoding the entire thymosin alpha peptide (synthesized by songon, shanghai, china.), partially complementary to each other (as indicated by the underlined sequences) were synthesized. thy - f : gtcgacatgggatccaacggttctgatgctgctgtagatacttcttctgagattactactaaagacctaa thy - r : ctcgagtcaagatctcccgttctcagcctcttcgacaacttccttcttctcctttaggtctttagtagta the 2 oligonucleotides were mixed and denatured by incubation at 94c for 10 minutes, and subsequently annealed at 60c for 10 minutes in presence of taqplus (bbi, toronto, canada). the desired double - strand dna was treated with t4 polynucleotide kinase (bbi, toronto, canada) and cloned into the cloning vector pucm - t (bbi, toronto, canada). the plasmid (pucm - t - t1, pt - t1) containing the t1 gene was digested with bamh i / xho i or bgl ii / xho i, respectively. the digested fragments containing the coding sequence of t1 were ligated with t4 dna ligase for 16 hours at 4c to produce the concatemer t1 gene of 2 repeats (pt - t12). when the plasmid was digested with isocaudamer bamhi (gctag c) and bgl ii (agatc t), the 2 fragments had similar termini. when the 2 fragments were catalyzed with t4 dna ligase the new sequence formed was ggatct, and this new site could not be digested by bamh i or bgl ii (see figure 2). using a pt - t12 plasmid, plasmids containing concatemer 4 repeats (pt - t14) of the t1 gene were prepared as described above. using a pt - t12 plasmid and a pt - t14 plasmid, the preparation of plasmids containing concatemer 6 repeats (pt - t1 6) was the same as pt - t12 plasmid. finally, a fragment containing the concatemer t1 gene of 6 repeats was constructed and inserted into expression vector pet-22b (+), between the restriction endonuclease recognition sites hind iii and nde i. a single bacterial colony containing pet-22b (+) -t1 was grown overnight at 37c in 5 ml lb medium with 50 g / ml ampicillin (bbi, toronto, canada). the overnight culture was inoculated into 250 ml lb medium in a 1 l flask with 50 g / ml ampicillin. after 2 hours of incubation, isopropyl-1-thio - beta - d - galactoside (iptg ; bbi, canada) was added to the culture at a final concentration of 1.0 mm, when the cell density had reached od600 nm = 0.6. after incubation at 37c for 8 hours, the bacterial cells were harvested by centrifugation (5000 g, 15 minutes) at 4c. the inducibility of the concatemer gene was determined by sodium dodecylsulfate - polyacrylamide gel electrophoresis (sds - page) (data not shown). the cell pellet was resuspended in 100 ml buffer a (50 mm tris - hcl [ph 8.0 ], 1 mm edta [ph 8.0 ], and 0.1 m nacl) and then sonicated (800 w, 20 minutes). after sonication, the sedimented material (containing the concatemer protein) was washed once first with 100 ml buffer b (50 mm tris - hcl [ph 8.0 ], 1 mm edta (ph 8.0), and 0.5% triton - x 100) and then with 100 ml buffer c (50 mm tris - hcl [ph 8.0 ], 1 mm edta (ph 8.0), and 2 m urea) by centrifugation. the sedimented material was then dissolved in 100 ml buffer d (50 mm tris - hcl [ph 8.0 ], 1 mm edta [ph 8.0 ], and 8 m urea) with stirring for 1 hour at 25c. the soluble protein was then dialyzed (dialysis membrane mwco 10 000, spectrum, tex, usa) with double distilled h2o and cryodesiccated. the concatemer protein the cleavage buffer contained 0.1 m trizma base and 2.0 m hydroxylamine - hcl. the ph of the cleavage buffer was adjusted with 5 m naoh solution to a ph of 9.0, and the reaction was performed at 45c for 4 hours. the reaction was terminated by adjusting the ph to 4.0 with concentrated formic acid and cooling to 4c. using sephadex - g25 (ge) column chromatography, the products of cleavage were desalted. column chromatography revealed 2 peaks ; one peak corresponded to the t1 monomer and the other contained the incompletely cleaved concatemer protein. the molecular mass of the t1 monomer was analyzed by gly - sds - page and mass spectrometry. spleens were removed from mice and dispersed through nylon mesh to generate a single - cell suspension. cells were pelleted (1500 g, 6 minutes), and the cell pellet was resuspended in erythrocyte lysis solution (0.15 m nh4cl, 0.02 m tris, sterilized with a 0.22 m filter) for 5 minutes to remove the red cells. after lysis, the cell suspension was incubated in a culture flask for 2 hours, and then the suspension was transferred to a new flask. splenic lymphocytes were incubated in rpmi 1640 medium with 5 g / ml concanavalin a (cona ; bbi, canada) for 12 hours, and then harvested by centrifugation (1500 g, 6 minutes). the cell pellet was resuspended in rpmi 1640 medium at a concentration of 1 10 cells / ml. a 96-well culture plate (costar, inc., calif, usa) was used for incubation of 180 l lymphocytes in rpmi 1640 medium. 50 g / ml of the proteins (cleaved t1 and synthesized t1) were added to the wells. after 24-hour or 48-hour incubation at 37c, 20 l of mtt (0.5 mg / ml), solution was added to each well, and the microplates were incubated for 4 hours at 37c. after incubation, the plates were centrifuged (1400 g, 18c, 10 minutes). the supernatants were removed, and 200 l of dimethyl sulfoxide (dmso) was added to each well and incubated for 15 minutes at room temperature. after incubation, the solubilized reduced mtt was measured colorimetrically at 570 nm using a benchmark microculture plate reader (bio - rad, calif, usa). the influence of t1 on mitochondrial activity was measured using mtt reduction, a common experimental method to quantify mitochondrial activity. the cells were seeded in a 96-well culture plate at 1 10 cells / ml. after 24-hour, 48-hour, and 72-hour exposures to t1 at a concentration of 0.1 mg / ml, the cells were incubated with mtt (0.5 mg / ml) for 4 hours at 37c. the formazan precipitate was then dissolved in 200 l dmso, and absorbance at 570 nm was measured using the benchmark microculture plate reader (bio - rad, calif, usa). the data are presented as mean sem, for at least 3 replicate experiments and evaluated using the student 's t - test. p - values below 0.05 were regarded as statistically significance (two - tailed test). hydroxylamine - induced cleavage at the asp - gly dipeptide site inserted between the 2 moieties of recombinant fusion proteins had been used for both analysis and preparation of mature protein. cleavage occurs at the asn - gly bonds and results from the tendency of the asp side chain to cyclize the formation of a substituted succinimide that is susceptible to nucleophilic attack by hydroxylamine. the infrequency of asn - gly bonds in most proteins results in the production of very large fragments that may overlap cnbr - produced fragments. in the complete amino acid sequence of t1 (see figure 1), only 1 asp was found at the c - terminus. hydroxylamine - induced cleavage is high specificity and safe, however, the efficiency of hydroxylamine cleavage is low. so we expressed a concatemer protein of 6 repeats of t1 to increase the yield of t1 after cleavage and to decrease the cleavage sites. the optimal conditions for inducing production of recombinant concatemer protein were at an iptg concentration of 1.0 mm 37c for 8 hours. under these conditions, the recombinant protein (~23 kd) comprised 31% of the total bacterial protein (see figure 4(a)). the concatemer protein was identified primarily in the inclusion bodies (see figure 4(b)). g-25 column chromatography was used to not only desalt but also remove the incompletely cleaved concatemer protein. because the molecular weight of the t1 monomer was ~3.1 kd however, the incompletely cleaved concatemer proteins, including 2t1 (~7 kd), 3t1 (~11 kd), 4t1 (~15 kd), 5t1 (~19 kd), and 6t1 (~23 kd), could not enter the g-25 gel because the gel only allows the entry of molecules < 5 kd. the gly - sds - page could satisfactorily separate polypeptides with molecular weights ranging from 3.5 to 68.0 kd with good linearity, and demonstrated the successful hydroxylamine - induced cleavage of the concatemer proteins (see figure 5). however, not all the concatemer proteins were cleaved, and the incompletely cleaved proteins could be completely cleaved by another reaction (data not shown). ms was performed to analyze the exact molecular weights of the products and the synthesized t1. the result showed that the products had only 1 peak of m / z = 1042.19, while the synthesized t1 had 2 peaks of m / z = 1037.80 and 1555.06 (see figure 6). the molecular weight of the synthesized t1 was found to be approximately 3108 da, it was assumed that the peptide might attach 2 or 3 protons [(1037.801) 3 = 3110.4 ; (1555.061) 2 = 3108.12 ]. therefore, the cleaved t1 was believed to attach 3 protons, and the molecular weight of the products was approximately 3123.57 [(1042.19 - 1) 3 = 3123.57 ]. the difference in the molecular weights between the cleaved t1 and the synthesized t1 was approximately 15. according to a previous research, the structural difference between the products and the synthesized t1 was a nh added to the n - terminus of the cleaved peptide (see figure 9). to determine the bioactivity of the t1 monomer on lymphocyte proliferation, the mitochondrial activity was compared in the presence with the absence of the t1. preliminary results showed that the peptides could stimulate the mice spleen lymphocyte proliferation. after a 24-hour treatment with t1 cleaved by hydroxylamine, mitochondrial activity was upgraded to 119.1% and after 48 hours, it was 110.4%.the numbers of the synthesized t1 were 117.5% and 116.0% at 24 hours and 48 hours, respectively (see figure 7). both of the peptide preparations could stimulate the proliferation of mice spleen lymphocytes to a similar degree. in this study, we used human liver tumor cells hepg2 and human lung tumor cells spc - a-1 as model, because the liver tumor and lung tumor are the most general tumor. to determine the role of mitochondria during incubation, the mitochondrial activity was examined. after the treatment with the cleaved t1 for 24 hours, 48 hours, and 72 hours, the mitochondrial activity of the spc - a-1 cells decreased to 82.4%, 27.1%, 11.6%, respectively, and the corresponding activities with synthesized t1 were 62.0%, 15.1%, and 9.8%, respectively. on the other hand, after the treatment for 24 hours, 48 hours, and 72 hours, the mitochondrial activity of hepg2 cells decreased to 86.6%, 18.4%, and 10.4%, respectively, and corresponding activities with the synthesized t1 were 59.2%, 13.1%, and 10.0%, respectively (see figure 8). both of the peptide preparations decreased the mitochondrial activity remarkably, as compared to the control. the difference in the activity between the 2 peptides was not significant ; however, the process of synthesizing t1 was rather intricate. t1 is mainly obtained by chemical synthesis and animal thymus extraction. however, in fact, t1 from an animal is often impure and may therefore cause heterogeneous allergy. although chemically synthesized t1 can reach a high level of purity, it is necessary to eliminate the side products at each step, including incorrect joining and dextral compounds. the longer the peptide is, the more intricate the chemical synthesis process becomes. t1 comprises 28 amino acid residues, and its chemical synthesis incurs a high production cost. prokaryotic expression systems allow for high expression of foreign genes and are widely used for producing large amounts of proteins or peptides. in this research, using a prokaryotic expression system bl21 (de3)/pet-22b (+), we have established a new method to obtain relatively pure and large quantities of t1. when the plasmid was digested with isocaudamer bamh i (gctag c) and bgl ii (agatc t), the 2 fragments had similar termini. when the 2 fragments were catalyzed with t4 dna ligase, the new sequence formed was ggatct, and this new site could not be digested by bamh i or bgl ii (see figure 2). therefore, the new 2 concatemer gene could not be destroyed when the 4 concatemer gene was constructed. the new site ggatct corresponded to the dipeptide gly - ser, located at the downstream of the t1 gene. when concatemer protein was cleaved with hydroxylamine, the downstream amino acid of t1 peptide was removed from this gly site. when the cleavage reaction proceeded, the upstream amino acid of t1 peptide was removed from another gly site, which was added in front of the t1 peptide. it is common knowledge that extraction of the micromolecular recombinant peptide from fermentation broth is difficult, when t1 is expressed alone. generally, a fusion protein is used under these circumstances, for example, a dsba fusion protein. however, the fusion protein must be removed because the peptide might be inactive, and this process incurs a high production cost. the concatemer protein of 6 repeats6t1was prepared in this research to avoid the use of a fusion protein when expressing the recombinant protein, but the number of the repeats was not unique. proteins with 4, 8, 12, and so forth repeats could also be constructed. the longer the concatemer protein is, the greater the yield of the t1 monomer is. on the other hand, when a concatemer protein with more repeats is cleaved, the amount of the incompletely cleaved protein also increases (see figure 5). however, these incompletely cleaved proteins can be cleaved by other 1 or 2 rounds of hydroxylamine cleavage. hydroxylamine was used to cleave the asn - gly peptide bond between the fusion protein and target protein. in this study, hydroxylamine was used to cleave the concatemer protein of 6t1 to release the t1 monomer. while chemical cleavage methods use relatively inexpensive and widely available reagents, cleavage conditions are often relatively harsh and amino acid side - chain modifications may occur. this is especially true when hydroxylamine is used to cleave the asn - gly peptide linkages as other asn or gln residues can be modified to their hydroxamic acid forms. because the t1 peptide does not contain gln, met, or other asn, chemical heterogeneity dose not occur by conversion of the gln or asn to their hydroxamate forms. moreover, there is no possibility of chemical heterogeneity by conversion of met to its oxidized forms. after hydroxylamine cleavage, a gly remained at the n - terminus of t1. however, the natural t1 had an acetylated nh - terminal (see figure 9). the molecular weight difference between the 2 groups was 15 da, but there was no significant difference between the 2 bands revealed by the gly - sds - page (see figure 5). hence, we used an ms to analyze the exact molecular weights of the 2 peptides. the result showed a difference was 15 da (31233108 da) (see figure 6), indicating that the concatemer protein was cleaved satisfactorily. to investigate the influence of the extra imino group on the t1, we compared the antitumor and immunomodulation activities between the synthesized t1 and the cleaved t1. furthermore, we found that t1 was able to depress the prolife ration of hepg2 cells by downregulating the oxidative stress (data not shown). jrnvall had analyzed 40 known -amino - acetylated polypeptide chains and postulated that acetylation occurs during an early posttranslational stage and may have particular importance in this regard. however, the structural or functional significance of the n - terminal acetylation of t1 is not known, and a protective function of acetylation with regard to proteolysis is possible. the change in the n - terminus of cleaved peptides might not influence the bioactivity of t1 ; however, it might influence the stability in vivo. in summary, t1 with a novel structure was prepared via the expression and hydroxylamine cleavage of a t1 concatemer protein. this novel t1 could perform proliferation of mice splenic lymphocytes activities, identical to the natural t1. this study would provide the basis for the preparation of active t1 and other peptides.	human thymosin alpha 1 (t1) is an important peptide in the development and senescence of immunological competence in human, and many studies have reported the expression of this peptide. in this study, we designed and synthesized the t1 gene according to the e. coli codon usage preference and constructed a 6t1 concatemer. the latter was inserted into an e. coli expression vector pet-22b (+), and transformed into e. coli bl21 (de3). after induction with iptg, the concatemer protein was successfully expressed in e. coli then cleaved by hydroxylamine to release the t1 monomer. gly - sds - page and mass spectrometry confirmed that the recombinant protein was cleaved as intended. the bioactivity of the t1 monomer was analyzed by lymphocyte proliferation and by mitochondrial activity in two different tumor cell lines. this study provides a description of the preparation of a bioactive t1, which may prove useful in future biomedical research.
contact dermatitis (cd) in the framework of occupational diseases remains prevalent among workers worldwide, impacting their quality of life and workability. the employees most affected by cd are hairdressers, healthcare workers, and metal workers because they are continuously exposed to harmful environments when working. as a result, employees with cd tend to use anti - inflammatory and immunomodulatory agents such as corticosteroids repeatedly. corticosteroids are effective and powerful agents for cd, but their doses should be restricted because of their adverse side effects. herbal medicines have recently emerged in the framework of complementary and alternative medicines (cam) for corticosteroids because they have relatively lower cost and safety. are frequently used as traditional or folk medicines for patients with abdominal pain, dysmenorrhoea, uterine haemorrhage, and inflammation in china, japan, and korea. a. argyi, chinese mugwort, is herbaceous perennial plant known in japanese as gaiyou and in chinese as aiye. a. princeps, japanese mugwort, is a perennial and very vigorous plant known as yomogi in japanese. recently, the leaves of a. argyi and a. princeps and their components have been shown to have antitumour [58 ], antifungal, anticoagulant, antidiabetic, and anti - inflammatory [12, 13 ] activity. based on these findings, we examined the effects of artemisia leaf extract (ale) on inflamed tissues in mice with cd and anti - inflammatory activities in raw 264.7 cells. specifically, the effects of ale on histopathological changes including ear swelling, epidermal hyperplasia, immune cell infiltration, and cytokine production such as interferon- (ifn-) gamma (), tumour necrosis factor- (tnf-) alpha (), interleukin- (il-) 6, and il-10 in ear tissues were assessed in mice with cd induced by topical application of 1-fluoro-2,4-dinitrobenzene (dnfb). the anti - inflammatory effects on production levels of prostaglandin e2 (pge2) and nitric oxide (no) and the expression levels of cyclooxygenase 2 (cox-2) and inducible nitric oxide synthase (inos) were investigated in raw 264.7 cells. artemisia leaf was purchased from gwangmyungdang (ulsan, korea). the artemisia leaf consisted of a mixture of a. argyi and a. princeps and was authenticated by professor jung - hoon kim, one of the authors of this study. twenty grams of artemisia leaf was immersed in 500 ml of methyl alcohol and sonicated for 15 min, after which they were extracted for 24 h. following extraction, the supernatant was transferred and the artemisia leaf was again extracted with 500 ml of methanol for 24 h. the two extracts were then combined and filtered through whatman no. 20 filter paper, after which they were condensed using a rotary evaporator (eyela, tokyo, japan). the evaporated extract was subsequently dried using a lyophilizer (labconco, kansas city, mo, usa), which yielded 1.04 g of freeze - dried powder (yield, 5.21%). mh2013 - 040) were deposited in the herbarium located in the school of korean medicine, pusan national university. six - week - old male balb / c mice were obtained from samtako (incheon, korea). all mice used in this experiment were housed in the cages under specific conditions, including a 12 h light / dark cycle and specific pathogen - free conditions. in addition, mice were provided with free access to standard rodent feed and water. we conducted all animal experiments according to institutional guidelines and all experimental procedures were approved by our animal care committee (pnu-2012 - 0140). cd was induced using our standard method as previously described. briefly, 0.1% dnfb (50 l) in vehicle composed of acetone and olive oil (4 : 1, aoo) was applied onto the shaved back of mice for three successive days (sensitization). mice were then treated by application of 0.2% dnfb (30 l) in vehicle onto the backside of their ears every two days. for topical treatment with drugs, dexamethasone (dex) and ale were dissolved in ethanol, filtered using a syringe filter (0.45 m), and finally diluted in vehicle (aoo : ethanol, 4 : 1). ale (30 or 300 g / ear) was topically applied onto the backside of ears for 7 days. a total of 36 mice were used in this study (nor group and dex group, 6 mice ; ctl group and ale group, 8 mice). mice were sacrificed with co2, after which ear pieces (5 mm in diameter) obtained via dermal punch were weighed using a microbalance and the thicknesses of both ears were measured with digimatic calipers (mitutoyo, kanagawa, japan) at the same time. obtained tissues were fixed in 4% formalin for 24 h and then dehydrated using ethyl alcohol. ear tissues (4 m) were subsequently resected, after which sections were stained with haematoxylin - eosin (h / e) and observed using a light microscope (50x). to evaluate hyperplasia in the epidermis and infiltration of immune cells, five nonoverlapping fields per slide the height from the basal lamina to the top of the stratum granulosum was quantified to evaluate the epidermal thickness. total immune cell numbers were quantified by counting immune cells in the same size counting grid. cytokine levels in ear tissues were evaluated using a mouse inflammation cytometric bead array (cba) kit (bd biosciences, san jose, ca, usa). briefly, to obtain tissue lysates, resected inflamed tissues were lysed using protein extraction solution (intron bio, daejeon, korea) and a homogenizer (next advance, ny, usa). next, 50 g of lysates was used to evaluate the levels of tnf-, ifn-, il-6, and il-10. raw 264.7 cells, the immortalized murine macrophage cell line, were cultured using dmem (hyclone, logan, ut, usa) containing foetal bovine serum (10%, fbs) and antibiotics (1%, penicillin - streptomycin). cells were seeded in 96-well plates at a density of 1 10 cells / well and then incubated overnight. next, cells were treated with the indicated concentrations of ale for 4 h, after which they were stimulated with 1 g / ml of lipopolysaccharide (lps) for 20 h. following stimulation, 100 l of supernatants was mixed with 100 l of griess reagent (2% sulphanilamide in 10% h3po4 and 0.2% of n-(1-naphthyl)ethylenediamine in distilled water) and then incubated at room temperature for 10 min. the absorbance at 540 nm was subsequently measured using a spectrophotometer (tecan, mnnedorf, switzerland). the production of pge2 was measured by enzyme - linked immunosorbent assay (elisa) using pge2 elisa assay kit (enzo life science, farmingdale, ny, usa). the culture supernatants were then collected, after which the optical densities were measured using a spectrophotometer (tecan, mnnedorf, switzerland) at a wavelength of 405 nm. briefly, ale and lps treated raw 264.7 cells were harvested, lysed, and homogenized as previously described. the protein concentration of each cell lysate primary antibodies against inos (482728, merck millipore, darmstadt, germany, 1:1000), cox-2 (sc-19999, santa cruz biotechnology, santa cruz, ca, usa, 1:1000), -actin (santa cruz biotechnology, santa cruz, ca, usa), and horseradish - conjugated secondary antibody (enzo life sciences, farmingdale, ny, usa, 1:3000) were used to detect specific protein levels. in addition, a west - q chemiluminescent substrate kit (gendepot, barker, tx, usa) and a las 4000 mini (ge healthcare, piscataway, nj, usa) were used to visualize the antigen - antibody complex. the mann - whitney u test was used for data obtained from in vivo experiments ; student 's t - test was used for data obtained from in vitro experiments, and prism 5 for window version 5.01 (graphpad software inc. at the end of experiment, the thicknesses and weights of both ears were evaluated. in ctl group, repeated treatment with dnfb elevated the levels of ear thickness and weight more than three times relative to the normal group. 30 and 300 g / ear of ale treatment effectively inhibited enlargement of ear thickness (figure 2(a)) and 300 g / ear of ale inhibited ear weight gain significantly (figure 2(b)). the effects of ale on epidermal hyperplasia, one of the major features of cd, and immune cell infiltration were investigated. marked increases in epidermal thickness (yellow bars) and infiltration of immune cells into inflamed tissues were observed in the nontreated cd mice. in addition, a large pustule (filled arrow) and vesicles were also observed (figure 3(a)). treatment with ale effectively inhibited epidermal hyperplasia and reduced immune cell infiltration compared to the ctl mice (figure 3(b)). we also checked the effects of ale on cytokine productions in inflamed tissues. in our results, elevated levels of tnf-, ifn-, and il-6 production were observed in ctl mice (figure 4). treatment with 300 g / ear of ale significantly lowered the production levels of tnf-, ifn-, and il-6, respectively. the il-10 production levels were not affected by induction of cd or treatment with ale, and only the dex treated group showed lower production of il-10 than the normal and control groups (figure 4). expression of inos was elevated in the lps treated ctl group compared to the normal group. treatment with more than 25 g / ml of ale lowered inos expression in a concentration dependent manner (figure 5(a)). in addition, treatment with lps elevated no production level more than nine times relative to neither lps nor ale treated group. this increase in no production was effectively inhibited by ale in a dose dependent fashion (figure 5(b)). the lps stimulated raw 264.7 cells showed marked increases in cox-2, representative cox enzyme, and treatment with ale inhibited cox-2 expression (figure 6(a)). in addition, the production of pge2, one of the major final metabolites, was markedly induced by lps stimulation. treatment with 50 mg / ml of ale inhibited pge2 production significantly (figure 6(b)). artemisia species have traditionally been utilized for amelioration of diseases including malaria, hepatitis, parasites, and various cancers worldwide. the anti - inflammatory effects of a. princeps in antigen - stimulated t cells and regulatory t cells have been reported by chang.. additionally, the major active components of artemisia species are sesquiterpenoids and flavonoids such as eupatilin and jaceosidin, which are extracted from most herbs in artemisia species, and these components have been reported to have anti - inflammatory effects in mice. based on the anti - inflammatory and immunomodulatory effects of artemisia species, we investigated whether ale can reduce inflammatory reactions in an animal model of cd to ameliorate its symptoms. the skin of cd patients tends to thicken because of chronic and repeated inflammatory reactions, which are closely related to epidermal and dermal hyperplasia. in our experiment, repeated application of dnfb induced ear swelling, which was effectively inhibited by topical application of ale (figure 2). given that the degree of ear swelling is recognized as an index of inflammatory reaction, these results imply that ale can exert anti - inflammatory action in animal models of cd. in our animal model of cd, repeated application of dnfb induced hyperplasia in the epidermis and massive infiltration of immune cells to the epidermis and into the connective tissues. in addition, spongiotic changes, vesicles, and pustules were seen in the control group (figure 3(a)(b)). topical application of ale effectively inhibited hyperplasia in the epidermis and infiltration of immune cells. in addition, the area of spongiotic changes and vesicles was diminished and pustules were rarely seen in the ale and dex group (figure 3). these findings indicate that ale can act as an anti - inflammatory agent in cd to reduce immune cell infiltration, resulting in inhibition of spongiotic changes, as well as pustule and vesicle formation. tnf- and ifn-, hallmarks of th1 skewing reaction, can stimulate keratinocytes, which subsequently proliferate, resulting in epidermal hyperplasia. these two cytokines are also closely related to infiltration of immune cells into the inflamed tissues. in the inflammatory cascade of cd, activated keratinocytes can release tnf- and il-6, which are growth promoting cytokines, resulting in accelerated immune cell infiltration and prolonged lifespan of immune cells. il-6 has also been reported to accelerate proliferation and migration of keratinocytes, leading to skin disorders, including psoriasis. in this study, topical treatment with ale effectively reduced production levels of tnf-, ifn-, and il-6 in ear tissues (figure 4). when combined with previous results, these findings imply that ale can inhibit hyperplasia in the epidermis and infiltration of immune cells via regulation of proinflammatory cytokines, finally leading to inhibition of enlargement of skin thickness. histopathological analyses revealed that many types of inflammatory cells, including macrophages, infiltrate into the dermis and epidermis in cd. macrophages also play an important role by releasing inflammatory mediators such as no, prostaglandins, and cytokines during initiation of cd. for these reasons, we also investigated anti - inflammatory effects of ale in vitro using a macrophage cell line, raw 264.7. treatment with up to 100 g / ml of ale had no effect on the viability of raw 264.7 cells (data not shown). ale significantly inhibited inos expression and no production in a dose dependent manner (figure 5). in addition, ale inhibited cox-2 expression and pge2 production induced by lps stimulation (figure 6). these findings indicate that ale inhibits inflammatory response in macrophages via regulation of no and pge2 production. as shown in figures 5 and 6, ale strongly inhibited inos expression and no production, while it had little effect on pge2 production or cox-2 expression. it is well known that intracellular signaling pathways such as erk, jnk, and p38 and transcription factors such as creb and nf-b play a central role in activation and inflammatory mediator production in macrophages. we checked the effects of ale on expression levels of signaling pathways and the nf-b pathway. treatment with ale prevented phosphorylation of p38 but had no effect on the nf-b pathway (data not shown). considering these results, the anti - inflammatory effects of ale may be closely related to the p38 signaling pathway, and the nf-b pathway may not participate in the anti - inflammatory mechanisms of ale. kim. reported immune - stimulatory effects of hot water extracts of the leaves of a. princeps. specifically, they found that extracts of a. princeps elevated production levels of no and tnf-, which is inconsistent with our results. many previous studies of a. princeps have shown anti - inflammatory or antiallergic effects, as well as antioxidative effects. in addition, components isolated from a. princeps such as eupatilin and jaceosidin have been reported to have inhibitory effects against ige - induced hypersensitivity and carrageenan - induced inflammation, respectively [16, 22 ]. however, kim. did not describe why their findings were inconsistent with those of previous results. in addition, water extracts of plant materials should also be checked for microbial contamination before use because they are more easily contaminated than methanol or ethanol extracts. overall, the results of this study suggest that the anti - inflammatory effects of ale in cd mice are closely related to regulation of the activation of immune cells, especially macrophages and cytokine production in inflamed tissues. in addition, ale effectively lowered the production levels of tnf-, ifn-, and il-6 in inflamed tissues. these anti - inflammatory actions prevented epidermal hyperplasia and immune cell infiltration. finally, ale effectively inhibited ear swelling induced by dnfb. these results imply that ale can be used for the treatment of patients with cd as a complement and alternative medicine (cam) to corticosteroids.	the leaves of artemisia argyi lev. et vant. and a. princeps pamp. are well known medicinal herbs used to treat patients in china, japan, and korea with skin problems such as eczema and itching, as well as abdominal pain and dysmenorrhoea. we investigated the anti - inflammatory effects of artemisia leaf extract (ale) using cd mice and raw 264.7 cells. the effects of ale on histopathological changes and cytokine production in ear tissues were assessed in mice with cd induced by 1-fluoro-2,4-dinitrobenzene (dnfb). moreover, the anti - inflammatory effects on production levels of prostaglandin e2 (pge2) and nitric oxide (no) and expression levels of cyclooxygenase 2 (cox-2) and inducible nitric oxide synthase (inos) were investigated in raw 264.7 cells. topical application of ale effectively prevented ear swelling induced by repeated dnfb application. ale prevented epidermal hyperplasia and infiltration of immune cells and lowered the production of interferon- (ifn-) gamma (), tumour necrosis factor- (tnf-) alpha (), and interleukin- (il-) 6 in inflamed tissues. in addition, ale inhibited expression of cox-2 and inos and production of no and pge2 in raw 264.7 cells. these results indicate that artemisia leaf can be used as a therapeutic agent for inflammatory skin diseases and that its anti - inflammatory effects are closely related to the inhibition of inflammatory mediator release from macrophages and inflammatory cytokine production in inflamed tissues.
service learning enhances student learning and engagement in undergraduate biology courses (1, 2), but obtaining student buy - in can be frustrating. to alleviate this issue, i had students design their own service - learning projects in my senior - level undergraduate neurobiology course. by carefully guiding students through project design early in the semester, i was able to ensure students produced successful projects that increased their understanding of course material and benefited the community organizations. there is no lab, but the service - learning component gives students one additional hour of credit. based on mercer university s service - learning guidelines, students are required to spend at least 30 hours planning and carrying out the project. course enrollment is typically 16 to 24 students, and i have taught it twice as a service - learning class. on the first day of class, i defined service learning, provided ideas for possible projects, and asked students to brainstorm ideas. over the first two weeks, students shared ideas every day in class, and i encouraged them to talk to one another about their ideas outside of class. students divided themselves into groups of two to five (depending on the needs of the project), based on interest and schedule. in week three, i met with each group to discuss the feasibility of the project. i encouraged them to make contact with their community partner immediately, and i facilitated these initial contacts when necessary. many students already had connections with their agencies, typically through volunteering or a service - learning project from another course. by four weeks into the semester, i asked each group to submit a project proposal. the proposals included a detailed description of the project, project goals and schedule, supplies needed, and a schedule of due dates for reflections (see appendix 1 for project and proposal guidelines). students came up with a diversity of projects, most of which involved teaching neuroscience to a group in the community. most projects lasted five to six weeks (usually weeks 712 of the semester), with one session per week of one to two hours. many chose to teach children, and projects included workshops for homeschoolers at the local museum of arts & sciences, at an after - school program for high school students, in several elementary and middle school classes, and at a rescue mission for homeless families. each group planned activities that demonstrated fundamental concepts in neuroscience that were appropriate for the age group they were teaching. their activities have included basic electrophysiological demonstrations of action potentials using spikerboxes from backyard brains (https://backyardbrains.com/), dissections of sheep brains, making models of brains and neurons, manipulation of the senses and discussions of the transduction of sensory stimuli, and watching and explaining videos of common neurological disorders. one group wanted to work with adults, and they chose to plan and lead two saturday morning workshops on neurodegenerative diseases at an assisted - living community. they presented the science underlying the most common neurodegenerative diseases, discussed new research on disease treatments, and led activities on nutrition and exercise. by the time groups began their projects (approximately week 7), students had a firm grasp of neuron function, synaptic transmission, and neuroanatomy, and we had begun discussing sensory and motor systems. as a result, they were ready to teach any of these topics. if a group proposed other topics, i met with them during their planning to ensure that they presented accurate information students wrote reflections after each visit. because the length and frequency of visits varied, i let each group decide on their due dates, as long as each reflection was turned in prior to the next visit. in their reflections, students wrote what they did, what they learned from their preparation and visit, and what they planned to do differently next time. as i read reflections, on the first day of class, i defined service learning, provided ideas for possible projects, and asked students to brainstorm ideas. over the first two weeks, students shared ideas every day in class, and i encouraged them to talk to one another about their ideas outside of class. students divided themselves into groups of two to five (depending on the needs of the project), based on interest and schedule. in week three, i met with each group to discuss the feasibility of the project. i encouraged them to make contact with their community partner immediately, and i facilitated these initial contacts when necessary. many students already had connections with their agencies, typically through volunteering or a service - learning project from another course. by four weeks into the semester, i asked each group to submit a project proposal. the proposals included a detailed description of the project, project goals and schedule, supplies needed, and a schedule of due dates for reflections (see appendix 1 for project and proposal guidelines). students came up with a diversity of projects, most of which involved teaching neuroscience to a group in the community. most projects lasted five to six weeks (usually weeks 712 of the semester), with one session per week of one to two hours. many chose to teach children, and projects included workshops for homeschoolers at the local museum of arts & sciences, at an after - school program for high school students, in several elementary and middle school classes, and at a rescue mission for homeless families. each group planned activities that demonstrated fundamental concepts in neuroscience that were appropriate for the age group they were teaching. their activities have included basic electrophysiological demonstrations of action potentials using spikerboxes from backyard brains (https://backyardbrains.com/), dissections of sheep brains, making models of brains and neurons, manipulation of the senses and discussions of the transduction of sensory stimuli, and watching and explaining videos of common neurological disorders. one group wanted to work with adults, and they chose to plan and lead two saturday morning workshops on neurodegenerative diseases at an assisted - living community. they presented the science underlying the most common neurodegenerative diseases, discussed new research on disease treatments, and led activities on nutrition and exercise. by the time groups began their projects (approximately week 7), students had a firm grasp of neuron function, synaptic transmission, and neuroanatomy, and we had begun discussing sensory and motor systems. as a result, they were ready to teach any of these topics. if a group proposed other topics, i met with them during their planning to ensure that they presented accurate information, i let each group decide on their due dates, as long as each reflection was turned in prior to the next visit. in their reflections, students wrote what they did, what they learned from their preparation and visit, and what they planned to do differently next time. as i read reflections, i made sure each group member was participating and gave groups help when necessary. overall, students were very engaged in their projects, and i received very positive feedback from community partners. i tried to see each group in action once, and i was impressed by their enthusiasm when leading activities. students felt a greater sense of accountability for learning the material because they had to teach it to their audience and because they did not want to embarrass themselves in front of their group. coordinating and discussing service - learning projects took class time (on average, 1530 minutes per week), which left less time for traditional lectures and in - class activities. however, i compared student performance on exams and papers between semesters in which i did and did not use service learning via unpaired t - tests. there were no significant differences in any of the measures, indicating that students learned course concepts just as well when service learning substituted for other activities. my students consistently made positive comments on service - learning evaluations (also see fig. 1) : overall, i think the service - learning part of this class was way more beneficial to me than being in a lab each week. teaching someone else about the subject as i was learning helped me reinforce the information and retain it better overall. i feel like this is one of the most direct ways we can utilize the things that we learn in college instead of complaining how we ll never use any of the things we learned in college. the things that i learn today could spark the curiosity and minds of people who might grow up to find a cure for alzheimer s or parkinson s. 1 = strongly disagree, 2 = disagree, 3 = neutral, 4 = agree, 5 = strongly agree. because students selected and designed their own projects, they were very proud of themselves and took ownership of the projects. once students saw how much work it took to design the project and coordinate the logistics with the organization, they felt more accountability for preparing and being on time for each visit. one in particular had so much fun, despite her initial trepidation, that she planned and carried out an 11-week anatomy & physiology workshop at the museum. the disadvantages of giving students so much flexibility in their projects is that it requires a lot of coordination at the beginning of the semester to make sure that every student is finding a group and that groups are making plans. although these provided some initial frustrations, i never had a problem arise once the group wrote a strong proposal. the benefits of having students choose their projects and become fully engaged in them outweighed these minor annoyances.	one of the challenges in teaching a service - learning course is obtaining student buy - in from all students in the course. to circumvent this problem, i have let students in my undergraduate neurobiology course design their own service - learning projects at the beginning of the semester. although this can be chaotic because it requires last - minute planning, i have made it successful through facilitating student communication in the classroom, requiring thorough project proposals, meeting with students regularly, and monitoring group progress through written reflection papers. most of my students have strong opinions about the types of projects that they want to carry out, and many students have used connections that they have already made with local organizations. almost all projects that students have designed to this point involve teaching basic concepts of neurobiology to children of various ages while simultaneously sparking their interest in science. through taking ownership of the project and designing it such that it works well with their strengths, interests, and weekly schedule, students have become more engaged in service learning and view it as a valuable experience. despite some class time being shifted away from more traditional assignments, students have performed equally well in the course, and they are more eager to talk with others about course concepts. furthermore, the feedback that i have received from community partners has been excellent, and some students have maintained their work with the organizations.
recently, there has been a considerable increase in the number of hepatectomies and mortality rates reduced to less than 5%. nevertheless, surgical approach to centrally located tumours close to the confluence of the hepatic veins is still a challenging issue. trisectionectomies are considered the first option but are associated with high mortality rates due to post - operative liver failure. recent studies showed that liver parenchymal damage may play a role in tumour cells and their micro - environment. thus, major hepatectomies with intense post - operative regeneration or segmentectomies that leave parenchyma without adequate venous drainage may not be the best treatment for hepatic malignancies. we performed a segment 8 hepatectomy with resection of a segment of the right hepatic vein followed by immediate reconstruction of the vein with a gore hybrid vascular graft (w. l. gore & associates, flagstaff, az, usa). it allowed to preserve all but one of the hepatic segments and provided optimal venous outflow to remaining segments. this is the first time this technique is performed, and it is a much easier and safer method than other previously reported techniques. a 54-year - old male patient with no comorbidities presented with liver metastasis of colorectal adenocarcinoma. he had undergone right hemicolectomy 1 year before and received chemotherapy with folfox followed by bevacizumab. computed tomography (ct) disclosed a 4.0 2.0 cm lesion in segment 8 involving the entire circumference of the right hepatic vein (which was still partially patent), extending medially to the middle hepatic vein and posteriorly to the retrohepatic inferior vena cava (ivc) (fig. 1). figure 1:(a) and (b) preoperative ct showing displacement of the tumour and its relationship with the right and middle hepatic veins and the ivc. (a) and (b) preoperative ct showing displacement of the tumour and its relationship with the right and middle hepatic veins and the ivc. as the vein was still patent and there was a concern that a considerable amount of parenchyma could be left with insufficient drainage, we proposed to reconstruct the vein with a graft. the most challenging step of reconstructing the right hepatic vein with a graft is the anastomosis of the graft with the vein in the transected area. we decided to use the gore hybrid vascular graft because it allows a sutureless anastomosis at one end of the graft as it has a constrained section with a deployment line that allows easy insertion into the vessel. intraoperative ultrasound (us) was performed to define tumour margins and its relationship with vascular structures. the right hepatic vein was dissected through the loose space between the vena cava and the liver and taped. transection started at the middle hepatic vein until it was released from the tumour, and proceeded posteriorly to release the tumour from the ivc. the remaining parenchyma was transected and the right hepatic vein was skeletonized for a length of 1 cm to allow graft placement (fig. 2). subsequently, the open end of the graft was anastomosed in a standard manner to the ivc (fig. 3). figure 2:(a) right hepatic vein sutured close to the ivc. (d) retrohepatic ivc, area to be anastomosed to one end of the graft. figure 3:the open end of the graft being anastomosed to the ivc (the graft was previously cut to the proper length, approximately 5 cm). (d) retrohepatic ivc, area to be anastomosed to one end of the graft. the open end of the graft being anastomosed to the ivc (the graft was previously cut to the proper length, approximately 5 cm). the constrained section of the graft was introduced ~2 cm inside the vein and the deployment line was pulled (fig. 4). figure 4:(a) specimen, (b) right hepatic vein and (c) graft. (a) specimen, (b) right hepatic vein and (c) graft. figure 5:final appearance : (a) intraoperative appearance and (b) schematic drawing. patient spent 1 day on intensive care unit and was discharged from hospital on post - operative day 6. u / l at 5 hours after the procedure and 72 u / l at discharge. double antiaggregant therapy was initiated on the second post - operative day with clopidogrel (75 mg orally daily) and aspirin (100 mg orally daily). one month after the procedure, clopidogrel was interrupted and aspirin maintained as single - antiplatelet therapy. doppler us on post - operative day 2 showed normal blood flow in the graft. contrast - enhanced ct scans performed on post - operative day 5 and 3 months after the procedure showed a properly positioned and patent graft (fig. figure 6:ct scan performed on the fifth post - operative day : (a) axial view, no intravenous contrast ; (b) axial view, intravenous contrast inside the graft and (c) coronal view, graft filled with contrast. ct scan performed on the fifth post - operative day : (a) axial view, no intravenous contrast ; (b) axial view, intravenous contrast inside the graft and (c) coronal view, graft filled with contrast. hepatectomies have evolved. in recent years, the goal is to preserve as much hepatic segments as possible. however, we are entering a new era, in which not only the liver segments, but also the hepatocytes are the focus of attention. the intention is to produce as little damage as possible to hepatocytes, as liver regeneration may create a micro - environment favourable for tumour cell proliferation and have negative impact on oncological outcome [36 ]. in this context, preservation of hepatic veins is crucial. better understanding of the role of the inferior right hepatic vein and that segment 6 has multiple veins forming numerous anastomoses with surrounding hepatic veins allowed the resection of segments 7 and 8 including the right hepatic vein without its reconstruction [2, 810 ]. however, atrophy of segment 6 and liver dysfunction / failure occur in most cases [2, 10 ]. we decided to resect segment 8 with right hepatic vein reconstruction using a vascular graft that allowed easy deployment of the graft into the vessel. it ensured maximal preservation of functional parenchyma without congestion, limiting post - operative regenerative activity and parenchymal dysfunction (as judged by low aminotransferase levels), with possible oncological benefits. we described a novel technique that allows preservation of liver segments with natural venous drainage, limiting parenchymal dysfunction. it is an easy and reproducible method for reconstructing hepatic veins, which may allow this type of reconstruction to be performed more frequently.	surgical resection is the treatment of choice for malignant liver tumours. nevertheless, surgical approach to tumours located close to the confluence of the hepatic veins is a challenging issue. trisectionectomies are considered the first curative option for treatment of these tumours. however, those procedures are associated with high morbidity and mortality rates primarily due to post - operative liver failure. thus, maximal preservation of functional liver parenchyma should always be attempted. we describe the isolated resection of segment 8 for the treatment of a tumour involving the right hepatic vein and in contact with the middle hepatic vein and retrohepatic vena cava with immediate reconstruction of the right hepatic vein with a vascular graft. this is the first time this type of reconstruction was performed, and it allowed to preserve all but one of the hepatic segments with normal venous outflow. this innovative technique is a fast and safe method to reconstruct hepatic veins.
histoid leprosy is an uncommon variant of lepromatous leprosy characterized by cutaneous / subcutaneous nodules and plaques present over apparently normal skin, with unique histopathological findings, characteristic bacterial morphology and very high bacillary load. the term histoid leprosy was coined by wade as a histological concept of bacillary - rich leproma composed of spindle - shaped cells in the absence of globi (so conspicuous in an ordinary leproma). it occurs in lepromatous patients who relapse after the dapsone monotherapy and resistance, or even de novo. responsible factors may include resistance to dapsone, irregular and inadequate therapy, or mutant organism (histoid bacillus). he had no complains of fever, epistaxis or slippage of sandals. on examination, multiple shiny papulonodular lesions were found over his forehead, ears, and cheeks [figure. bilateral ulnar, greater auricular, common peroneal, anterior tibial, and posterior tibial nerves were thickened. on histopathological examination, sections showed extensive cellular infiltration in the dermis mainly composed of macrophages, lymphocytes and plasma cells. hematoxylin and eosin stain showed histiocytes arranged in a storiform pattern with classical histiocytic granulomas [figure. 2 ] and heavy bacillary load on wade fite stain. slit skin smear revealed acid fast bacilli with a bacteriological index of 6.[figure. 6 ] wade fite stain revealed numerous solid staining acid fast bacilli arranged discretely and in clumps inside the dermis, including the grenz zone, and surprisingly also inside the epidermis, inside vacuoles in the prickle cell layer and in the stratum granulosum.[figure. 35 ] the presence of bacilli at various levels inside the epidermis suggested their movement upward along with epidermal cell maturation. multiple shiny succulent papulonodular lesions over the forehead, cheeks, and chin histopathology showing extensive cellular infiltration in dermis mainly composed of macrophages, lymphocytes and plasma cells with histiocytes arranged in a storiform pattern with classical histiocytic granulomas wade fite stain revealed numerous acid fast bacilli arranged in clumps inside the epidermis wade fite stain revealed numerous solid staining elongated slender acid fast bacilli arranged discretely in vacuoles in the stratum corneum, prickle cell layer and in the stratum granulosum wade fite stain revealed numerous solid staining elongated slender acid fast bacilli arranged discretely and in clumps inside the epidermis and in in dermis slit skin smear showing numerous acid fast bacilli histoid leprosy presents with shiny, skin colored or erythematous papules, nodules or plaques arising abruptly over normal skin, mostly after inadequate or irregular treatment or because of dapsone resistant mutant strains, but may occur de novo. in a recent indian report, histoid leprosy was found to constitute 1.8% of all leprosy cases and occurred de novo in 12.5% of cases. there are scant reports of detection of mycobacterium leprae inside the leprous epidermis and they could be missed / underreported unless specifically looked for. suggested that dermal bacilli could be gradually transferred to the epidermal layers through phagocytic activity of young basal cells, and finally eliminated, possibly through the intact skin. transepidermal elimination of m. leprae in lepromatous leprosy and attributed it to rapidly growing dense granulomas in the upper dermis. the presence of bacilli in all layers of epidermis indicates that the bacilli that are taken up by the basal cells from the upper dermis can move upward inside the epidermal cells and are ultimately eliminated from the stratum corneum into the environment. the very large area of skin of about 1.62 m in an adult indian and the average turnover time of about 28 days seems to provide an opportune environment for bacillary multiplication and elimination, as even 1 g of lepromatous tissue is estimated to carry up to 7 billion bacilli. it is also known that the bacilli once eliminated could remain viable for several days or weeks. as shown in polymerase chain reaction studies, 60% of untreated mycobacterium patients had bacilli in the keratin layer, and 80% had m. leprae deoxyribonucleic acid in skin washings. interestingly, on the other hand, the reports of initial / single leprosy lesions developing at the site of tattooing, vaccination scar or trauma suggesting that the bacilli could gain entry through traumatized skin. the recent report of linearly distributed skin lesions suggesting pseudo - isomorphic phenomenon of koebner in a histoid leprosy patient points toward cutaneous inoculation of the bacillus. dendritic cells are antigen - presenting cells that phagocytose microorganisms and particles and are represented by langerhans cells in the epidermis. they have a role in the cutaneous response to leprosy, which may be a determinant of the course and clinical expression of the disease. future studies on their contribution, if any, to the carriage of and response to the epidermal bacilli might help unravel a few unexplored aspects of leprosy. the transepidermal exit of the m. leprae demonstrated in this case, along with previous reports mentioned above, indicate that skin could possibly be a portal for both exit and entry of the bacillus causing the occurrence of pseudo - isomorphic koebner phenomena and should be given due recognition for its contributory role in leprosy transmission. suggested that upper dermal lepra bacilli in multibacillary patients could be phagocytosed by basal cells and gradually move up through the epidermis to be finally eliminated, possibly also from intact skin.	histoid leprosy is a rare form of multibacillary leprosy with distinct clinical and histopathological features. it is a variant of lepromatous leprosy with a very high bacillary load. it appears in patients as relapse after dapsone monotherapy and resistance or rarely, de novo. although leprosy is slowly declining the exact mode of transmission is unclear. at least until recently, the most widely held belief was that the disease was transmitted by contact between cases of leprosy and healthy persons. transmission by the respiratory route is also gaining ground. there are other possibilities such as transmission through insects, which can not be completely ruled out. however, the present case report possibly suggests the role of skin as a portal of both exit and entry for the bacillus in histoid leprosy transmission. de novo form of histoid leprosy has numerous solid staining bacteria inside the epidermis. the reports show that these bacilli can be eliminated from the intact epidermis, which indicate an unusual role of the skin in the transmission of leprosy.
prostate cancer is the most frequently diagnosed solid organ cancer in men and is the second most common cause of cancer death. psa screening and testing has been shown to reduce mortality from prostate cancer and increase life expectancy. however, this has caused a tendency towards over - detection of low risk prostate cancer and, as a result, treatment of both indolent and aggressive prostate cancer. radical treatment is associated with significant morbidity, predominantly with incontinence and erectile dysfunction ; as such, it is vital that patients are accurately staged and appropriately counselled before making their treatment decision. currently, investigations for those with elevated psa revolve around transrectal ultrasound guided prostate biopsies. this involves taking 12 biopsy cores in a systematic approach as supported by aua and eau guidelines [4, 5 ]. this is due to the risk of seeding intestinal flora into the prostate and the limited antibiotic penetration of prostatic tissue. the risk of major infection has been reported as high as 5.5% in some studies. another limitation is the relatively high proportion of false negative results that occur with transrectal biopsies, having been shown to miss up to 30% of prostate cancers. even if prostate cancer is detected on transrectal biopsy, a significant proportion is upgraded after template biopsy or after histological analysis of the radical prostatectomy specimen [8, 9 ]. this is due to the complex, heterogeneous and multifocal nature of prostate cancer, as well as the challenging aspect of sampling the anterior part of the prostate transrectally. as a result, there may be a proportion of patients who have been falsely reassured by being diagnosed with low - grade disease and thus chose their treatment based on incomplete information. transperineal template - guided prostate biopsies (template biopsies) have been shown to overcome some of these limitations. this is due to the greater number of biopsy cores taken, and due to the fact that the anterior portion of the gland is accessed and biopsied with greater certainty. as a result subsequently, the rate of detection of template biopsies in those who have previously had a negative transrectal biopsy has been reported to be as much as 39%. this study aims to evaluate the use of transperineal template - guided prostate biopsy presently in use at our unit. we also aim to investigate the detection rate of prostate cancer in those with previously negative transrectal biopsies and the upgrade rate of those on active surveillance for gleason 3 + 3 = 6 prostate adenocarcinoma following transperineal template - guided prostate biopsy. all data from patients having undergone a transperineal template - guided prostate biopsy were prospectively collected over a 22-month period. these scans were performed and reported in a manner consistent with the standards and recommendations approved by a european consensus meeting using the prostate imaging reporting and data system (pirads) score (table 1). patients who had previously received brachytherapy or had undergone template biopsies for anorectal abnormalities were excluded. pi - rads score as adapted from the esur mr guidelines 2012 transperineal template - guided prostate biopsies were performed as ambulatory day - case surgery under general anaesthesia by a consultant urologist. patients were given prophylactic antibiotics (gentamicin 240 mg intravenously and metronidazole 500 mg rectally) at induction of anaesthesia and placed in the dorsal lithotomy position. multiple transperineal prostatic biopsy cores were taken in a systematic fashion using a 5 mm brachytherapy template grid as previously described. the total number of cores varied with the size of the prostate ; however, a minimum of 24 named sectors were biopsied. four regions were sampled anteriorly and four regions were sampled posteriorly from each of 3 transverse planes (figure 1). if the prostate was found to have a large volume and further sampling was needed, then further regions were sampled from a fourth transverse plane at the surgeon 's discretion. schematic of the prostate identifying the three transverse planes and the four sagittal planes that make up the 24 named sectors that are biopsied. transperineal template - guided prostate biopsies were performed as ambulatory day - case surgery under general anaesthesia by a consultant urologist. patients were given prophylactic antibiotics (gentamicin 240 mg intravenously and metronidazole 500 mg rectally) at induction of anaesthesia and placed in the dorsal lithotomy position. multiple transperineal prostatic biopsy cores were taken in a systematic fashion using a 5 mm brachytherapy template grid as previously described. the total number of cores varied with the size of the prostate ; however, a minimum of 24 named sectors were biopsied. four regions were sampled anteriorly and four regions were sampled posteriorly from each of 3 transverse planes (figure 1). if the prostate was found to have a large volume and further sampling was needed, then further regions were sampled from a fourth transverse plane at the surgeon 's discretion. schematic of the prostate identifying the three transverse planes and the four sagittal planes that make up the 24 named sectors that are biopsied. a comparison between patients with positive template biopsy results and negative template biopsy results is presented on table 2 (excluding those on active surveillance and those post - radiotherapy). there is no significant difference between demographic data ; however, the mri results indicate that a positive template biopsy is associated with a significantly greater pirads score than negative template biopsies (p 50 ml had an aur rate of 20% (p = 0.039). we found that the use of a single dose of tamsulosin 400 mcg at the time of template biopsy significantly reduced the rate of aur, with an nnt score of 13 to prevent one episode of retention. the use of tamsulosin has already been shown to help improve international prostate symptom scores (ipss) and maximum flow rates in transrectal biopsies in a randomized prospective study. our data shows that a single dose can also be used to help reduce the rate of aur in template biopsies and so improve morbidity. another indication for the use of template biopsies is to provide evidence of unifocal disease. if this is the case, then the patient can be considered for focal therapy. due to the complex, heterogeneous and multifocal nature of prostate cancer, focal therapy relies on accurate patient selection. as a result, however, according to the national institute of clinical excellence (nice) guidelines and the european association of urologists (eau) guidelines, cryotherapy can only be offered in those who are unsuitable for surgery or radiotherapy, furthermore high - intensity - focused ultrasound (hifu) should only be offered as part of a trial. consequently, our unit did not look into this indication for template biopsies and further studies would need to be performed to evaluate this further. our unique series complements previously published data on transperineal template - guided prostate biopsies, showing it to be safe and accurate. our study suggests that due to the relatively high detection rate one must consider template biopsies after one negative transrectal biopsy if there is clinical suspicion. in addition, those with gleason 3 + 3 = 6 prostate cancer who are considering active surveillance should be offered template biopsies so more accurate staging can be done and appropriate counselling performed. however, to counter the increased risk of aur we have found a single dose of tamsulosin to be beneficial. all procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee, and with the 1964 helsinki declaration and its later amendments or comparable ethical standards.	introductionwe aim to present transperineal template - guided prostate biopsy (template biopsy) outcomes at a tertiary referral centre. furthermore, to identify the detection rate of prostate cancer in those with a previous negative transrectal ultrasound guided prostate biopsy and the upgrade rate of those on active surveillance for gleason 3 + 3 = 6 prostate adenocarcinoma.material and methodswe conducted a prospective study of 200 consecutive men who underwent template biopsy over a 22-month period in a tertiary referral centre, using a standard 24 region template prostate biopsy technique. indications and histology results, as well as complications, were recorded.resultsmedian age was 67 years and median psa was 10 ng / ml. overall detection rate was 47%. 39.5% of cases with previous negative transrectal biopsies were found to have prostate adenocarcinoma. 47.5% of cases on active surveillance for gleason 3 + 3 = 6 prostate adenocarcinoma were upgraded. the most frequent complication was acute urinary retention at a rate of 12.5%, however, the use of a single prophylactic dose of tamsulosin was found to be beneficial, with 13 cases needed to treat to prevent one episode.conclusionstemplate biopsies are safe and efficacious with an overall detection rate of 47% in the present series. due to the high detection rate, one must consider template biopsy following one negative transrectal biopsy where there is persistent clinical suspicion. furthermore, those considering active surveillance for gleason 3 + 3 = 6 disease should be offered template biopsy to confirm the grade of their disease.
fat at birth is unique to humans among primates, suggesting that a recently evolved genetic component accounts for this difference. body fat accumulated during intrauterine development in humans is thought to serve as an energy source to support growth of the large human newborn brain in the early neonatal period. as such, subcutaneous however, high or low body fat at birth is associated with increased morbidity in the first year of life as well as an increased susceptibility to poor metabolic and/or cardiovascular health later in life. while maternal fuels such as glucose and triglycerides impact fetal fat accretion, genetic factors also contribute. a locus between ccnl1 and lekri on chromosome 3 (3q25.31) was previously shown to be associated with birthweight, but our recent genome - wide association study (gwas) performed in a multi - ancestry cohort of newborns revealed this locus was much more strongly associated with newborn sum of skinfolds (p=1.52 10), a measure of newborn adiposity, than with birthweight (p=5.72 10). the goal of the present study was to determine whether this same locus is associated with adiposity at later points during childhood. the hyperglycemia and adverse pregnancy outcome (hapo) study was an international multi - center epidemiologic study that examined levels of maternal glucose tolerance and risk of adverse pregnancy outcomes. briefly, eligible women underwent a 75 g oral glucose tolerance test between 24 and 32 weeks gestation. using standardized methods and procedures, maternal height, weight, and blood pressure cord blood was collected for measurement of newborn glucose, c - peptide and dna extraction. using standardized methods and equipment, neonatal anthropometric measurements were obtained within 72 h of birth, including head circumference, birth length, birthweight and skinfold thickness measured at the flank, triceps and subscapular regions. hapo participants and their offspring from the belfast, northern ireland center were invited to participate in the hapo family study in which offspring were examined at the ages of 2 and 6 years. measurements included weight, height, head circumference, mid - arm circumference, and triceps, subscapular, and suprailiac skinfold thickness. for genetic analyses, individual skinfold measurements and their sum examining newborn size, hapo newborns from four ancestries were genotyped using illumina genomewide single nucleotide polymorphism (snp) arrays (illumina, inc., san diego, ca, usa). details of genotyping platform selection, genotype calling algorithms, quality control and imputation procedures, and estimating population substructure were previously reported. we examined the association of four snps at this locus with sum of skinfolds, a measure of adiposity, among belfast hapo offspring at 2 and 6 years of age using linear regression analyses (snptestv2) with an additive model, adjusting for ancestry (principal components 1 and 2), newborn gender, gestational age at delivery, parity, and maternal age, body mass index (bmi), height, mean arterial blood pressure, smoking status and alcohol intake at the time of oral glucose tolerance test. all other analyses were conducted in sas 9.3 (sas institute, cary, nc, usa). genotype and phenotype data were available at birth and age 2 for 293 children and at birth and age 6 for 350 children (table 1) ; 270 children were common between the two cohorts. four snps at the chromosome 3q25.31 locus that are in strong linkage disequilibrium (lowest pairwise r=0.93), rs10049088, rs1482853, rs17451107 and rs900400, were previously shown to be the snps most highly associated with sum of skinfolds in a cohort of hapo newborns. the association and direction of effect between one of these snps, rs900400, and sum of skinfolds as well as individual skinfold thicknesses in the cohorts of 2- and 6-year - olds from the belfast hapo family study are shown in table 2. a positive association was observed between rs900400 and newborn sum of skinfolds among the 293 children in the cohort of 2-year olds (p=4.8 10 ; =0.025), and 350 children in the cohort of 6-year olds (p=2.8 10 ; =0.024). in contrast, at age 2 years and 6 years no association was observed between sum of skinfolds and rs900400 (p=0.06, =0.015 at age 2 ; p=0.86, = 0.002 at age 6). although rs900400 demonstrated a marginal, albeit non - significant, association with sum of skinfolds at age 2 years, the direction of effect was opposite to that observed in the newborns. similar analyses were performed examining the association of rs900400 with measurements of the flank / suprailiac, triceps and subscapular skinfolds (table 2). a positive association was observed between rs900400 and individual skinfold measurements at birth among the 293 children in the cohort of 2-year olds (triceps, p=3.4 10 ; subscapular, p=2.0 10 ; flank, p=5.0 10), and 350 children in the cohort of 6-year olds (triceps, p=1.8 10 ; subscapular, p=5.0 10 ; flank, p=1.0 10). for the suprailiac and subscapular skinfold thicknesses, an association with rs900400 was not seen at either age 2 (p=0.14 and p=0.52, respectively) or age 6 (p=0.70 and p=0.98, respectively). however, in the cohort of 2-year olds, rs900400 was weakly associated with triceps skinfold thickness at age 2 (p=0.03).. there was no evidence for association of rs900400 with triceps skinfold thickness at age 6 (p=0.97). excess adiposity at birth, which is recognized as a risk factor for peri- and postnatal complications as well as childhood obesity and adverse metabolic health outcomes during adulthood, is influenced by both environmental and genetic factors. multiple maternal factors influence the intrauterine environment, including maternal bmi, height, blood pressure, nutrition, alcohol consumption, smoking and socioeconomic background, with maternal metabolic factors also having an important impact on fetal adiposity. in addition to maternal metabolism, fetal genetics also plays an important role in fetal fat accretion. we and others have previously used gwas to identify genes associated with birthweight and/or newborn sum of skinfolds. a highly significant and reproducible finding was the association of snps within the chromosome 3q25.31 locus with newborn adiposity as reflected by newborn sum of skinfolds or ponderal index. in this report, we now demonstrate that association of this locus with measures of adiposity is no longer evident at ages 2 and 6 years, consistent with the recent observation that this locus is no longer associated with birthweight by age 2 months. association of this locus with sum of skinfolds, driven primarily by skinfold thickness at the triceps, approached significance in the cohort of 2-year olds, but this association was in the opposite direction to that observed in newborns. in the cohort of 6-year olds, there was no evidence of association of rs900400 with any of the individual skinfolds. together with the results of previous studies, these new findings suggest that genetic variation within the 3q25.31 locus contributes to fetal fat accretion but not to early childhood adiposity. as in newborns, both low and normal birthweight newborns with rapid postnatal weight gain during the first 2 years of life have higher body fat and lower lean body mass and, during adulthood, higher body weight and fat. prevention of rapid postnatal growth in such individuals may prevent development of an obese adult phenotype. genetic variation is another important determinant of childhood adiposity. through gwas and related approaches, association of variants within 22 different genetic loci with childhood bmi or body weight has been demonstrated, but snps in the chromosome 3q25.31 locus have not been reported. consistent with that, meta - analyses performed by the early growth genetics (egg) consortium (www.egg-consortium.org) in european ancestry children failed to demonstrate association of rs900400 with childhood obesity (defined as bmi 95 percentile ; p=0.90). similarly, a previous analysis of data from the giant consortium showed no evidence of association of the 3q25.31 locus with adult bmi. despite the presence of high - quality genotype and phenotype data, our study had certain limitations. we have examined a small cohort of offspring from only one hapo ancestry and, as a result, our power to demonstrate an association at age 2 and 6 years was limited. thus, we can not rule out an association of snps at the chromosome 3q25.31 locus with early childhood adiposity ; however, if this locus is associated with childhood adiposity, the association is much weaker than that observed during fetal life and does not alter our conclusion that the chromosome 3 locus primarily influences fetal fat accretion. similarly, we were not able to assess directly whether this locus is associated with measures of obesity in older children and adolescents, but previous studies of the genetics of childhood obesity have failed to demonstrate an association. in summary, we have demonstrated that snps within the chromosome 3q25.31 locus, which exhibit strong association across multiple ancestry groups with measures of newborn adiposity, do not have a clear association with early childhood adiposity among european ancestry hapo offspring.	increased newborn adiposity is associated with later adverse metabolic outcomes. previous genome - wide association studies (gwas) demonstrated strong association of a locus on chromosome 3 (3q25.31) with newborn sum of skinfolds, a measure of overall adiposity. whether this locus is associated with childhood adiposity is unknown. genotype and sum of skinfolds data were available for 293 children at birth and age 2, and for 350 children at birth and age 6 from a european cohort (belfast, uk) who participated in the hyperglycemia and adverse pregnancy outcome gwas. we examined single nucleotide polymorphisms (snps) at the 3q25.31 locus associated with newborn adiposity. linear regression analyses under an additive genetic model adjusting for maternal body mass index were performed. in both cohorts, a positive association was observed between all snps and sum of skinfolds at birth (p=2.3 104, =0.026 and p=4.8 104, =0.025). at the age of 2 years, a non - significant negative association was observed with sum of skinfolds (p=0.06 ; = 0.015). at the age of 6 years, there was no evidence of association (p=0.86 ; =0.002). the 3q25.31 locus strongly associated with newborn adiposity had no significant association with childhood adiposity suggesting that its impact may largely be limited to fetal fat accretion.
little is known about longus colli tendinitis (lct), a rare inflammatory condition believed to be caused by the deposition of calcium hydroxyapatite crystals in the longus colli muscle. also known as calcific tendinitis of the longus colli muscle or retropharyngeal tendinitis, lct is characterized by the acute presentation of severe neck pain, dysphagia, torticollis, and/or a painful restriction of neck movement. its presentation may resemble more serious conditions requiring immediate treatment, such as meningitis, retropharyngeal abscess, foreign body aspiration, or traumatic injury. patients also typically present with mild leukocytosis, which should prompt investigation to first rule out infectious etiologies. characteristic radiographic findings of the neck include calcifications in the superior longus colli muscle tendons at the level of c1 or c2, which confirm the diagnosis. the incidence of lct has been reported to be 1.31 per 100,000 person - years but may actually be higher due to the belief that the disorder is underdiagnosed. therefore, we would like to make practitioners aware that lct is an important etiology for neck pain that must always be included in the differential diagnosis for acute neck symptomatology. a 44-year - old female patient with a history of gastric ulcers, gastritis, gastric polyps, schatzki ring, and hiatal hernia presented with the complaint of progressive dysphagia to solid foods. this dysphagia first developed after an incident of food aspiration 4 weeks prior to presentation. three weeks after this event, she experienced persistent odynophagia with left - sided occipital headaches and neck pain radiating to the left ear and mastoid process. physical examination was remarkable for decreased neck range of motion with flexion and extension and bilateral neck soft tissue tenderness to palpation. after an infectious etiology had been ruled out, an upper gastrointestinal series with barium swallow confirmed the patient 's known schatzki ring and hiatal hernia. a ct scan, mri, and mra of the brain were negative for abnormalities ; however, a ct scan of her neck was positive for calcified longus colli tendons just interior to the anterior ring of her c1 vertebrae (fig 1, fig 2). the patient underwent a trial of oral nonsteroidal anti - inflammatory drugs (nsaids) and corticosteroids that quickly improved her dysphagia and odynophagia, which soon also resolved. this case presentation emphasizes the importance of having a vast differential diagnosis for relatively common complaints, such as neck pain and dysphagia. it is important to carefully contemplate these differentials in order to not misdiagnose less common yet benign etiologies for these complaints, such as lct. the longus colli muscle is a prevertebral muscle of the anterior neck that consists of a superior oblique, inferior oblique, and vertical portion. the longus colli muscle assists in cervical flexion and ipsilateral side - bending and rotation. the mean annual crude incidence of lct was found to be 0.50 cases per 100,000 person - years, with a standardized incidence of 1.31 for the age - matched population. it most commonly affects both males and females between the ages of 30 and 60 years, as seen in our 44-year - old female. the pathophysiology behind lct is explained by a reactive inflammatory process induced by calcium hydroxyapatite crystal deposition on the longus colli tendon. these calcifications, which are usually present in the superior oblique portion of the longus colli muscle at the c1c2 levels, are the cause of symptom development. the most common presenting symptoms are neck pain (94%), limited neck range of motion (45%), odynophagia (45%), neck stiffness (42%), dysphagia (27%), sore throat (17%), and neck spasm (11%). our patient displayed all of these signs and symptoms except for sore throat and neck spasm. laboratory findings typically include mildly elevated white blood cell count, erythrocyte sedimentation rate, and c - reactive protein, but this case featured only an isolated white blood cell count of 11.88. ct is the gold standard for diagnosing lct, as it is readily available and can detect both prevertebral edema and calcium hydroxyapatite crystal deposition at the longus colli tendon [4, 7 ]. mri can show prevertebral edema and corresponding effusion, but it is difficult to recognize calcific deposits [4, 7, 8 ]. this patient demonstrated longus colli calcifications in the anterior ring of the c1 vertebrae found on ct. the differential diagnosis between lct and other diseases showing similar clinical features includes meningitis, retropharyngeal abscess, neoplasm, cervical disc herniation, and traumatic fracture dislocation. the most important mimickers of lct are meningitis and retropharyngeal abscess [4, 7 ]. it is critical to thoroughly work up the case in order to make a correct initial diagnosis and avoid unnecessary interventions. unlike meningitis and retropharyngeal abscesses, which are often associated with high morbidity, antibiotics use, and surgical intervention, lct is a benign, self - limiting disease. nsaids are the first - line treatment for lct and often alleviate symptoms, but in severe cases, corticosteroids and opioids may be necessary to abate acute symptom exacerbations [4, 6, 8 ]. symptoms typically resolve within a few days of treatment initiation, and the patient often becomes symptom - free after 13 weeks [6, 9 ]. our patient required both oral nsaids and corticosteroids, which quickly improved her dysphagia and odynophagia within 1 day of treatment initiation. lct is a rare, unusual clinical entity believed to be underdiagnosed due to its often nonspecific presentation. increased awareness of the disease and its progressive presentation would perhaps yield more timely identification to avoid diagnostic errors and unnecessary patient and physician anxiety. therefore, it is imperative that practitioners be aware of lct as an important etiology for various presentations of unexplainable neck pain, dysphagia, and odynophagia. dominic m. colella and fiorela caldern sandoval wrote the manuscript, david w. powers and nimal patel revised the manuscript, and javier sobrado critically revised the manuscript and is the guarantor.	longus colli tendinitis (lct) is an acute inflammatory condition with symptoms typically consisting of acute neck pain and stiffness with or without dysphagia. once more severe etiologies for these symptoms are ruled out, this self - limiting condition usually resolves spontaneously with nonsteroidal anti - inflammatory drugs and corticosteroids. we present a case of lct that presented as acute neck pain, dysphagia, and odynophagia that rapidly resolved once diagnosed and treated with anti - inflammatory agents. though exceedingly rare, lct must be considered in the differential diagnosis of acute neck pain, dysphagia, and odynophagia when more common etiologies do not correlate with the clinical presentation.
the stratus td - oct had a scanning rate of 400 a - scans per second and generated a standard scanning pattern that consisted of six radial, concentric, 6-mm - long b - scans centered on the fovea and offset by 30. image quality often depended on the cooperation of the patient and the skill of the operator, since tracking was not yet available and higher - density scans took longer to acquire. over the past 15 years as oct technology has evolved, td - oct has given way to spectral - domain oct (sd - oct). spectral - domain oct, a type of fourier - domain oct (fd - oct) with scanning rates of approximately 27,000 to 70,000 a - scans per second, resulted in denser, more varied scanning patterns, better image quality, better reproducibility, and large volumetric datasets that could be viewed using both the traditional cross - sectional approach and an en face approach. compared with the more advanced sd - oct technology now available in the clinics, the capabilities of the stratus td - oct instrument now seem primitive, yet at the time, the images obtained using td - oct imaging provided a view of macular neovascularization (mnv) and exudation that would forever change the way we managed patients. the b - scan image of a normal macula shows layers of differing reflectivity that approximate the major cellular layers of the retina and choroid. however, features of the choroidal anatomy were poorly visualized using td - oct imaging. thus, by using td - oct, it was possible to obtain a crude optical biopsy of the macula and distinguish between a normal retina / rpe contour and an abnormal one, which was all that was needed for the retinal specialist to identify exudation. moreover, one of the most commonly used quantitative parameters derived from oct datasets was the retinal thickness measurement, which was obtained by segmenting the internal limiting membrane (ilm) and a boundary representing the rpe. this information was used to generate retinal thickness maps, and these maps proved useful for identifying and describing deviations from normal anatomy and how these deviations changed over time. however, it was the viewing of individual b - scans that provided the retina specialist with a powerful strategy for following disease progression and response to treatment. while it came as no surprise that oct imaging was extremely useful for detecting the macular edema expected in cases of diabetes and retinal vein occlusions, it was a surprise to most retina specialists that intraretinal fluid was commonly found as an early and prominent component of the exudation associated with neovascular age - related macular degeneration (nvamd). the classification system previously developed for nvamd was based on leakage patterns seen on dye - based angiography, and this leakage was thought to result in the accumulation of fluid under the retina and under the rpe. this made intuitive sense since the neovascularization was thought to arise under the retina from the choroidal circulation, so why would there be fluid in the retina ? in retrospect, the detection of intraretinal fluid turned out to be one of the most novel and important observations made when using td - oct for imaging nvamd. at the time, one explanation for the presence of intraretinal edema was that the subretinal exudation had extended into the retina, but the extension of subretinal fluid into the retina was not commonly seen in other conditions associated with subretinal fluid such as central serous chorioretinopathy (cscr) and retinal detachment. moreover, eyes with early cscr and subretinal fluid, prior to the onset of fibrosis and photoreceptor disorganization, tended to have good best - corrected visual acuity while eyes with nvamd and similar amounts of subretinal fluid always seemed to have worse vision due to the intraretinal fluid. an alternative explanation for the intraretinal exudation was that this fluid came from established mature retinal vessels, and the exudation was in response to vegf. after all, intraocular injections of vegf into primate eyes had previously been shown to cause retinal edema and hemorrhages. thus, the presence of macular fluid came to be appreciated as a useful surrogate measure for the presence of excess vegf. another explanation for intraretinal fluid was that the neovascularization could be arising from within the retina, and this neovascularization could be directly responsible for the intraretinal macular fluid. in the early days of oct, this hypothesis seemed far - fetched, but now we know that type 3 mnv (known as retinal angiomatous proliferation) can arise from within the retina and is associated with intraretinal edema. while type 3 mnv accounts for a minority of neovascular lesions and macular edema is a common feature of all mnv, the most likely explanation for the presence of intraretinal fluid is that vegf induces exudation from the mature retinal vessels in nvamd. thus, lessons learned from early oct imaging of nvamd included an appreciation that intraretinal fluid was one of the first signs of vegf - mediated exudation (figs. late fluorescein angiographic (fa) images and time - domain optical coherence tomography (oct) diagonal b - scans from a patient treated with intravitreal ranibizumab in the phase ii study, then followed in the extension study with observation and no additional retreatments with ranibizumab. (c) after eight injections over 140 days, the fa leakage had significantly diminished, and va improved to 20/80. (e) after an additional 22 months without any ranibizumab injections, va improved to 20/32 and there was no evidence fa leakage. late fluorescein angiographic (fa) images and time - domain optical coherence tomography (oct) diagonal b - scans from a patient treated with intravitreal ranibizumab in the phase ii study, then followed in the extension study with observation and retreatment with ranibizumab. (a, c, e, g, i, k) late fa images. (b, d, f, h, j, l) diagonal oct b - scans. (c) two weeks later, va improved to 20/160, and the fa image showed significant reduction in leakage with focal areas of blocked fluorescence at the margin of the lesion consistent with hemorrhages. (d) corresponding oct b - scan with significant resolution of macular edema 2 weeks after the first injection. (e) after 140 days and 10 injections from baseline, va improved to 20/100, and late fa image showed no leakage with near - complete resolution of the hemorrhages. (f) corresponding oct b - scan showed resolution of the macular edema with evidence of macular atrophy as seen by the hypertransmission of light into the choroid (arrow). (g) after 10 months without treatment, va was stable at 20/100, but the late fa image showed minimal leakage. (h) corresponding oct b - scan showed the appearance of small intraretinal cysts indicative of recurrent macular fluid (i) after 4 months without treatment, va remained unchanged at 20/100 but the patient was complaining of increasing distortion. (j) corresponding oct b - scan showed increased macular edema, and a ranibizumab injection was performed. (k) five months after retreatment, va improved to 20/63 and the fa image showed no leakage. (l) corresponding oct b - scan showed minimal macular thickening nasal to the fovea. overall, oct - guided retreatment improved va, decreased fa leakage, and decreased the macular fluid. not surprisingly, oct imaging also revealed that macular fluid accumulated in the space between the rpe and the neurosensory retina. serous exudate appeared on oct imaging as a homogeneous hyporeflective space, while proteinaceous exudation appeared less homogeneous and was shown to contain reflective fibrinous membranes (fig. this proteinaceous material has been referred to as subretinal hyperreflective material (shrm) or subretinal hyperreflective material exudate (shre) and is different from the hyperreflective fibrosis that can form in more advanced neovascular lesions. the shrm / shre of particular concern is the material that accumulates between the retina and rpe as seen on oct imaging (fig. neovascular lesions with shrm / shre have been associated with worse visual acuity (va) outcomes due to the formation of fibrotic scars and photoreceptor atrophy. usually, type 2 mnv, which grows in the subretinal space, has been shown to be associated with a larger volume of subretinal fluid compared with sub - rpe lesions or type 1 mnv. while these type 1 neovascular lesions have been associated with less subretinal fluid, they are associated with detachments of the rpe known as retinal pigment epithelial detachments (peds) (figs. 4, 5), and early on, oct imaging was shown to be a useful technique for identifying and characterizing peds. while the presence of a ped and blood was shown to serve as a poor prognostic sign in neovascular amd, an even worse development, often associated with hemorrhage, was the formation of a rpe tear. optical coherence tomography imaging of rpe tears was shown to reveal an area of rpe discontinuity with the free edge of the rpe often curled under the ped. adjacent to the tear, oct imaging has revealed areas with increased choroidal reflectivity with improved visualization of choroidal vessels due to the absence of the rpe. typically, the overlying retina was found to be intact, but could be separated from the area of atrophy by subretinal fluid. late fluorescein angiographic (fa) images and time - domain optical coherence tomography (oct) diagonal b - scans from a patient treated with intravitreal ranibizumab in the phase ii study, followed in the extension study by observation and then retreatment with ranibizumab. (a, c, e, g, i, k) late fluorescein angiographic images. (b, d, f, h, j, l) diagonal oct b - scans. (b) corresponding baseline oct image showed subretinal fluid (arrow) and a retinal pigment epithelial detachment (ped, arrowhead). (c) after 140 days and six ranibizumab injections, va improved to 20/50, and the fa image showed a marked reduction in leakage. (d) corresponding oct b - scan showed resolution of the subretinal fluid with a residual low - lying ped (arrowhead). (e) after 3 months without an injection, va decreased to 20/100, and the fa image showed a subtle increase in leakage. (g) three months after the ranibizumab injection, va improved to 20/80, and the fa image showed a marked decrease in leakage. (h) corresponding oct b - scan showed only small intraretinal cysts, and no injection was given. (i) after 6 months without treatment, va slowly decreased to 20/100 and the patient then complained of distortion. (k) over the next 12 months, the patient received two more ranibizumab injections, and the va improved to 20/50. (l) corresponding oct b - scan showed marked improvement in the macular fluid. macular neovascularization (mnv) with subretinal hyperreflective material / exudate (shrm / shre). (a, c, e) horizontal spectral - domain optical coherence tomography (sd - oct) b - scans. (a, b) patient with mnv secondary to age - related macular degeneration was seen in clinic and found to have macular fluid on sd - oct imaging. visual acuity ((c, d) two weeks later, the patient returned complaining of decreased vision. sd - oct imaging showed increased macular fluid and increased shrm / shre (arrows). the first injection of a vascular endothelial growth factor (vegf) inhibitor was given. (e, f) one month after the third of three monthly injections, va improved to 20/30 with resolution of the macular fluid and srhm / shre. a fourth anti in all these examples of macular fluid, whether the fluid was in the retina, under the retina, or under the rpe, oct imaging was the ideal technique for detecting the excess fluid and determining whether it had changed. since most of the macular fluid occurred in response to excess vegf, the stage was now set for the convergence of an ideal imaging technique with an ideal treatment that would inhibit exudation and reduce the accumulation of fluid. at the same time that the stratus oct was introduced into the clinic, the first vegf inhibitor, previously known as rhufabv2 and now known as ranibizumab (lucentis ; genentech / roche, south san francisco, ca, usa), entered into a phase i clinical trial. in this phase i single - injection, dose - escalation, safety trial, oct imaging was not available and fa imaging was used to characterize the neovascular lesions only at baseline. from this phase i trial, a maximum tolerated dose of 0.5 mg was identified based on dose - limiting inflammation that occurred at the 1.0-mg dose. while safety and va outcomes were encouraging, it was n't until the phase ii study that oct imaging was first used at the bascom palmer eye institute. one phase ii trial studied the safety and tolerability of monthly repeated dosing of 0.3- or 0.5-mg ranibizumab through 6 months, and the second phase ii trial explored a dose - escalation regimen in which the ranibizumab dose was sequentially increased from 0.3 to 2 mg and injected as frequently as every 2 weeks in eyes with nvamd. while both phase ii trials demonstrated va improvement associated with ranibizumab therapy, oct imaging unambiguously demonstrated that the improvement in va was correlated with the resolution of macular fluid (rosenfeld pj, unpublished data, 2003). moreover, in an extension program at the end of the phase ii studies, retreatment with ranibizumab was permitted at the investigators ' discretion once the fixed dosing was stopped. investigators could base their retreatment on changes in va, fa imaging, or oct imaging. as these patients were being followed off treatment, oct imaging identified the earliest recurrence of macular fluid in some patients even before fa imaging detected leakage and before va was affected (figs. some patients needed no additional treatment for up to 2 years while some patients frequently developed recurrent macular fluid. the fluid that was initially detected by oct imaging, if untreated, would then increase over time, resulting in subsequent vision loss. thus, a small amount of fluid led to even more fluid and served as a harbinger of disease progression. as the macular fluid gradually reaccumulated in these subjects in the extension study, retreatment with ranibizumab once again resulted in resolution of the macular fluid ; and in those patients with decreased va associated with the recurrent fluid, va improved once the fluid resolved. these were the first observations of how oct imaging of macular fluid could serve as a surrogate marker for the presence of excess vegf, how oct imaging unambiguously demonstrated the profound effect of anti - vegf therapy on macular fluid, and how oct monitoring of macular fluid might serve as a strategy to determine the appropriate dosing interval used for the clinical management of nvamd. these data were presented at the retina subspecialty day before the american academy of ophthalmology meeting in 2004 and at other scientific meetings in an attempt to convince skeptical colleagues that oct imaging rather than fa imaging could be used in the management of nvamd patients. to convincingly demonstrate the benefits of oct imaging for managing nvamd patients, genentech supported an investigator - sponsored trial known as the prospective oct imaging of patients with neovascular amd treated with intra - ocular ranibizumab (pronto) study. in the 2-year pronto study, nvamd patients received three monthly injections of 0.5-mg ranibizumab and continued to receive monthly injections as long as there was persistent macular fluid based on oct imaging. however, once oct imaging revealed a fluid - free macula, patients were followed monthly without injection until at least one of several retreatment criteria were met. these criteria included a va loss of at least five letters with oct evidence of fluid in the macula, an increase in oct central retinal thickness of at least 100 m, new macular hemorrhage, or a new area of classic mnv identified by fa imaging. the purpose of these criteria was to determine if oct imaging was the most sensitive strategy for detecting the recurrence of macular fluid. after the first year of the study, it was obvious that oct imaging was able to detect the need for retreatment long before leakage was detected on fa imaging, before vision loss occurred, and before hemorrhage appeared. at the time, retina specialists questioned why the oct threshold for retreatment was set at an increased central retinal thickness measurement of 100 m, and the reason for this threshold was to prove a point. we wanted to show our skeptical colleagues that when a small amount of fluid appeared and it was left untreated, even more fluid and vision loss would result. once we proved our point, the threshold for retreatment was lowered in the second year of the study. a zero tolerance strategy was developed, and the protocol was amended to allow for retreatment once any fluid was detected after a dry macula was achieved. after 2 years, the va outcomes would turn out to be very similar to the outcomes observed following monthly dosing with ranibizumab in the phase ii and phase iii trials, but unlike the situation with a fixed monthly dosing regimen, these patients in the pronto study required approximately half the number of injections. the pronto study served as a model for subsequent studies investigating oct - guided retreatment using a pro re nata (prn) regimen for nvamd. however, several of the smaller studies failed to appreciate two key features of the pronto study : the requirement for monthly exams and the amended requirement that retreatment be given if any macular fluid was detected by oct imaging once the macula was fluid - free. moreover, retreatment was not decided by changes in va, but only by changes in oct imaging. while none of the phase iii pivotal trials that led to food and drug administration (fda) approval for any of the anti - vegf drugs used a pure oct - guided prn dosing regimen, monthly dosing versus oct - guided prn dosing has been studied in several large, well - controlled prospective clinical trials. the comparison of age - related macular degeneration treatments trials (catt) and the inhibition of vegf in age - related choroidal neovascularization (ivan) trial showed similar va outcomes after 2 years when the same anti - vegf drug was dosed monthly or discontinuously. however, the most convincing of these comparative trials is the phase iii, double - masked, multicenter, randomized, active treatment - controlled study of the efficacy and safety of 0.5 mg and 2.0 mg ranibizumab administered monthly or on an as - needed basis (prn) in patients with subfoveal neovascular age - related macular degeneration (harbor) trial. the harbor trial used a prn regimen similar to one employed in the pronto study. the two doses of ranibizumab were compared using a monthly versus an oct - guided prn dosing regimen in which patients received three monthly injections followed by monthly retreatment as long as there was persistent fluid. once the macula became fluid - free, patients were followed monthly and retreated if any macular fluid was detected by oct imaging. this study not only showed that monthly dosing and oct - guided prn dosing were equivalent, but did not even rely on a fundus exam when making the retreatment decision. the totality of the data suggest that a rigorously implemented oct - guided prn dosing strategy appears to be as beneficial as a fixed monthly dosing regimen, and as a result, this strategy has gained global acceptance. the greatest advantage of an oct - guided prn dosing strategy is that it improves and maintains va while significantly decreasing the number of injections needed. another dosing strategy for anti - vegf drugs that has gained global acceptance is a regimen known as an oct - guided treat - and - extend dosing. this regimen also relies on oct imaging to determine when the macula has become fluid - free, but then patients continue to be injected at each subsequent visit. however, once the macula is fluid - free, the patient is injected and the visit intervals are slowly extended to determine when and if macular fluid recurs. if oct imaging detects the recurrence of fluid at a visit, then a treatment is given and the interval is shortened. usually, visit intervals are lengthened or shortened by 2-week intervals depending on the status of macular fluid. while this oct - guided strategy mandates that an injection be given even when the macula is dry, the advantage over other strategies is that it minimizes the overall number of visits. to date, only two prospective studies, known as the lucentis compared to avastin study (lucas) and treat - and - extend in amd (trex - amd) study, have used this treat - and - extend strategy, and the results appear to be similar to the outcomes from larger monthly dosing trials. despite the limited prospective clinical trial data, clinicians and patients appear to prefer the treat - and - extend dosing regimen as a compromise between frequent monthly visits and monthly injections. on occasion, oct monitoring of neovascular lesions has shown the importance of even more frequent injections with anti - vegf drugs when monthly dosing proved unable to prevent the growth and worsening exudation of the neovascular lesions. thus, oct monitoring allows clinicians to follow a goldilocks strategy when managing patients, which is not too many injections, not too few injections, but just the right number of injections for that patient. during the early to mid-2000s when oct was being accepted into practices as an essential tool for monitoring patients with nvamd, clinicians became empowered by their ability to monitor the real - time effectiveness of different therapeutic agents, whether it was verteporfin photodynamic therapy (pdt), intravitreal steroids, or anti - vegf therapy. during this time, two anti - vegf drugs for intraocular delivery were in clinical trials : ranibizumab (lucentis, genentech / roche), an antibody fragment that binds all isoforms of vegf - a ; and pegaptanib sodium (macugen ; osi / eyetech, new york, ny, usa), an aptamer that binds vegf165 and larger isoforms. as mentioned previously, oct imaging was useful in demonstrating the anatomic improvements that followed ranibizumab therapy, and selected sites performed oct imaging during the phase iii ranibizumab trials for neovascular amd known as the minimally classic / occult trial of the anti - vegf antibody ranibizumab in the treatment of neovascular amd (marina) and the anti - vegf antibody for the treatment of predominantly classic choroidal neovascularization in age - related macular degeneration (anchor). in these trials, ranibizumab was dosed monthly for 2 years, and the oct images that were obtained on a subset of subjects from these studies did confirm the drying effect of ranibizumab therapy. in contrast, oct imaging was never used in the pegaptanib trials, and after pegaptanib was approved in late 2004 and became commercially available in early 2005, clinicians used oct imaging on patients undergoing treatment with pegaptanib. not surprisingly, the disappointing va outcomes following pegaptanib therapy were found to correlate with persistent macular fluid following treatment, thus empowering clinicians to make an objective assessment of the drug 's efficacy. as a result of this discrepancy between the oct outcomes when using pegaptanib versus other anti - vegf drugs, oct imaging confirmed the need to suppress all vegf isoforms to obtain the maximum drying effect from anti - vegf therapy. optical coherence tomography imaging in the marina and anchor trials with ranibizumab also revealed that the major cause of severe vision loss was not associated with persistent macular fluid or a scar ; rather, severe vision loss was more commonly associated with a fluid - free macula. for the first time, there was an appreciation that macular atrophy was a major cause of vision loss in treated eyes with nvamd, rather than the more typical cause of severe vision loss prior to anti - vegf therapy, which was the formation of a disciform scar. the formation of macular atrophy after anti - vegf therapy was confirmed in the catt and ivan trials after 2 years, and in these trials, the monthly dosing groups had a higher incidence of macular atrophy compared with the discontinuous treatment groups. while this observation has raised questions about whether anti - vegf therapy is responsible for the macular atrophy in some patients or whether the atrophy appears as a feature of normal disease progression and a consequence of suppressing neovascularization, it has become abundantly clear that oct imaging is an invaluable tool for not only monitoring the presence of macular fluid, but also for monitoring the anatomic integrity of the outer retina and rpe as macular atrophy evolves (fig. macular atrophy developing after treatment with an inhibitor of vascular endothelial growth factor (vegf). (a, d, g, f) spectral - domain optical coherence tomography (sd - oct) fundus images at sequential visits. (b, e, h, k) horizontal sd - oct b - scans. (c, f, i, l) vertical sd - oct b - scans. (a, b, c) one month after an anti - vegf injection for a hemorrhagic fibrovascular retinal pigment epithelial detachment secondary to age - related macular degeneration. the oct fundus image showed a small focus of atrophy as seen in (a) (arrow). (d, e, f) two months after the second injection, va was 20/25, but the area of atrophy was enlarging as shown in (d) (arrow) with evidence of hypertransmission shown on the horizontal (e) and vertical (f) b - scans. disruption of the outer retinal layers can be appreciated in the areas with the hypertransmission defects. (g, h, i) a fourth injection was given 2 months after the third injection, and 2 months after the fourth injection, the patient returned with a va of 20/25, but an even larger area of atrophy could be appreciated as shown in (g) (arrow) with evidence of even more hypertransmission as shown on the horizontal (h) and vertical (i) b - scans. (j, k, l) three months after the fifth injection, the area of atrophy was slightly larger (j) (arrow) and prominent hypertransmission defects were appreciated as shown in (k) and (l). disruption of the normal outer retinal structures was seen in the areas with the hypertransmission defect. during the clinical development of ranibizumab and pegaptanib, there were not only questions about the appropriate dose, dosing interval, and long - term effectiveness of these drugs ; there was also uncertainty about the safety of repeated intravitreal injections and whether patients would tolerate intravitreal injections as a long - term drug delivery strategy. due to these concerns about the safety and tolerability of repeated intravitreal injections, we investigated a third vegf inhibitor known as bevacizumab (avastin ; genentech / roche), a full - length humanized antibody against vegf - a sharing a common clonal ancestry with an anti - vegf murine monoclonal antibody that gave rise to ranibizumab ; and like ranibizumab, bevacizumab was known to bind all the isoforms of vegf - a. since intravenous bevacizumab was being developed as a therapy for colon cancer, we decided to initiate a self - funded study using the same intravenous strategy for the treatment of nvamd, and once again, oct imaging proved pivotal in these bevacizumab studies. the systemic avastin in neovascular amd (sana) study was initiated once bevacizumab was approved for colon cancer in february 2004. twenty - four hours after the first systemic dose of bevacizumab in the first patient, oct imaging unambiguously showed a profound benefit from intravenous bevacizumab. by may of 2005, oct imaging had confirmed that systemic bevacizumab had a significant and sustained drying effect on the macula, and this drying effect was associated with va improvement. however, during the course of the sana study, the fda added a black box warning describing the small but significant risk of thromboembolic events from the use of high - dose intravenous bevacizumab. while none of the patients in the sana study experienced a thromboembolic event, they did experience mild hypertension that was easily controlled with oral medication. due to these systemic safety concerns and the appreciation from the ranibizumab and pegaptanib studies that patients were able to tolerate repeated intravitreal injections, we decided to offer patients an off - label intravitreal dose of bevacizumab as salvage therapy. this dose was 500- to 1000-fold lower than the dose given intravenously, thus minimizing the systemic risk. moreover, this intravitreal dose was capable of binding the same amount of intraocular vegf as the intravitreal dose of ranibizumab. once again, oct imaging played a pivotal role in demonstrating the benefits of bevacizumab. when first used as salvage therapy in a nvamd patient with a lesion that was enlarging despite treatments with pdt and pegaptanib, oct imaging showed a remarkable improvement in the amount of macular fluid after intravitreal bevacizumab, and this improvement was associated with va improvement. since ranibizumab had not yet been approved, and would not be commercially available for another year in july 2006, the off - label use of intravitreal bevacizumab spread globally and became the treatment of choice in 2005 due to its perceived clinical efficacy, its widespread availability, its superiority over pegaptanib, and its low cost. in july 2006 when ranibizumab was commercially available for the treatment of nvamd, clinicians had a choice between two drugs manufactured by genentech that had a similar genetic lineage but differed with respect to size, affinity for vegf, and cost. with the widespread use of oct imaging, clinicians were now empowered to see for themselves that ranibizumab and bevacizumab were superior to pegaptanib and that ranibizumab appeared similar to bevacizumab with respect to its drying effect on the macula and improvement in va following treatment. without oct imaging, it is unlikely that bevacizumab and oct - guided retreatment would have gained worldwide popularity. in 2011, a fourth drug, known as aflibercept (eylea ; regeneron pharmaceutical, tarrytown, ny, usa), was fda approved for the treatment of nvamd. aflibercept, a fusion protein combining the fc portion of an antibody with vegf - binding domains from vegf receptors 1 and 2, has an even higher affinity for vegf compared with ranibizumab and a molecular size in between those of bevacizumab and ranibizumab. moreover, it has the theoretical advantage of neutralizing placental growth factor, as well as all the isoforms of vegf - a. in the phase iii aflibercept trials known as the vegf trap - eye : investigation of efficacy and safety in wet amd (view1 and view2) trials, intravitreal injections of aflibercept every 2 months during the first year were shown to be equivalent to monthly injections of ranibizumab. in the second year of the trials, subjects received a mandatory injection every 3 months, with oct imaging performed during the intervening monthly visits. if there was evidence of recurrent or persistent macular fluid using oct imaging, then injections could be administered during these intervening visits. this modified - prn regimen resulted in similar va and oct outcomes when aflibercept and ranibizumab were compared, but subjects receiving aflibercept required slightly fewer injections using oct - guided therapy. once again, oct imaging proved valuable for detecting macular fluid and determining the need for retreatment in this prospective clinical trial setting. since its approval, aflibercept has gained popularity as an anti - vegf drug with the ability to increase the retreatment interval while maintaining improved va and a fluid - free macula in nvamd patients. the perception that aflibercept provides increased durability arises not only from the dosing interval of every 2 months used in the phase iii studies, but also from numerous research papers showing that when patients are switched from ranibizumab or bevacizumab to aflibercept, there is a tendency for investigators to report improved macular anatomy and extended fluid - free intervals based on oct imaging. a fifth drug, known as conbercept (lumatin ; chengdu kanghong pharmaceutical group, chengdu, china), gained china fda approval in 2013. conbercept, which is a fusion protein similar to aflibercept and combines the binding domains of vegf receptors 1 and 2 with an fc portion of an antibody, has a molecular weight comparable to that of bevacizumab, but it is currently used only in china. once again, oct imaging correlated with the va benefits observed during the conbercept clinical trials. now that td - oct has been surpassed by sd - oct in the clinics, the next step in the commercial evolution of oct imaging for the management of nvamd is the development of oct angiography (octa). optical coherence tomography angiography utilizes novel scanning patterns and algorithms to image blood flow in the retina and choroid without the use of exogenous dyes. not only can blood flow be imaged, but also the scans provide detailed structural information comparable to or better than the images currently available from commercial sd - oct instruments. different octa platforms are being developed that use different scanning patterns and algorithms, but they can be broadly divided into sd - oct and swept - source oct (ss - oct) instruments. each platform will have certain advantages and disadvantages, but whether octa has advantages over conventional oct imaging in the management of nvamd patients remains to be determined. the ability to noninvasively visualize the appearance and growth of mnv should augment the routine oct strategy of monitoring for the presence or absence of macular fluid when deciding to initiate anti - vegf therapy and when to re - treat. since exudation is the metric by which we currently measure vegf activity and exudation can arise from both the neovascularization and the normal vasculature, there should be certain advantages of using octa to directly monitor changes in the neovascular lesion rather than just monitoring the level of exudation. even if octa imaging does not influence our decision on when to treat, it will surely add to our understanding of disease onset, progression, and response to treatment. for example, octa can visualize the presence of type 1 mnv in eyes that were diagnosed with dry, intermediate amd. the presence of nonexudative neovascular lesions raises new questions about whether we can predict when exudation will arise and if there is a role for anti - vegf therapy even before exudation occurs. if octa can now replace fa and icga imaging for most mnv, then octa imaging will provide all the structural and flow information needed for the diagnosis and management of mnv. with respect to their development, oct imaging and antiangiogenic therapies have been inextricably linked. if oct imaging had not been available when anti - vegf therapies were developed, then clinicians and patients would have been chained to fixed - dosing regimens without the ability to easily and noninvasively assess whether the exudation from the neovascularization had been adequately controlled. in the era of pdt before anti - vegf therapy, patients underwent invasive, time - consuming fa imaging every 3 months to determine whether leakage was present and treatment was needed. when intravitreal pegaptanib was approved, injections were given per protocol every 6 weeks, but when patients lost vision and additional oct imaging was performed, the presence of persistent or worsening macular fluid was confirmed. in the era of oct imaging and pan - vegf blockade, oct imaging unambiguously confirmed the effectiveness of anti - vegf therapy in resolving macular fluid. optical coherence tomography imaging empowered clinicians to personalize therapy by evaluating different drugs and dosing regimens. clinicians were able to customize therapy to either minimize the number of injections or minimize the number of visits. without the confidence that came from oct imaging and the ability to demonstrate the beneficial effect of anti - vegf therapy within 24 hours of treatment, it is unlikely that intravenous and then intravitreal bevacizumab therapy for the treatment of exudative eye diseases would have been developed in 2005, and it is unlikely that the global ophthalmologic community would have been convinced to rapidly adopt intravitreal bevacizumab as a viable treatment strategy. while significant cost savings have resulted from the use of oct compared with fa imaging, greater cost savings have resulted from the use of oct - guided retreatment to avoid monthly dosing, and even greater cost savings have resulted from the use of off - label bevacizumab in lieu of the more expensive drugs. in the united states alone, over the past 10 years, the combined medicare cost savings from the use of oct and bevacizumab have exceeded $ 20 billion when conservatively extrapolated from published utilization data for all forms of exudative eye diseases. globally, four intravitreal drugs dominate the market for anti - vegf therapy in neovascular amd, diabetic macular edema, and retinal vein occlusions : bevacizumab, ranibizumab, aflibercept, and conbercept. despite differences in molecular size, affinity for vegf, and intravitreal half - life, the most likely explanation for the effectiveness of these four drugs is that they bind all the biological isoforms of vegf - a whereas pegaptanib inhibited only vegf165 and larger isoforms. thus, this was the first example of how oct imaging was able to distinguish between different therapeutic targets. as new therapies and drug delivery strategies are developed to treat exudative eye diseases, oct will once again play an important role. for example, anti - vegf drugs are being delivered through implantable devices, as topical therapy, and as oral medication. treatments that combine intravitreal anti - vegf therapy along with inhibitors of platelet - derived growth factor (pdgf), angiopoietin 2, and tissue factor are being investigated, and topical squalamine is being combined with anti - vegf therapy in clinical trials as well. optical coherence tomography continues to be an integral part of all these trials, but the phase iii trials for the pdgf inhibitor known as e10030 (fovista ; ophthotech corp., princeton, nj, usa) have incorporated a novel inclusion requirement for their treatment - nave subjects with nvamd ; that requirement is the presence of shrm (clinicaltrials.gov identifier : nct01944839, nct01940900, nct01940887). while improvement of va remains the ultimate outcome that will determine the success or failure of any new treatment, there 's so much more to the macula than va, which measures only central foveal function. unlike dye - based angiography, oct imaging can identify macular abnormalities involving the photoreceptors, the rpe, and the choroid that can help explain visual function changes such as reduced reading speed, reduced microperimetric thresholds, and delayed dark adaptation. while oct is clearly the gold standard imaging strategy for predicting the success of any antiangiogenic treatment, it is also a valuable strategy for studying a wide range of anatomic changes that correlate with reduced visual function and disease progression. in summary, oct is held in such high regard that clinicians will never be convinced of a treatment 's efficacy until oct imaging demonstrates an unambiguous anatomic benefit.	purposeto explain the pivotal role optical coherence tomography (oct) imaging had in the development of antiangiogenic therapies for the treatment of neovascular age - related macular degeneration (nvamd).methodsa historical literature review was combined with personal perspectives from the introduction of oct imaging and the early clinical use of vascular endothelial growth factor (vegf) inhibitors.resultsat the time that oct emerged, the gold standard for imaging of nvamd was fluorescein angiography (fa), a time - consuming, dye - based, invasive technique that provided en face images of the retina and was used to characterize leakage, perfusion status, and the types of macular neovascularization (mnv). in comparison, oct imaging was a fast, safe, noninvasive technique that complemented fa imaging by providing cross - sectional images of the macula. oct was able to visualize and quantify the macular fluid that was associated with the presence of excess vegf, which was identified by intraretinal fluid, subretinal fluid, and fluid under the retinal pigment epithelium (rpe). clinicians quickly appreciated the benefits of oct imaging for following macular fluid after anti - vegf therapy. by observing the qualitative and quantitative changes in macular fluid depicted by oct imaging, clinicians were empowered to compare anti - vegf drugs and move from fixed - dosing regimens to patient - specific dosing strategies requiring fewer injections.conclusionsoptical coherence tomography imaging was adopted as a vegf - meter, a method to detect excess vegf, and evolved to become the gold standard imaging strategy for diagnosing nvamd, assessing treatment responses to anti - vegf drugs, deciding when to re - treat, and evaluating disease progression.
hyperuricemia is a condition that is significantly associated with markers of metabolic syndrome such as dyslipidemia, glucose intolerance, high blood pressure, and central obesity, which are accepted as risk factors for developing cardiovascular disease. hyperuricemia is probably associated with glucose intolerance due to various mechanisms ; however, the most important is the association between insulin and renal resistance to absorption of urates. hyperuricemia has been associated with insulin resistance ; however, there are few studies where the association of hyperuricemia - insulin resistance and beta cell function is evaluated. a modest positive association between concentrations of uric acid and incidence in type 2 diabetes mellitus was observed in a cohort of a chinese population. it was reported recently in another cohort that uric acid is a risk factor for development of dm2. at the same time, another study demonstrated that serum uric acid values may be useful as predictors of dm2 in adults who are glucose intolerant. cohort studies support the fact that uric acid is a risk factor for developing dm2 ; in a meta - analysis by kodama., the authors concluded that the variability of the results and control of confounding variables should be considered in the final analysis of competitive models for interpreting the results regarding the role of uric acid as a risk factor for developing dm2. in a study that reported beta cell function with homa in subjects with hyperuricemia, failure of beta cells to compensate for the variation in insulin sensitivity these studies from different population samples established an association between serum uric acid and prevalence of dm2, however a temporal effect between uric acid and different phases of insulin secretion can not be shown by these data. the role of uric acid as a continuous variable and its relationship to insulin secretion without hyperuricemia in patients with dm2 in a model such as the clamp has not been evaluated. the aim of this study was to determine the relationship between serum concentrations of uric acid with insulin secretion in adults with dm2 without hyperuricemia using the hyperglycaemic clamp technique. a cross - sectional analytical study was carried out in 45 subjects of both genders. subjects were between 40 and 60 years of age and were classified as overweight or grade i obesity and diagnosis of dm2 100%. in the present study, hyperglycemic clamp (200 mg/100 ml) technique was used to stimulate insulin secretion, and a positive relationship was demonstrated between the serum concentration of uric acid and the total phase of insulin secretion. the first phase of secretion was the most important one and it influenced in an important way on the positive correlation ; this was probably caused by a compensatory response of the beta cell in subjects with dm2 without hyperuricemia. serum concentration of uric acid showed a positive relationship with the total phase of insulin secretion ; even in states prior to hyperuricemia, uric acid can play an important role in the function of the beta cell in patients with dm2.	to determine the relationship between serum concentrations of uric acid and insulin secretion with hyperglycaemic clamp technique among adults with type 2 diabetes mellitus (dm2) without hyperuricemia, we carried out a cross - sectional study on 45 patients of both gender. we observed correlation between uric acid with male gender r = 0.710 (p = 0.001). also correlation between uric acid and total insulin secretion was positive r = 0.295 (p = 0.049). as well as a positive correlation adjusted for body mass index was demonstrated for the first, second, and total phases of insulin secretion, respectively, r = 0.438 (p = 0.022), r = 0.433 (p = 0.022), and r = 0.439 (p = 0.024). serum concentration of uric acid showed a positive relationship with the total phase of insulin secretion ; even in states prior to hyperuricemia, uric acid can play an important role in the function of the beta cell in patients with dm2.
the short and long - term benefits of early intervention for childhood developmental disorders have been demonstrated in numerous studies (16) and an increasing urge has been developed for their early identification (6). however, it is now well - known that relying on a clinician s clinical instincts and experience can be misleading in the early differentiation between normal and abnormal development (1, 610). several studies have supported the observation that parents can give accurate information about their child s development (1013) and thus there is increasing tendency for production and application of questionnaires that are based on parents reports and some authors have confirmed their use (14). in the present study the asq, a parent - report questionnaire, was chosen because it has been proven to be a valid and/or reliable screening test, even in its translated and culturally adapted versions in different populations of children (7, 1422). however, few studies have examined the psychometric properties of asq in cultures outside the united states. this test has not been validated and standardized before in iran. this study was conducted for the purpose of cultural adaptation, validation and standardization of the asq questionnaire for 460 months - old iranian children and children with similar socio - cultural backgrounds, such as all those living in the middle east. the questionnaire was translated to the persian language and then back - translated by two independent native translators who also had experience in the field of child development. by comparing the two, the discrepant parts were identified and corrected. the resulting persian questionnaire was then assessed in terms of content validity by a panel of seven, including four pediatricians, a psychologist, a speech pathologist, and a psychometrist. it was tried hard to maintain the meaning of the original items, however some changes were inevitable for improving clarity of meaning in the persian language or for adapting culturally. a pilot study was carried out on 100 parents of 460 month - old iranian children, recruited by convenient sampling in tehran, in order to determine the degree of clarity of items, cultural appropriateness, to detect ambiguous items and to identify dilemmas in the process of test implementation. after identifying and resolving problematic issues in the previous phase, the revised version of the persian test was performed for 38 children, two from each of the 19 age groups, once by the mother and once by the father, in order to determine the inter - rater reliability. in order to perform the test on a national sample, we trained a group of physicians from selected cities in the country, which were selected purposively, trying to provide a widespread and an evenly - spread selection, covering the main socio - cultural and geographic divisions of the country, and also based on the presence of skilled physicians in health care centers willing to supervise the implementation of the research. for implementation of the test at the national level the sample was determined to be 11000 children but turned out to be 10516 children actually. our inclusion criteria were 460 months age, iranian nationality, lacking gross developmental disorders, and parents educational level at least elementary school.. informed consent was acquired from parents of children. for assessment of psychometric properties of the test, the cronbach s alpha coefficient was calculated for each of the five domains and then totally, in all nineteen age groups for determining reliability. in order to determine the construct validity of the questionnaires using factor analysis, first the kmo (kaiser - meyer - olkin measure of sampling adequacy) was measured and was calculated to be 0.865 meaning the sampling was adequate for performing the factor analysis. in order to prove the correlation matrices between test items did not equal zero, the bartlett s test of sphericity was utilized. therefore, performing the factor analysis based on correlation matrices between items of the questionnaire was explicable. then, factor analysis was performed using the principal components analysis (pc) method. in order to determine that the items of the asq questionnaires were saturated with significant factors, after extraction of significant factors and performing the varimax rotation, the factor loadings (correlation of test items with the extracted factors) were calculated and the percentage of explained variances was determined. to arrive at cut - off values for the iranian sample, the mean scores minus 2sds for each domain in every age - specific questionnaire were determined (15). we were unable to assess the criterion - referenced validity of the questionnaire using a gold standard diagnostic developmental test because no such tests were available in iran. the minimum number of children belonged to the 60-month s age group and the maximum number belonged to the 12-month olds. in terms of the cronbach s alpha values of reliability, the lowest values for reliability appeared at 22-months in the problem - solving (0.55) and at 18-months in the social - personal domains (0.55). conversely, the highest values were detectable at 14-months in the gross motor (0.80) and at 36 and 60 months in the fine motor domains (0.80). the highest and lowest total reliability values belonged to the 36-months (0.86) and 18 months (0.76) questionnaires, respectively. the inter - rater reliability values obtained were 0.88, 0.91, 0.88, 0.89, and 0.86 for the communication, gross motor, fine motor, problem - solving, and social - personal domains, respectively, and 0.93 totally. the results of the principal component analysis (pc) are demonstrated in table 1 which shows the calculated factor loadings with values larger than 0.3, along with the rotated eigen value and the percentage of explained variance by each significant extracted factor. these figures for factor loadings of the test items demonstrate that the extracted factors are capable of assessing the five different developmental domains in iranian children. table 2 demonstrates the results of the iranian children s mean scores in comparison to the mean scores in the normative sample and those derived from three studies conducted in norway, spain (galicia), and korea. the results of only 10 age groups have been presented here because in the normative sample, data have been provided only on these 10 age groups. also, for the purpose of easier comparison, the standard error (se) results in the korean study have been converted and demonstrated as standard deviation (sd) results (using the formula : se= sd/n). in table 2, when we consider significant differences that are occurring repeatedly uni - directionally between iranian mean scores and those of at least three of the four other countries, the results are : - iranian children demonstrate significantly higher scores at 12 months in all domains except the gross motor ; at 16 months in the communication, problem - solving, and social - personal ; at 8 months in the problem - solving and social - personal and at 20 months in the communication and problem - solving domains. iranian children demonstrate significantly lower scores at 24 and 36 months in the gross and fine motor, at 48 months in the problem - solving and at 60 months in the fine motor and problem - solving domains. in the communication domain, the iranian mean scores are significantly higher at 12, 16, 20, and 24 months ; in gross motor they are significantly lower at 24 and 36 months ; in fine motor they are significantly lower at 12, 24, 36, and 60 months ; in problem - solving s they are significantly higher at 8, 12, 16, and 20, and significantly lower at 48 and 60 months ; and finally in the social - personal domain they are significantly higher at 8, 12, and 16 months. at other age ranges than those mentioned above, significant differences between iranian figures and those of the other four countries seem to be randomly distributed and no specific pattern can be detected. when comparing only with the normative sample results, at 8 and 12 months, iranian children demonstrated significantly higher scores in at least three domains (including the fine motor, problem - solving and social - personal domains). whereas at 36 months they showed significantly lower scores in the same domains as well as the gross motor. at other age ranges, the significant differences did not seem to follow any specific pattern. when considering each domain separately across all age groups, one considerable result is the significantly lower iranian mean scores in the fine motor domain in six different age groups of 16, 20, 24, 30, 36 and 60 months. in terms of cultural and linguistic appropriateness for iranian children, several items underwent modifications, most of which were in the communication domain. a similar process of item modifications took place in two studies performed on korean (21) and turkish children (22). conversely, in two separate studies performed on norwegian and dutch children no needs for modifications in the contents of the test were reported (18, 23). unlike the authors of the turkish study who have concluded that despite the modifications, their study supports the idea of cultural independence of the test (25), we believe that considering the several modifications that were needed in order to prepare asq for implementation in the iranian society as well as the korean and turkish studies, it can not be considered totally culture - free, especially considering countries outside the western zone. in terms of validity and reliability, this finding is consistent with several other studies (7, 1423) which have generally shown the test validity and /or reliability lying in the range of 70% to 100%. in the two studies that have covered the full age range of 4 to 60 months, the figures reported for sensitivity, specificity and reliability respectively have been 75.8%, 87.5%, and 94% in the us study (15), and 94%, 85%, 82.1%(test - retest reliability) and 87%(inter - rater reliability) in the turkish study (22). one pattern that can be noted when comparing the mean scores of the iranian children with that of the other four countries is that iranian children seem to be at a higher developmental level than children of the other four countries are at ages below 24 months, after which they seem to decline developmentally and demonstrate lower developmental status. a similar phenomenon was reported after developmental assessment of filipino infants using the griffiths mental development scales and comparing the results with those of british infants. the researchers suggested that differences in such factors as : genetics, nutrition and breastfeeding, environmental stimulation and mother - child interaction, socio - economic status and maternal intelligence quotient played a role (24). in another study, scores obtained on the revised bayley scales of infant development ii by children from low - income families were compared with those of the normative sample. this study showed that in infants, scores were consistent in the two groups, whereas the toddlers of the former group obtained lower mental, motor and behavioral scores. black concluded that this was either explained by a lack of enriching care - giving practices for toddlers in low - income families which did not development is guided by species - specific self - righting processes that protect infants from fulfill the requirements of toddlerhood, or the canalization theory in which early many environmental influences (25). in addition, it must also be noted that in iran, the ministry of health has continuously promoted exclusive breast - feeding from about 20 years ago.. it may be possible that after complete weaning, that is at about 24 months, the iranian childrens nutritional requirements are not ideally met which may also affect their development. another pattern that was noted when comparing the mean scores is that iranian children seem to be at a better - off situation in terms of the communication, problem - solving and social - personal domains respectively, especially before 24 months. the domain showing the least signs of significant differences with children in other countries appears to be the gross motor domain, and the fine motor domain seems to be the domain at which iranian children are the weakest developmentally. our finding is not consistent with the findings of the korean study, which showed significantly lower mean scores in the communication and problem - solving domains at several age intervals (21). our finding is also inconsistent with the findings of the dutch study performed only at 48-months, which showed that problem - solving scores were higher in the us sample (17), while fine motor scores were higher in the norwegian samples (24). a major strength of this study was that it was based on prospective data acquired from a very large and random nation - wide sample including the diverse cultures and socio - economic groups existing in the iranian population and that it included all of the 19 age - specific questionnaires of the asq. the major limitation of this study however, was our inability to determine concurrent validity of the test with a gold standard due to inaccessibility, as explained before. the reliability of asq ranged from 0.76 to 0.86, and the validity was satisfactory. the developmental status of iranian children tended to be higher in the communication, problem - solving and personal - social domains, especially under the age of 24 months, after which it seemed to deteriorate, especially in the gross and fine motor domains. ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors.	backgroundto provide the validated and standardized form of the persian version of the ages and stages questionnaires as an appropriate developmental screening tool for evaluation of iranian children s development.methods:this was a cross - sectional study. translation and back - translation, content validity determination, cultural and lingual modifications, pilot study on 100 parents and inter - rater reliability determinations were performed, respectively. the national and final stage was carried out 11000, 460 month - old children in selected cities throughout the country in order to determine the validity, standard deviation, reliability, sensitivity, specificity, and mean scores points of the test.results:the reliability, determined by cronbach s alpha ranged from 0.76 to 0.86 and the inter - rater reliability was 0.93. the validity determined by factor analysis was satisfactory. the mean scores of iranian children were identified and compared with those of the normative sample as well as with three other populations of children. the developmental status of iranian children was higher in the communication, problem - solving and personal - social domains, especially under the age of 24 months, after which their developmental status seems to deteriorate, especially in the motor domains.conclusion:the persian version of the asq has appropriate validity and reliability for screening developmental disorders in iran.
ethiopia is one of the few countries with the highest number of people living with hiv / aids globally. according to ethiopian demographic and health survey (edhs) 2011, federal ministry of health (fmoh) and hiv / aids prevention and control office (hapco) estimated that adult hiv prevalence was 1.5% a fee - based art program in 2003, and a free antiretroviral therapy (art) program in early 2005 was started. subsequently, a number of initiatives have been undertaken to expand the availability of art in ethiopia. task shifting and decentralization of the service to increasing numbers of both health centers and hospitals was done since august 2006. the provision of antiretroviral treatment has decreased morbidity and mortality in people living with hiv [37 ]. there have been several enabling factors for rapid scale - up of art in resource - limited settings. despite recent progress in improving access to art, limited uptake, poor retention, and difficulties in accessing care remain a serious concern for art programs [3, 4, 810 ]. treatment discontinuation raises some of the concern about drug resistance, which incomplete adherence does, and negates much of the benefit sought by those implementing treatment programs [11, 12 ]. patients with clinical aids who discontinue art will likely die within a relatively short time. long - term retention of patients in treatment programs has received far less attention perhaps because most large - scale treatment providers have few resources available to track missing patients [4, 10 ]. as a result, much attention has focused on patient day - to - day adherence to antiretroviral medications. there are also concerns regarding high rates of lost to followup and early mortality in a number of sub - saharan african art programs [1416 ]. high rates of attrition from treatment programs thus pose a serious challenge to program implementers and constitute an inefficient use of scarce treatment resources [17, 18 ]. attrition from these antiretroviral treatment programs is mainly due to the death and loss to followup of the patient [4, 8, 19 ]. there is thus a legitimate concern that a focus on numbers of people initiated on art may compromise quality of care. the feasibility of implementing art in resource limited countries has been demonstrated in multiple studies, with one - year outcomes often comparable to those in developed countries [4, 5, 1921 ]. little is known about the longer - term sustainability of such outcomes in such programs. the aim of this study is, therefore, to describe the art program practices and clinical outcome as measured by attrition (mortality and loss to followup) and retention care at the university of gondar (uog) hospital, art clinic, from march 2005 to august 2010. examination of these data helps to understand the challenges that must be overcome to ensure the long - term success of treatment services in the treatment program in ethiopia. a retrospective cohort analysis was conducted at the uog hospital, art clinic, north - west ethiopia. unpublished report by uog showed that the hospital is a leading referral hospital in north - west ethiopia serving an estimated of five million people. summary of medical records of the hospital showed that as of january, 2012, about 9,019 patients were enrolled at the aids care clinic, out of which 3, 516 adult clients started on art in the clinic. as part of the government program, the clinic provides care and support to people living with hiv free of charge using the national and who guidelines [2, 22 ]. the art clinic has begun using electronic database in 2006 to monitor the performance of art program. patients at the art clinic are referred by art clinician to case managers for adherence and managing art risk factors. case managers are trained high school graduates with modest experience of community health practice. they are required to conduct planned outreach activities to trace those lost to follow - up and onsite community support. after art initiation, all adult, nonpregnant, aids patients in the treatment program from march 2005 through august 2010 were eligible. patients were excluded if art initiation or termination date was missing, and/or if dates were wrongly recorded such as, art initiation date after art termination date. patients who failed to return on their designated appointments date initial tracing was attempted by telephone by the clinic nurses on the day following the missed appointment. patients who could not be traced and did not subsequently return to the clinic during the study period were classified as lost to followup (ltf). any death confirmed and recorded in the patient card on art was assumed to be related to hiv / aids, unless and otherwise specified. patients who notified clinic staff of their intention to transfer to another facility were given a written referral form and classified as transfer out (to). retention to care was defined as those patients under the study known to be alive and receiving art regularly at uog hospital art clinic as of august 30, 2010. attrition was defined as discontinuation of art for reasons related to death or loss to followup. transfers to another health facility, when reported, were not regarded as attrition because these patients who transferred were assumed to be retained in another facility. however, actual retention to the facility was considered transfer - outs as the patient were assumed to be no more a part of the art program. data were retrieved from patients ' art card by trained nurses working in the art clinic using uniform data abstraction format prepared for the study. whenever relevant information was missing, the art database was consulted. observation was censored at the date of ltf or transfer out or at the end of the study period, 30 august 2010. mortality and ltf overtime were evaluated by declaring data at each calendar year, considering time of entry and exit to determine the contribution of time spent in the cohort. every year entry and exit observations were used to calculate person - time, event counts, and incidence rates at each calendar times. the overall rate was calculated in the same fashion at the end of the follow - up time. then mortality and lost to follow - up rates were calculated by dividing deaths or ltf by person - year at each calendar time to get incidence rates per 100 person - years of observation. we estimated time to death and ltf and retention to care by kaplan - meier failure and survival functions, respectively. to get insight into the shape of the survival function for each group and give an idea of whether or not the groups were proportional, we used the kaplan - meier curves for all the categorical predictors. we tested equality across strata to explore whether or not to include the predictor in the final model. for the categorical variables, we used the log - rank test of equality across strata. for the continuous variables, we used the cox proportional hazard model with a single continuous predictor. we considered including the predictor if the test had a p value of 0.20 or less. for our model building, we considered the model which included all the predictors that had a p value of less than 0.20 in the univariate analyses. covariates predetermined to be in the univariate analysis were entered into full multivariable cox proportional hazard model. the following baseline covariates were entered into the model : age, gender, marital status, occupation, disclosure status, type of initial regimen, the presence of tuberculosis, place of residence, functional status, who stage, and cd4 count. this study obtained ethical clearance from the institutional review board of the university of gondar, ethiopia. no personal identifiers were declared in the data set prepared for analysis and in the study report. between march 2005 and august 2010, 3194 patients were initiated on art, out of which 3012 were considered in the analysis (figure 1). sociodemographic and clinical baseline patient characteristics of the 3012 aids patients who were on art is presented on tables 1 and 2. the median age was 32 years (iqr 2740) ; 55.2% were female, and 45% of the patients were separated, divorced, or widowed. majority of the patients were christian by religion (92.4%), and came from urban areas (96.1%). thirty - five percent and 54% of the patients were uneducated and unemployed, respectively. when patients initially presented, 75% disclosed their hiv status to one or more person they knew. eligibility for art was determined by cd4 count in 55.3%, and both by cd4 and clinically in 39% of the patients. the majority of the patients (84%) were presented with who stages iii and iv. the most common type of initial regimen used was stavudine, lamuvudine, and neverapine (36%) or efaveranze (19%). during 6,386.2 patient - years of followup, 216 (7.2%) died, 551 (18.4%) ltf, 430 (14.3%) transferred to other health facility, and 1815 (60.3%) were retained in the program. survival analysis with kaplan meier estimates of retention into care was 87.5% (95 ci : 86.388.7) at 6 month, 80.7% (95 ci : 79.282.1) at 12 months, 73.8% (95 ci : 72.175.5) at 24 months, 69.3% (95 ci : 67.271.2) at 48 months, and 61.4% (95 ci : 47.372.7) at the end of the follow - up period (5.5 years). attrition was mainly due to ltf (31.4%, 95% ci : 19.548.1) followed by death (10.4%, 95% ci : 9.012.1). the majority of the deaths and lost to follow - ups occurred at 6 and 12 months of followup. fifty - six percent of deaths and 46% of lost to follow - ups occurred in the first year of treatment (table 3). as shown in table 4, the overall incidence of mortality and ltf rates were 3.4 per 100 person - years (95% ci : 3.03.9), and 8.4 per 100 person - years (95% ci : 7.89.2), respectively. mortality and ltf were highest in the first 3 months, 8.3 per 100 person - years (95% ci : 6.510.7) and 18.7 per 100 person - years (95% ci : 15.822.2), respectively ; from art initiation to 6 months, these rates were 7.7 per 100 person - years (95% ci : 6.49.4) and 18.8 per 100 person - years (95% ci : 16.721.4), respectively ; between 6 and 12 months after art initiation, these rates declined to 4.0 per 100 person - years (95% ci : 2.95.3) and 12.1 per 100 person - years (95% ci : 10.314.4), respectively. trend of mortality, ltf and retention in care is presented in figure 2 and table 3. the rate of mortality declined from 3.3 per 100 person - years (95% ci : 1.48.0) in 2005 to 0.3 per 100 person - years (95% ci : 0.2, 0.5) in 2010. this decline in the incidence of mortality was statistically significant (test for trend, p 200 cells/l, mortality decreased from 2.7 per 100 person - years (95% ci : 2.13.4) at the baseline to 2.3 per 100 person - years (95% ci : 1.63.0) after 24 months of followup. cox proportional hazards model demonstrated that being male (hr = 3.26 ; 95% ci : (2.19, 4.88), ambulatory and bedridden functional status (hr = 2.20 ; 95% ci : (1.30, 3.70) and (hr = 12.88 ; 95% ci : (8.19, 20.26), resp.). cd4 count < 200 cells/l (hr = 5.02 ; 95% ci : (2.03, 12.39), and the presence of hiv - tuberculosis coinfection at art initiation (hr = 2.91 ; 95% ci : (2.11, 4.02) were significantly associated with mortality. in contrast, cd4 count < 200 cells/l (hr = 1.33 ; 95% ci : (0.95, 1.88) and ambulatory functional status (hr = 1.65 ; 95% ci : (1.22, 2.23) were significantly associated with ltf. as shown in figure 3, who stages iii and iv were significantly associated with mortality in bivariate analysis (hr = 2.6 with 95% ci : (1.6, 4.5) ; when adjusted for other variables, the direction of the relationship changed and turned out insignificant, which resulted from the small number of deaths occurring at who stages i and ii (table 5). this study adds to the limited number of researches on art outcome assessments from sub - saharan africa regarding concerns related to high rates of ltf, early mortality, and challenges of retention of aids patients in long - term care [8, 16, 20, 21, 24, 25 ]. the analysis of 3012 patients initiating art in 5.5 years in a referral teaching hospital in north - west ethiopia art program demonstrated that more than 60% of patients initiated on art were retained. in addition the study shows that the majority of the deaths and ltf occurred in the first 3 months of followup. this study thus provides longer assessment of aids clinic outcomes over those reported from similar studies, which reported outcomes of up to two years [4, 15, 19 ]. retention in long - term care is complex, especially in low- and middle - income countries [8, 25, 27 ]. the findings of this study document the difficulties in retaining patients in care for life - long treatment due to ltf. patient retention in care in this program (61.4%) is comparable to the findings of other sub - saharan countries [4, 15 ]. for example, assefa., in the study of outcomes of the art services in the 55 health facilities in ethiopia, found out that art programs in ethiopia are able to retain on average 68% (95% ci : 5185) of their patients in 2 years. likewise, a recent systematic review in sub - saharan africa art programs by fox and rosen (2010) demonstrated that these programs were able to retain about 60% of their patients at the end of 2 years. the art program in this hospital, therefore, has a better picture of the effectiveness of art delivery at 5.5 years as compared to previous studies, which suggested that less than 50% of the patients were retained at a shorter follow - up time [20, 28 ]. the probability of surviving 5.5 years is more than 50% and hence the chance of surviving to the last followup was high. this program, therefore, has better effectiveness in retention of patients in care after 5.5 years, suggesting a better success, although there is no clear threshold at which retention in care is considered adequate. we observed a low overall mortality rate of 3.4 per 100 person - years, comparable to studies done in africa. there was also relatively lower death rate of 7.7 per 100 person - years in the first 6 months of art compared to other similar art clinics [14, 16, 29 ]. for example, the death rate observed in arbaminch hospital in ethiopia demonstrated an overall mortality rate of 9.1 per 100 person - years. the low mortality rates observed in our cohort may, in part, be due to access to tertiary care level diagnostics and hospital care. as a program expands, its ability to accurately ascertain patient deaths deteriorates, and high observed ltf may be associated with poor mortality ascertainment [14, 30 ]. nonetheless, mortality is not regarded as a major reason for patient attrition in large art programs in developing countries. the greater threat to the success of many african art program is the observation of high levels of ltf, insofar as this outcome reflects patients who have truly left care. ltf increases throughout the follow - up period resulting in decline in retention of care. rate of ltf of 8.4 per 100 person - years in our cohort is comparable to that observed in the south africa studies [9, 16 ], but relatively higher as compared to the rate observed in aids clinical trial group (actg) analysis, which is 5.1 per 100 person - years of observation. the size and pace of art scale - up may have contributed to the observed ltf. the program has grown in size dramatically, with our cohort increasing enrolment about 8-fold from 2005 to 2010. in contrast to these individuals, patients who are truly ltf are likely to be nonadherent to treatment and at higher risk of death [31, 32 ]. in addition, they face increased risk of drug resistance to art, undermining the long - term effectiveness of treatment programs [9, 33 ]. the finding of this study should be interpreted with caution and has got a number of limitations. to start with consequently, deaths which occurred at home might have not been documented in the patient card. this may underestimate the rate of mortality. besides, patients ' card was the main source of information in our study. in the data collection, however, we learned that some of the data lack accuracy and completeness. second, there were a high number of patients who were ltf or transferred to another art site following the country - wide roll - out of art in our study. though we assumed that those transfered out were retained in another health facility, our assumption may not hold true as these patients might not be in care. the actual retention in our study considering this scenario of transfer out as a means of attrition reduces retention to the level of 46%. besides, some ltf might have died, although we have little reason to believe these misclassifications would be anything more than random. lastly, the study is institutional based and more than 96% of the study cohort was from urban residence that might be able to reach to get the service than the rural people. this may overestimate retention, though residence was not a predictor for both mortality and ltf in our cohort. as a result, future research should be targeted to population level and rural areas of the country. in conclusion, this study highlights that patient retention in chronic aids care remains a challenge during scalingup of the program. though overall mortality rate has declined, attrition from the program is mainly due to increase in rates of ltf and early mortality. facilities must set up simple, standardized monitoring system to track the numbers of patients starting therapy every quarter and determine the treatment outcomes. in addition, future research should also be directed towards understanding the contextual reasons and outcomes of patients ltf and transferred to other treatment facilitates. this information could also be potentially useful in the development of strategies designed to increase patient followup and long - term retention in treatment programs.	background. there has been a rapid scale up of antiretroviral therapy (art) in ethiopia since 2005. we aimed to evaluate mortality, loss to followup, and retention in care at hiv clinic, university of gondar hospital, north - west ethiopia. method. a retrospective patient chart record analysis was performed on adult aids patients enrolled in the treatment program starting from 1 march 2005. we performed survival analysis to determine, mortality, loss to followup and retention in care. results. a total of 3012 aids patients were enrolled in the art program between march 2005 and august 2010. at the end of the 66 months of the program initiation, 61.4% of the patients were retained on treatment, 10.4% died, and 31.4% were lost to followup. fifty - six percent of the deaths and 46% of those lost to followup occurred in the first year of treatment. male gender (adjusted hazard ratio (ahr) was 3.26 ; 95% ci : 2.194.88) ; cd4 count 200 cells/l (ahr 5.02 ; 95% ci : 2.0312.39), tuberculosis (ahr 2.91 ; 95% ci : 2.114.02) ; bed - ridden functional status (ahr 12.88 ; 95% ci : 8.1920.26) were predictors of mortality, whereas only cd4 count < 200 cells/l (hr = 1.33 ; 95% ci : (0.95, 1.88) and ambulatory functional status (hr = 1.65 ; 95% ci : (1.22, 2.23) were significantly associated with ltf. conclusion. loss to followup and mortality in the first year following enrollment remain a challenge for retention of patients in care. strengthening patient monitoring can improve patient retention aids care.
conventional open surgery is the preferred treatment for tumor resection, as is for intrahepatic bile duct stones, especially in patients who have undergone previous upper abdominal surgery. in recent decades, continuous innovation and improvement of laparoscopic equipment and technology have led to the evolution of laparoscopic hepatectomy from a challenging procedure performed by highly skilled surgeons in handful medical centers to one that is widely performed. however, few laparoscopic hepatectomy procedures have been reported for patients with previous upper abdominal surgery. because severe adhesions secondary to previous surgery can lead to higher rates of complication and conversion to open surgery, the aim of this study was to evaluate the feasibility and safety of laparoscopic hepatectomy in these patients. prior to data collection, the study protocol was evaluated and approved by the ethical committees for human subjects at sir run run shaw hospital affiliated to medical college of zhejiang university. three hundred and thirty - six patients who had undergone laparoscopic hepatectomy at our hospital from march 2012 to june 2015 were included in this retrospective study. we divided these patients into two groups : an experimental group of those who had undergone previous upper abdominal surgery (ps group, n = 42) and a control group of those who had not (ns group, n = 294). previous upper abdominal surgery was defined as surgery performed above the umbilical region, including hepatobiliary and pancreatic surgery, splenectomy and gastrectomy, and surgery involving the small intestine, colon, kidney, and upper abdominal retroperitoneum. in this study, laparoscopic hepatectomy included malignant or benign primary hepatic tumors, metastatic hepatic carcinoma, and intrahepatic duct stones. inclusion criteria were : (1) primary hepatic malignant tumors with no extrahepatic metastasis ; metastatic hepatic tumors that metastatic focuses were limited to one lobe of liver ; benign hepatic tumors that were obviously symptomatic, at high risk for rupture with life - threatening consequences, or with unclear diagnosis ; intrahepatic duct stones with obvious stricture or dilation of the bile duct and liver atrophy ; (2) child - pugh score a or b ; (3) well - preserved general condition. patients with severe portal hypertension or american standards association (american society of anesthesiologists) grade iii or future liver remnant insufficiency (< 30% in the nonsclerotic liver or < 40% in the liver with cirrhosis) were excluded. briefly, a 10 mm camera trocar was inserted subumbilically in the ns group and in where adhesions were expected to be minimal in the ps group. pneumoperitoneum was established with carbon dioxide to a pressure of 1215 mmhg (1 mmhg = 0.133 kpa). under laparoscopic guidance, three more ports were established : a 10 mm main port in the xiphoid for surgical manipulation and two 5 mm accessory ports on the right side of the abdomen. for major liver resection, the hepatic hilum was dissected, and liver parenchymal transection was carried out using a peng 's multifunctional operative dissector (hangzhou shuyou medical instrument co., ltd., hangzhou, zhejiang, china) along with ischemia line. vascular clips or polypropylene sutures were used to control the divided vascular and biliary structures. the specimen of the liver was retrieved and placed in a plastic bag after enlargement of one of the port sites. all patients received prophylactic antibiotics immediately prior to the operation and had the same postoperative care by a professional nursing team. postdischarge transhepatic arterial chemoembolization and other adjuvant therapies were recommended for patients diagnosed with hepatocellular carcinoma or other malignant tumors. follow - up included physical examination, laboratory tests, tumor markers, and imaging examination every 3 months. statistical analysis was performed using ibm spss statistics for windows, version 19.0 (ibm corp., armonk, ny, usa). continuous variables were expressed as median (range) while categorical variables were expressed as number (percentage). whitney u - test while categorical variables were compared using the chi - square test or fisher 's exact test prior to data collection, the study protocol was evaluated and approved by the ethical committees for human subjects at sir run run shaw hospital affiliated to medical college of zhejiang university. three hundred and thirty - six patients who had undergone laparoscopic hepatectomy at our hospital from march 2012 to june 2015 were included in this retrospective study. we divided these patients into two groups : an experimental group of those who had undergone previous upper abdominal surgery (ps group, n = 42) and a control group of those who had not (ns group, n = 294). previous upper abdominal surgery was defined as surgery performed above the umbilical region, including hepatobiliary and pancreatic surgery, splenectomy and gastrectomy, and surgery involving the small intestine, colon, kidney, and upper abdominal retroperitoneum. in this study, laparoscopic hepatectomy included malignant or benign primary hepatic tumors, metastatic hepatic carcinoma, and intrahepatic duct stones. inclusion criteria were : (1) primary hepatic malignant tumors with no extrahepatic metastasis ; metastatic hepatic tumors that metastatic focuses were limited to one lobe of liver ; benign hepatic tumors that were obviously symptomatic, at high risk for rupture with life - threatening consequences, or with unclear diagnosis ; intrahepatic duct stones with obvious stricture or dilation of the bile duct and liver atrophy ; (2) child - pugh score a or b ; (3) well - preserved general condition. patients with severe portal hypertension or american standards association (american society of anesthesiologists) grade iii or future liver remnant insufficiency (< 30% in the nonsclerotic liver or < 40% in the liver with cirrhosis) were excluded. briefly, a 10 mm camera trocar was inserted subumbilically in the ns group and in where adhesions were expected to be minimal in the ps group. pneumoperitoneum was established with carbon dioxide to a pressure of 1215 mmhg (1 mmhg = 0.133 kpa). under laparoscopic guidance, three more ports were established : a 10 mm main port in the xiphoid for surgical manipulation and two 5 mm accessory ports on the right side of the abdomen. for major liver resection, the hepatic hilum was dissected, and liver parenchymal transection was carried out using a peng 's multifunctional operative dissector (hangzhou shuyou medical instrument co., ltd., hangzhou, zhejiang, china) along with ischemia line. vascular clips or polypropylene sutures were used to control the divided vascular and biliary structures. the specimen of the liver was retrieved and placed in a plastic bag after enlargement of one of the port sites. all patients received prophylactic antibiotics immediately prior to the operation and had the same postoperative care by a professional nursing team. postdischarge transhepatic arterial chemoembolization and other adjuvant therapies were recommended for patients diagnosed with hepatocellular carcinoma or other malignant tumors. follow - up included physical examination, laboratory tests, tumor markers, and imaging examination every 3 months. statistical analysis was performed using ibm spss statistics for windows, version 19.0 (ibm corp., armonk, ny, usa). continuous variables were expressed as median (range) while categorical variables were expressed as number (percentage). whitney u - test while categorical variables were compared using the chi - square test or fisher 's exact test the cohort consisted of 336 patients divided into ps (n = 42) and ns (n = 294) groups. the ps group was significantly older than the ns group (65 years [3880 years ] vs. 54 years [1685 years ], p < 0.001). moreover, the alkaline phosphatase of patients in ps group was significantly higher than that in ns group (111 u / l [46551 there were no significant between - group differences in other demographic variables, including sex, presence of cirrhosis or hepatitis - b, and liver function. characteristics and preoperative liver function of patients with laparoscopic hepatectomy continuous data are presented as median (range), mann whitney u - test ; categorical data are presented as n (%), chi - square test. akp : alkaline phosphatase ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; -gt : gamma - glutamyltransferase ; pt : prothrombin time ; tb : total bilirubin ; ps : previous upper abdominal surgery ; ns : nonprevious upper abdominal surgery ; alb : albumin. there was no significant difference in median operative duration between the ps group and the ns group (180 min [50400 min ] vs. 160 min [25500 min ], p = 0.869). median intraoperative blood loss was same between the ps group and the control group (200 ml [201500 ml ] vs. 200 ml [52000 ml ], p = 0.907). in the ps group (n = 42), six cases were converted to open surgery because of the presence of severe adhesions. of 37 conversions to open procedure in the ns group (n = 294), 25 were because of severe adhesions, six because of bleeding, four because of inadequate margins, and two because of severe gas embolism. the rate of intraoperative transfusion was identical between the two groups (14.3% vs. 14.3%, p = 1.000). with regard to the pringle maneuver, the ns group tended to have a shorter clamping time, but the difference was not significant (13 min [525 min ] vs. 15 min [1248 min ], p = 0.294). the overall complication rate was significantly lower in the ns group than in the ps group (17.0% vs. 31.0%, p = 0.030), but no between - group difference was observed in clavien - dindo grade. the duration of postoperative hospital stay, the rate of unplanned re - admission within 3 months, and the postoperative liver function were similar between groups [table 3 ]. perioperative outcomes of patients with laparoscopic hepatectomy continuous data are presented as median (range), mann whitney u - test ; categorical data are presented as n (%), chi - square test ; categorical data are presented as n (%), fisher s test. na : not available ; 3-month re - admisssions : unplanned re - admission within 3 months ; pm : pringle maneuver ; ps : previous upper abdominal surgery ; ns : nonprevious upper abdominal surgery. liver function of patients with laparoscopic hepatectomy on postoperative day 1 data are presented as median (range), mann akp : alkaline phosphatase ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; -gt : gamma - glutamyltransferase ; pt : prothrombin time ; tb : total bilirubin ; ps : previous upper abdominal surgery ; ns : nonprevious upper abdominal surgery ; alb : albumin. liver function of patients with laparoscopic hepatectomy on postoperative day 3 data are presented as median (range), mann - whitney u - test. akp : alkaline phosphatase ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; -gt : gamma - glutamyltransferase ; pt : prothrombin time ; tb : total bilirubin ; ps : previous upper abdominal surgery ; ns : nonprevious upper abdominal surgery ; alb : albumin. the extent of liver resection did not differ significantly between the two groups [table 5 ]. the extension of liver resection of two groups had no differences significantly (= 2.636, p = 0.104). in the ps group, 13 left hemihepatectomies, and 4 right hemihepatectomies were performed. in the control group, of the 56 patients who underwent left hemihepatectomy, three underwent concomitant caudate lobe resection. extent of liver resection of patients with laparoscopic hepatectomy hemi (or greater) hepatectomy was defined as major liver resection ; less than hemihepatectomy was defined as minor. ps : previous upper abdominal surgery ; ns : nonprevious upper abdominal surgery. of the 336 patients in both groups, 111 (101 in the ns group and 10 in the ps group) although the preoperative alpha - fetoprotein level was significantly higher in the ns group, the median maximum tumor diameters (2.7 cm [1.37.5 cm ] in the ps group and 4.0 cm [0.816.2 cm ] in the control group, p = 0.188) did not differ significantly between groups. r0 resection was achieved in all patients except one, in the ns group, whose resection was r1 [table 6 ]. hcc - related outcomes of patients with laparoscopic hepatectomy continuous data are presented as median (range), mann whitney u - test ; categorical data are presented as n (%), fisher s test. na : not available ; rfa : radiofrequency ablation ; tace : transhepatic arterial chemoembolization. one patient received tace and targeted radiotherapy ; hcc : hepatocellular carcinoma ; ps : previous upper abdominal surgery ; ns : nonprevious upper abdominal surgery ; afp : alpha - fetoprotein. the ps group patients (n = 42) were divided into two subgroups by type of previous surgery, laparoscopic (n = 13) or open (n = 29). there were no significant between - group differences in any perioperative outcome, including operative duration, intraoperative blood loss, and rates of transfusion and overall complications [table 8 ]. subgroup analysis of patients of previous upper abdominal surgery group by type of previous surgery subgroup analysis of patients with previous upper abdominal surgery continuous data are presented as median (range), mann whitney u - test ; categorical data are presented as n (%), fisher s test. na : not available ; 3-month readmissions : unplanned re - admission within 3 months ; pm : pringle maneuver. the ps group patients (n = 42) were divided into two subgroups by type of previous surgery, laparoscopic (n = 13) or open (n = 29) there were no significant between - group differences in any perioperative outcome, including operative duration, intraoperative blood loss, and rates of transfusion and overall complications [table 8 ]. subgroup analysis of patients of previous upper abdominal surgery group by type of previous surgery subgroup analysis of patients with previous upper abdominal surgery continuous data are presented as median (range), mann whitney u - test ; categorical data are presented as n (%), fisher s test. na : not available ; 3-month readmissions : unplanned re - admission within 3 months ; pm : pringle maneuver. in the past, repeat surgery was considered as a contraindication for a laparoscopic procedure. we believe there is an even greater challenge associated with any type of laparoscopic hepatobiliary surgery after previous upper abdominal surgery. bleeding of vascular - rich adhesions can lead to poor visualization, and disruptions in the hilar structure and immobility of liver result in a prolonged operation and greater blood loss. several groups have reported that the morbidity and mortality rates of open repeat liver resections vary from 22% to 28% and 0% to 5%, respectively. as the innovation of laparoscopic technique, laparoscopic hepatectomy has the advantages of inducing less surgical trauma and resulting in a shorter recovery time and hospital stay. in theory, a laparoscopic procedure decreases the stress response and protects postoperative immunity, which are also benefits of a minimal incision and which correspond to the popular concept of rapid rehabilitation. although, there have been some concerns about the ability of laparoscopic hepatectomy to cure malignancy. however, cherqui. reported 93% 3-year survival in patients with peripheral hepatocellular carcinoma undergoing laparoscopic resection, with an acceptable 30% overall recurrence rate, and similar results were reported by sarpel. moreover, many literature reviews have demonstrated no difference in the treatment of malignant tumors in terms of surgical margin between laparoscopic and open hepatectomy, and some experts even have suggested that laparoscopic left lateral lobe resection is now a standard procedure. in this study, although the age of ps group was significantly older than that of ns group, there were no other significant between - group differences in short - term outcomes. only overall complications were significantly higher in the ps group than in the ns group. only two cases with gas embolism were reported in ns group. in the subgroup analysis, the type of previous surgery (laparoscopy vs. laparotomy) had no effect on short - term postoperative outcomes. therefore, previous upper abdominal surgery is not the contraindication for a laparoscopic procedure as before, though many patients are still undergoing follow - up, and data on long - term outcomes of oncologic integrity are collected sequentially. in conclusion, laparoscopic hepatectomy in patients with previous upper abdominal surgery is controversial, and the ethical issues that would be associated with a randomized controlled trial prevent resolution of the issue in this manner. however, it is gaining greater acceptance, and a number of retrospective studies support laparoscopic hepatectomy as a feasible and safe procedure for patients who have undergone previous upper abdominal surgery and regardless of whether the previous surgery was laparoscopic or open. this study was supported by the grant from the fundamental research funds for the central universities (no. this study was supported by the grant from the fundamental research funds for the central universities (no.	background : laparoscopic liver resection has become an accepted treatment for liver tumors or intrahepatic bile duct stones, but its application in patients with previous upper abdominal surgery is controversial. the aim of this study was to evaluate the feasibility and safety of laparoscopic hepatectomy in these patients.methods:three hundred and thirty - six patients who underwent laparoscopic hepatectomy at our hospital from march 2012 to june 2015 were enrolled in the retrospective study. they were divided into two groups : those with previous upper abdominal surgery (ps group, n = 42) and a control group with no previous upper abdominal surgery (ns group, n = 294). short - term outcomes including operating time, blood loss, hospital stay, morbidity, and mortality were compared among the groups.results:there was no significant difference in median operative duration between the ps group and the ns group (180 min vs. 160 min, p = 0.869). median intraoperative blood loss was same between the ps group and the control group (200 ml vs. 200 ml, p = 0.907). the overall complication rate was significantly lower in the ns group than in the ps group (17.0% vs. 31.0%, p = 0.030). mortality and other short - term outcomes did not differ significantly between groups.conclusions:our study showed no significant difference between the ps group and ns group in term of short - term outcomes. laparoscopic hepatectomy is a feasible and safe procedure for patients with previous upper abdominal surgery.
recently, a possible disparity in the degree to which the right and left hand are susceptible to optical illusions when grasping or pointing at objects has received considerable attention (de grave. 2009 ; gonzalez. the interest is motivated by the two - visual systems model for action and perception, which proposes that movement control and perception are dissociated, not only in relation to the underlying neuro - anatomical separation of the dorsal and ventral pathways, but also with respect to the visual information that they rely upon (milner and goodale 1995, 2008 ; de wit. the empirical support for this dissociation is partly grounded in intriguing but controversial observations that the perception of objects is much more affected by illusory configurations (and hence visual context) than movements directed toward those objects (bruno. perception gathers knowledge about the environment predicated on the use of allocentric information that specifies objects and their properties relative to other objects of the environment. action controls the movements that are directed to the objects by exploiting egocentric information that specifies the object relative to the body. the information - based distinction, however, also implies that an illusory bias in object - directed movements (however small) points to the exploitation of both egocentric and allocentric information for movement control, indicating that the visual systems for perception and action normally do not operate in total isolation. in this regard, gonzalez. (2006) reported that hand aperture during grasping with the left hand, but not with the right hand, was affected by ponzo and ebbinghaus size illusions. this asymmetry in illusory bias between right- and left - handed grasping was found in right - handed as well as in left - handed participants, indicating that the difference occurred irrespective of handedness. these observations are pertinent in that they may suggest another instance of interactive contributions from the two visual systems in movement control. yet, the empirical evidence for an asymmetrical illusory bias between the right- and left - hand is contentious. (1997) reported that the bias in pointing movements directed towards the vertex of mller - lyer configurations was the same for one sample of participants who pointed with their right hand and a second sample of participants who pointed with their left hand. likewise, de grave. (2009), using a within - participant comparison, found that the brentano illusion affected the accuracy of pointing movements of right - handers with their right and left hand to the same extent. yet, in a study involving patients with unilateral brain damage, radoeva. (2005) found that patients with left - hemisphere damage who grasped shafts embedded in mller - lyer configurations with their (unaffected) left hand had a larger illusory bias in hand aperture than patients with right - hemisphere damage who grasped with their right hand. however, because right - handed grasps were always directed to the (unaffected) left visual hemifield and vice versa, the different illusory influences for the two hands can also be attributed to the visual hemifield in which the object was presented. in addition, no illusion - related differences in hand aperture between the right and left hand were found in a group of right - handed control participants. finally, adam. (2010) recently reported that aiming movements with the left hand were more sensitive to visual context than movements with the right hand. rather than employing the illusion paradigm, these authors built on earlier observations that the presence of a linear array of placeholders (one of which is the target) violated the relationship between movement time and object distance (i.e., fitts s law ; adam. this effect of visual context (i.e., placeholders) on movement time suggests involvement of allocentric sources of information for controlling reaching movements. adam. (2010) showed that the violation of fitts s law was greater for reaches with the left hand. these discrepancies in both the findings and methodology of previous work warrant further scrutiny. in order to account for an asymmetrical illusory bias for the right and left hands two explanations need to be distinguished. first, the visuomotor networks for action in the dorsal pathway may be more strongly dissociated from the networks for perception in the ventral pathway in the left hemisphere than in the right hemisphere. that is, the encapsulated visuomotor networks for action in the dorsal pathway may have evolved preferentially in the left hemisphere (gonzalez. 2006 ; perenin and vighetto 1988), perhaps as a consequence of human language taking up or co - opting networks in the left hemisphere located between the dorsal and ventral pathways, whereas in the right hemisphere these networks were retained for visual perception (radoeva. all the same, a stronger dissociation between the two pathways in the left hemisphere may limit their interaction, leaving right - handed movements less prone to the use of allocentric illusion - inducing information than movements with the left hand. the evolutionary argument should be distinguished from an experience - dependent explanation concerning the interactive contributions of the two visual systems. gonzalez. (2008), for example, found that unfamiliar awkward grips were much more susceptible to a size - contrast illusion than the precision grips that participants habitually used to grasp small objects. this finding points toward new or less - practiced actions being more reliant on allocentric information than well - practiced automatized actions, perhaps suggesting less encapsulation of the dorsal pathway in early stages of motor learning (gonzalez. if the asymmetry in illusory bias is experience - dependent then its direction should depend on handedness : in participants with right hand preference the illusory bias should be larger in the less practiced grasps with the left hand, while participants with a left hand preference should show a larger bias when grasping with their right hand (cf. the possibility that the observed asymmetry in illusory bias between the right and left hand in fact results from the visual hemifield in which the object is presented, should also be considered. on the same evolutionary perspective (corballis. 2000), networks for visual perception occupy more cortical space in the right hemisphere as compared to the left hemisphere. and also milner and goodale (1995, p. 112 150151) originally argued that hemispheric asymmetry is much more apparent for ventral than for dorsal pathways. indeed, perception research suggests that susceptibility to illusions is higher when they are presented to the left visual field as compared to the right visual hemifield (e.g., clem and pollack 1975 ; rasmjou. 1999), although the evidence is not unambiguous (e.g., bertelson and morais 1983). it is noticeable therefore that the studies that did report an asymmetrical bias for right - handed and left - handed grasping did not systematically control for the visual hemifield in which the illusory object was presented (gonzalez. hence, it can not be ruled out that the different illusion effects for right - handed and left - handed grasping simply reflect differences in visual perception depending on visual hemifield rather than hand use. in the study by radoeva. (2005), for example, participants with damage to the right hemisphere only grasped with their right hand to objects embedded in a mller - lyer configuration that were presented in the right visual hemifield, and conversely, participants with left hemispheric damage solely grasped with their left hand to objects in the left visual hemifield. reaching movements with the left hand were only made to targets presented in the left visual hemifield, while right - handed reaches were only made to targets presented in the right visual hemifield. reaches with the left hand to targets in the left hemifield appeared more vulnerable to visual context. (1997) reported that participants tended to show a somewhat larger illusory bias when pointing to the vertex of mller - lyer configurations located in the left visual field. by contrast, the between - participants comparison for right - handed and left - handed pointing movements did not reveal an asymmetrical illusory bias. in the study that we report, we asked participants in an action task to reach and grasp a shaft embedded in a mller - lyer figure with either their right or left hand. additionally, we asked them in a perception task to match hand aperture to the length of the shaft, again using their right or left hand. we reasoned that the observation of an illusory bias would point to the exploitation of allocentric or contextual information, which presumably reflects contributions of the visual perception system in the ventral pathway. we expected a more reliable illusory effect to emerge for the grasp (i.e., hand aperture) compared to the reach (i.e., reaching movement time), because a mller - lyer figure induces an illusion of size and not of distance. however, given previous reports that allocentric information may be differentially involved in reaches performed by the left and right hand (adam. we specifically tested the proposition that the visuomotor networks in the dorsal pathway of the left hemisphere are more encapsulated (i.e., more dissociated from the visual perception networks in the ventral pathway) than those in the right hemisphere. if correct, then an asymmetry in illusory bias in grasping (and perhaps reaching) should occur with the bias being larger for left - handed movements than for right - handed movements (i.e., showing greater reliance on allocentric information in left - handed movements). we explicitly aimed to disentangle any asymmetrical bias between the right and left hands from influences of handedness (or hand preference) and visual hemifield. hence, right - handed as well as left - handed participants performed both the action and perception task with the shaft presented in the centre of the visual field, in the right visual hemifield (i.e., participants fixated to the left of the shaft) and in the left visual hemifield (i.e., participants fixated to the right of the shaft). the proposition of a left hemispheric specialization for visuomotor networks predicts that an asymmetrical illusory bias in action between the hands would be independent of handedness (instead, an enhanced illusory bias for the non - preferred hand would point to a dependency on experience) and fixation location. alternatively, the proposition of lateralized ventral pathways subserving perception predicts that the illusory bias in perception (and perhaps action, see franz. 2009) would be larger for objects presented in the left visual hemifield than in the right visual hemifield, irrespective of the hand that is used or handedness. twelve male right - handed (age range 1824 years) and twelve male left - handed (1724 years) undergraduate students from the university of hong kong volunteered to participate in the experiment.1 criteria for inclusion were a score of > 50 for right - handers and of 50 for right - handers and of 0.19). of the remaining t - tests (including those for the perception task) all but one were significant (p s 0.06). the lack of a significant illusory bias suggests that differences in reach movement times (if any) are not caused by illusion inducing aspects of the stimulus configurations.fig. 2percentage of illusory bias in reach movement time (and se) as function of hand used and location fixation. the left panel presents the bias in reach movement time bias for the left - handers, while the right panel presents the bias for the right - handers percentage of illusory bias in reach movement time (and se) as function of hand used and location fixation. the left panel presents the bias in reach movement time bias for the left - handers, while the right panel presents the bias for the right - handers optical illusions normally impinge conscious perception of objects to a far greater extent than the movements directed to those objects. the illusory bias in the perceptual length estimate was clearly larger than in the grasping or reaching movements. hence, perception seemed more reliant on allocentric or contextual information, whereas movement control was influenced more by egocentric or context - independent information instead. this is consistent with the two - visual systems model, which proposes that perception and action are dissociated in that they are supported by separate neuroanatomical pathways and, among other distinctions, exploit different sources of information (e.g., milner and goodale 1995, 2008). yet, the present study also showed that there is no strict one - to - one mapping between function (i.e., perception and movement control) and information use. that is, movements were not always immune to illusion - inducing allocentric information, suggesting interacting contributions from the two visual systems to movement control. gonzalez. (2006) argued that because of the encapsulation of visuomotor networks for action in the left - hemispheric dorsal stream, the interactive influences of visual perception on movements controlled by the left hemisphere should be reduced relative to movements controlled by the right hemisphere. accordingly, they reported that right - handed grasps were less susceptible to optic illusions than grasps performed with the left hand (see also adam. replication of this asymmetrical illusory bias for the hands in pointing, however, has not always been possible (de grave. our results showed that the illusory bias was similar for grasping movements with the right and left hand and only occurred for movements that were directed at non - foveated objects irrespective of visual hemifield. in fact, the difference in illusory bias between grasping and reaching movements corroborates evidence that adaptations to the target size usually manifest more in grasping movements than in reaching movements (e.g., paulignan. taken together, we found no evidence that movements performed with the right hand are less prone to the use of allocentric illusion - inducing information than movements with the left hand (see also de grave. 2009). since the illusion effects emerged irrespective of whether the participants used their preferred- or non - preferred hand, also the hypothesis that experience leads towards a stronger reliance on egocentric information (gonzalez. 2008 ; van der kamp. that said, the precision grips required in the present study were relatively automatized movements, even for the non - preferred hand. this may have reduced the likelihood that an asymmetric illusory bias for the preferred and non - preferred hand occurred (gonzalez., we found no indication in our data that the left hand is more susceptible to visual illusions than the right hand, implying that the interactive contributions of the visual systems for perception and action are similar for the two hands. this means that there is no reason at least for the simple grasping task toward mller - lyer targets used the present study to suggest that movement control is lateralized or more strongly encapsulated in either the left or right hemisphere (gonzalez. 2005).3 obviously, the generality of this claim would be further enhanced if future research replicates the current findings in the context of other geometrical illusions, such as the ebbinghaus and ponzo illusions. likewise, a greater role for allocentric illusion - inducing information in grasps to the left visual hemifield in comparison to grasps to the right hemifield was expected from the proposition of a right hemispheric specialization of visual perception networks in the ventral stream (corballis. 1997) found, by comparing two groups of patients with unilateral brain damage, that grasps directed to the left visual hemifield resulted in a stronger illusory bias than grasps directed to the right hemifield. by contrast, although in both the perception and the action task of the current study, the illusion unquestionably affected hand apertures when the shaft was not foveated, the magnitude of the biases did not differ between the hemifields (for a similar finding see bertelson and morais 1983). this is not to deny right hemispheric dominance in visual perception. yet, if one takes the right hemisphere to be relatively dominant among left lateralized persons (corballis. 2000), then the tendency towards a smaller illusory bias in the perception task for the left - handed participants compared to right - handed participants might be inconsistent with this conjecture. at the same time, the absence of the handedness effect in the action task underlines the dissociation between visual perception and movement control. the illusory bias in the grasping movements clearly differed between foveated and non - foveated objects, indicating that participants relied much more on allocentric information when making estimates of or acting upon objects in the peripheral visual field (see also gentilucci. one explanation for this difference is that the magnitude of the illusion depends on gaze. instructions to attend to the shaft and ignore the tails of mller - lyer illusion have been reported to destroy the illusory bias in perceptual size estimates (coren and girgus 1972 ; festinger. (2009) recently claimed that the differential effects of the mller - lyer illusion on action and perception were related to systematic differences in patterns of gaze. they argued that fixation of the different regions of the mller - lyer figure is associated with the detection of egocentric (i.e., regions surrounding the shaft) and allocentric information (i.e., regions surrounding the tails). for instance, van doorn. (2009) found that the more time the participants spent viewing the shaft, the smaller the illusory bias across perception and action tasks. similarly, in the present study, the fixation of the shaft may have significantly reduced the size of the illusion. in fact, the illusory bias of 6.8% for foveated objects in the perception task is at the lower end of the biases that are typically reported (i.e., 518.8%, see bruno and franz 2009). it is, however, a significant bias, and hence, an enhanced exploitation of egocentric information due to fixation on the shaft in itself can not provide a full explanation for the observed difference in the illusory bias for foveated and peripheral objects. alternatively, the larger illusory bias for objects in the peripheral visual field may also point to greater reliance on allocentric information for acting upon non - foveated objects. this would run counter to claims that perception and action exploit different information from the peripheral visual fields (milner and goodale 1995). (1997) observed a large illusory bias for pointing towards mller - lyer figures in the peripheral field, whereas thompson and westwood (2007 : see also binsted and elliott 1999) found no enhanced influence of the illusion on pointing accuracy. however, we think that they indicate that the difference between central and peripheral fields in itself is not crucial here. instead, it is to be expected that grasping (or pointing at) non - foveated objects is less automatized and more awkward than grasping foveated objects,4 and hence, may be more reliant on allocentric information (see gonzalez., we examined the hypothesis that the control of movement of the left hand would be more likely to entail the use of allocentric information than movements performed with the right hand. the hypothesis was derived from the idea that within the left hemisphere, which controls the right hand, the visuomotor networks in the dorsal pathway are more strongly dissociated from the visual perception networks in the ventral pathway than in the right hemisphere. we did not find evidence to support this hypothesis ; the mller - lyer illusion influenced the grasping movements of the right and left hand to the same extent. we also did not find evidence that the illusory effects were different dependent on the visual hemifield toward which the movements are directed. yet, there was a consistent and pronounced effect of the illusion when grasping objects in the peripheral visual field, suggesting that allocentric information becomes more important in the control of grasping movements directed to nonfoveated objects.	we investigated whether the control of movement of the left hand is more likely to involve the use of allocentric information than movements performed with the right hand. previous studies (gonzalez. in j neurophys 95:34963501, 2006 ; de grave. in exp br res 193:421427, 2009) have reported contradictory findings in this respect. in the present study, right - handed participants (n = 12) and left - handed participants (n = 12) made right- and left - handed grasps to foveated objects and peripheral, non - foveated objects that were located in the right or left visual hemifield and embedded within a mller - lyer illusion. they were also asked to judge the size of the object by matching their hand aperture to its length. hand apertures did not show significant differences in illusory bias as a function of hand used, handedness or visual hemifield. however, the illusory effect was significantly larger for perception than for action, and for the non - foveated compared to foveated objects. no significant illusory biases were found for reach movement times. these findings are consistent with the two - visual system model that holds that the use of allocentric information is more prominent in perception than in movement control. we propose that the increased involvement of allocentric information in movements toward peripheral, non - foveated objects may be a consequence of more awkward, less automatized grasps of nonfoveated than foveated objects. the current study does not support the conjecture that the control of left - handed and right - handed grasps is predicated on different sources of information.
scientific workflows attempt to automate repetitive computation and analysis by chaining together related processes. automating repetitive time - consuming tasks allows scientists to keep pace with ever - growing volumes of data (1). furthermore, workflows can aid in the reproducibility of scientific computations by providing a formal declaration of an analysis. reproducibility is central to the scientific method, and detailed workflow provenance information ensures an analysis can be reproduced and extended (1). it has been suggested that in addition to references, scientific publications should include documentation of computational methods that allows readers to easily reproduce their results, increasing reader engagement with scientific publications (2). researchers need to access exponentially growing amounts of dna and protein sequence data along with a large number of bioinformatic analytic tools (3). the data is distributed in different formats and in heterogeneous data structures and information systems. analytic tools are made available to scientists in a variety of ways including as downloadable tools for local installation, web pages and web services. by enabling researchers to work effectively through multiple interconnected tools and handling multiple data formats and large data quantities, workflows with embedded data pipelines and analysis scripts are a powerful alternative to using standalone, off - grid applications (4). the bioextract server (5) is a web - based system that offers researchers a flexible environment for analyzing genomic data. the bioextract server provides : (i) a flexible querying and retrieval interface to national center for biotechnology information (ncbi) nucleotide and protein databases, european molecular biology laboratory (embl - bank) non - redundant nucleotide databases, and the european bioinformatics institute 's (ebi) universal protein resource (uniprot) and uniprot reference clusters (uniref) databases ; (ii) the ability to filter search results and use them as input into analytic tools ; (iii) the facility to save search results as data extracts that are automatically integrated into the system as searchable data resources ; (iv) access to analytic tools including a large list of curated web services, as well as generic access to soap - based web services and command line tools residing on the user 's local workstation ; (v) the ability to save a series of bioextract server tasks (e.g. query a data source, save a data extract and execute an analytic tool) as a workflow ; and (vi) the opportunity for researchers to share their data extracts, analytic tools and workflows with collaborators. queries are constructed by first checking the box for one or more data sources and composing a query via a web form. figure 1 presents an example of querying ncbi nucleotide and protein databases for the r2r3-myb genes in pinus taeda (loblolly pine). queries executed against data sources residing at the bioextract server respond quickly. for example, a query of viridiplantae returns in a matter of seconds. response times for queries against data sources residing at other sites (e.g. ncbi) are consistent with response times for queries made directly at those sites. figure 1.bioextract server queries are executed through the query tab of the system by checking the box for the desired data resource(s), constructing the query using the query form, and submitting the query. bioextract server queries are executed through the query tab of the system by checking the box for the desired data resource(s), constructing the query using the query form, and submitting the query. the execution of the query results in the creation of a data extract that is displayed on the extracts tab of the system. individual records may be viewed locally or through an external link to the original data source. in addition, data extracts may be filtered, exported and used as input into analytic tools. while most of the bioextract server 's functionality is available to all users, the ability to save a data extract is available only to users who have registered with the system. users who are signed into the system as registered users (figure 1, top right) also have the option of saving a data extract simply by clicking the save extract button on the extracts tab and entering the name and description of the extract. once saved, the researcher 's privately owned data extracts are integrated into the bioextract server platform and are listed with other available data resources on the query tab for the registered user only. data extracts may also be created within the system by providing a list of sequence record identifiers (ids) such as accession numbers. on the query tab, researchers are able to fetch sequence records by entering or uploading a list of ids and specifying from what data source the records should be retrieved. all data extracts created within the system are privately owned by the researcher and are only made available to others by explicitly sharing them with a group. groups tab ; (ii) creating a group (under additional actions) ; (iii) clicking on the new group ; (iv) selecting the extracts tab for the new group ; and finally (v) clicking the add elements button to select the extract to share. a number of well - established and unique bioinformatic analytic tools are made available through the bioextract server, with the majority integrated as curated web services. researchers access analytic tools through the list of available tools on the tools tab. the source of the analytic tool 's input(s) may be : (i) the current data extract (i.e. the records listed on extracts tab) ; (ii) the output from a previously executed tool ; or (iii) private data provided by the researcher (uploaded or entered in a text box). analytic tool parameters may be selected or modified before execution and resulting output files may be viewed, downloaded and used as input into subsequently executed analytic tools. the bioextract server offers researchers the ability to add additional analytic tools to their workspace. users may select such tools from a large list of web services, including emboss soaplab, biomoby and kegg, with the integration of biomart (www.biomart.org) currently in development. the system also offers generic support for other soap - based web services and lets users integrate local command line tools residing on their own workstations through the use of a client - side java applet. customize your tools functionality, which allows users to provide detailed descriptions of the tools as well as a help link to additional information. as researchers perform tasks (e.g. execute a query, save a data extract or execute an analytic tool) within the bioextract server, their actions are recorded and saved in the background. the series of recorded tasks may be saved as a workflow through the workflow tab after the user provides a name and description of the workflow. workflows are represented within the bioextract server as directed acyclic graphs with the graph nodes representing specific tasks and the arcs representing the data dependencies. reuse of workflows is realized through the easily modified workflow inputs, queries and analytic tool parameter settings. copying a workflow for future editing is accomplished by executing the workflow and providing a name and description for the copy. once a task within a workflow has been executed, specific task information may be accessed by clicking on the executed node within the workflow graph. additionally, a provenance report related to an executed workflow provides detailed information such as a general description of the workflow, analytic tool descriptions, parameter settings, input / output data, query information and saved data extract descriptions. bioextract server workflows may be shared with the research community in a variety of ways. first, researchers can export a workflow as an xml file and share it with collaborators. when a workflow xml file is imported into the bioextract server by a registered user, a new instance of that workflow is created and privately owned by the user. myexperiment is a virtual research environment designed to provide a mechanism for collaboration and sharing workflows. this was achieved by adopting a social web approach that is tailored to the particular needs of the scientist (6). bioextract server workflows imported into myexperiment may be additionally annotated through their system and executed directly through their platform. third, along with data extracts and analytic tools, workflows can be shared with collaborative groups through the bioextract server groups tab. to explain how a simple workflow is created within the bioextract server, consider the task of carrying out a multiple sequence alignment analysis using a known protein sequence record as input. to complete this analysis, a researcher would : sign into the bioextract server by clicking the sign in link in the upper right corner of the screen.execute the ncbi blastp tool (7) located under the similarity search tools node in the available tools tree on the tools tab. the execution of blastp results in the creation of a data extract that can be viewed on the extracts tab.execute the xmknr analytic tool, a simple shell script that employs vmatch (8) (www.vmatch.de) to remove duplicate sequences, found under the edit tools node. the input into this tool is specified as use records on extract page formatted as fasta. its execution results in the modification of the data extractexecute the clustalw (9) tool to create the multiple sequence alignment with the input specified as use records on extract page formatted as fasta.navigate to the workflow tab and click the create and import workflows node within the researchers are able to annotate the workflow with information that will assist others in understanding decisions made during its creation.the multiple sequence alignment could serve as input for subsequent steps ; for example, phylogenetic or motif analyses. sign into the bioextract server by clicking the sign in link in the upper right corner of the screen. execute the ncbi blastp tool (7) located under the similarity search tools node in the available tools tree on the tools tab. the execution of blastp results in the creation of a data extract that can be viewed on the extracts tab. execute the xmknr analytic tool, a simple shell script that employs vmatch (8) (www.vmatch.de) to remove duplicate sequences, found under the edit tools node. the input into this tool is specified as use records on extract page formatted as fasta. its execution results in the modification of the data extract execute the clustalw (9) tool to create the multiple sequence alignment with the input specified as use records on extract page formatted as fasta. navigate to the workflow tab and click the create and import workflows node within the researchers are able to annotate the workflow with information that will assist others in understanding decisions made during its creation. the multiple sequence alignment could serve as input for subsequent steps ; for example, phylogenetic or motif analyses. more information related to the creation of workflows (including this video demonstration) can be found through the follow link : http://bioservices.usd.edu/bioextract/videos/blastn-clustalw-example.html. the top node denotes a tool that has completed execution, the middle node represents an executing tool, and bottom node represents an idle tool waiting to execute. the top node denotes a tool that has completed execution, the middle node represents an executing tool, and bottom node represents an idle tool waiting to execute. to further illustrate the advantages of the bioextract server platform this workflow represents typical steps undertaken in the primary analysis and comprehensive annotation of a set of transcript sequences. such sets are currently abundantly generated from full - length cdna sequencing projects or assembly of est sequences, and with increasing read lengths of novel sequencing technologies, there will be similar assemblies of rna - seq data from many species and sampling conditions. using ncbi blast (7), museqbox (10) and linux shell scripting, the workflow partitions the input transcript set successively according to matching targets. specifically, mrna markup identifies potential vector or bacterial contaminants (sequencing artifacts), potential chimeras (assembly artifacts), likely full - length protein - coding mrnas, as well as matches from a comprehensive protein database and a protein domain database. what remains after these identifications include presumably novel transcripts for further analysis with other programs. while conceptually simple, implementation and maintenance of the programs in individual labs would be hugely time- and cost - ineffective compared to a publicly available workflow system. for illustration, the workflow is implemented with a sample configuration consisting of a transcript set of arabidopsis est assemblies derived from plantgdb (www.plantgdb.org) and target databases consisting of univec (www.ncbi.nlm.nih.gov/vecscreen/univec.html) for vector contaminants, an escherichia coli genome for determining bacterial contamination, the arabidopsis reference protein set tair10 (www.arabidopsis.org), a comprehensive protein subject database containing viridiplantae entries from uniref90 (www.uniprot.org/help/uniref) and the conserved domain database (www.ncbi.nlm.nih.gov/structure/cdd/cdd.shtml). the final step of the workflow produces a summary report detailing how many sequences were matched during each step as well as how many potentially novel sequences remain. the workflow is presented graphically at bioextract.org / scripts/. when the user clicks the mrna markup label within the list of workflows, a workflow graph will be displayed. the user can click any node in the workflow graph to acquire more information regarding the analytic step it represents as well as the output it produces. there exist many biological data and analytic tool integration systems that offer powerful and comprehensive workflow creation functionality. taverna (11), kepler (12), triana (13), ugene (14) and trident (15) are some examples of workflow management systems that are primarily stand - alone applications requiring installation of client software. through a drag - and - drop user interface, these systems allow researchers to develop complex workflows from distributed and local resources. the taverna workbench, part of the mygrid project (16), is arguably the most well - known bioinformatic workflow development system. it is an application that gives users the ability to easily integrate the growing number of molecular biology tools and databases available on the web, especially web services. genepattern (17), mobyle (18) and galaxy (19) are web - based workflow management systems. genepattern is a genomic analysis platform that provides access to tools for gene expression analysis, proteomics and common data - processing tasks. a web - based interface provides easy access to these tools and allows the creation of multi - stepanalysis pipelines. mobyle provides a flexible web environment for defining and running bioinformatics analysis by embedding management features allowing users to combine tools using a hierarchical typing system. galaxy is an open source workflow system with which researchers are able to create complex workflows that can be shared, cloned, annotated and edited. the primary objective of the galaxy system is to provide support for accessible, reproducible and transparent science (19). the bioextract server is entirely web - accessible, and while allowing completely flexible customization including integration of locally accessed scripts and tools, the interface and functionality of the site are specifically designed for the novice and implementation detail - uninterested user whose primary need is ease of use and quality results. these design criteria seek to explicitly circumvent the gap between workflow developers and the user community, which from our experience is still substantial. future enhancements to the bioextract server will focus on improving workflow reproducibility, flexibility and provenance information. functionality is currently being developed that will provide better feedback regarding a tool 's compatibility with the user - selected input file format. in order for workflows to be reproducible, it is imperative that the required analytic tools be reliable. this underscores the need for curation of analytic tools to ensure they are both available and well defined (4). biocatalogue is a comprehensive, curated registry of biological web services that aims to improve process reliability and validation through individual, automatic and community curation (3). recognizing that the application of web services within a computational experiment can compromise its reproducibility (19), version information will be incorporated into all of the bioextract server 's publicly accessible analytic tools. the intent of a workflow to the research community is an important aspect of a computational experiment. providing researchers with the capacity to annotate and search workflow provenance information, advances the ability to understand the decisions made during the experimental design and execution. the bioextract server provenance information will be expanded to allow for additional user annotations and ad hoc querying of provenance data. national science foundation (dbi-0606909 to v.b and c.m.l.) funding for open access charge : national science foundation.	the bioextract server (bioextract.org) is an open, web - based system designed to aid researchers in the analysis of genomic data by providing a platform for the creation of bioinformatic workflows. scientific workflows are created within the system by recording tasks performed by the user. these tasks may include querying multiple, distributed data sources, saving query results as searchable data extracts, and executing local and web - accessible analytic tools. the series of recorded tasks can then be saved as a reproducible, sharable workflow available for subsequent execution with the original or modified inputs and parameter settings. integrated data resources include interfaces to the national center for biotechnology information (ncbi) nucleotide and protein databases, the european molecular biology laboratory (embl - bank) non - redundant nucleotide database, the universal protein resource (uniprot), and the uniprot reference clusters (uniref) database. the system offers access to numerous preinstalled, curated analytic tools and also provides researchers with the option of selecting computational tools from a large list of web services including the european molecular biology open software suite (emboss), biomoby, and the kyoto encyclopedia of genes and genomes (kegg). the system further allows users to integrate local command line tools residing on their own computers through a client - side java applet.
lung cancer is the leading cause of cancer among men in india. according to international agency for research on cancer, 43,500 indian patients are diagnosed with lung cancer every year and 37,500 die of the disease. adjuvant chemotherapy is generally indicated for patients with resected stages ib through iiia non - small - cell lung cancer (nsclc). elderly patients may have a poor tolerance to chemotherapy due to impaired organ function and frequent co - morbidities. patients with poor performance status (ps 2 due to nsclc and/or coexisting illnesses) are often considered unfit for conventional chemotherapy. alteration of the major cell - signaling and regulatory pathways either by overexpression or gene mutation is a frequent event in lung cancer. the epidermal growth factor receptor (egfr) is a promising target for anticancer therapy because it is overexpressed in a variety of tumors, including nsclc. the egfr is overexpressed in 40 - 80% of patients with nsclc, and is associated with poor prognosis. high levels of egfr expression and deregulation promote tumor growth by increasing cell proliferation, motility, adhesion, invasive capacity, and by evading apoptosis, and therefore have been associated with poorer prognosis in several studies. egfr - tyrosine kinase inhibitors (tkis) inhibit the intracellular tyrosine kinase domain of the egfr, and therefore block the signal transduction pathways implicated in the proliferation and survival of cancer cells. there was lack of correlation with egfr overexpression and response to egfr inhibitors. in a landmark study, it was shown that patients whose tumors demonstrated a mutation in the kinase domain of egfr responded to such inhibitors. gefitinib is an egfr - tki and has recently been approved in several countries for use in advanced or metastatic nsclc. gefitinib monotherapy in patients with advanced nsclc has been investigated in two large, multicenter, randomized phase ii trials : iressa dose evaluation in advanced lung cancer (ideal) 1 and 2 and the iressa survival evaluation in lung cancer study (isel). the study showed an increased efficacy with gefitinib in the subgroup of patients of asian origin and those who were non - smokers. however, there is very little data about the efficacy of the drug in patients of indian origin. we analyzed patients receiving oral gefitinib for advanced nsclc to understand the clinical response in the indian patients. the study included all patients with histology - proven nsclc, who had failed previous chemotherapy regimens, or those chemotherapy - naive patients who were either not willing or unfit for conventional chemotherapy. gefitinib was administered at 250 mg / day until disease progression, unacceptable toxicity, or death occurred. patients who were diagnosed from january 2006 to january 2010 were included, and they were studied till january 2011. data were collected on demographics, smoking status, type of tumor, tumor stage at the time of diagnosis, eastern cooperative oncology group (ecog) ps, previous local treatment, previous chemotherapy status and response, time of starting gefitinib from the time of diagnosis, duration of gefitinib treatment, time to progression, symptom improvement (si) and outcome after gefitinib. detailed information about previous chemotherapy was included, i.e., number of lines, drugs administered, and duration of treatment, treatment - free intervals, and best objective response to each line. response was evaluated according to response evaluation criteria in solid tumors (recist) parameters. patient 's initial evaluation of response was performed after the first month of treatment by chest x - ray and/or computed tomography (ct) scan of thorax. the relationship between patients characteristics and likelihood of response or disease control (i.e., response or stabilization) was tested using a chi - square test in the univariate analysis. overall survival was defined as the period between the date of diagnosis and the date of last follow - up or date of death. time to progression was defined as the period between initiation of gefitinib treatment and the date of progression or date of last follow - up. the relationship between patients characteristics and likelihood of response or disease control (i.e., response or stabilization) was tested using a chi - square test in the univariate analysis. overall survival was defined as the period between the date of diagnosis and the date of last follow - up or date of death. time to progression was defined as the period between initiation of gefitinib treatment and the date of progression or date of last follow - up. the relationship with survival was studied by kaplan - meier analysis. a p value of < 0.05 was considered to be statistically significant. sixty - three patients with nsclc, diagnosed from january 2006 to january 2010, were included in this study. the median age of patients was 63 years (range 39 - 86 years). majority of the patients (n = 55, 87%) had advanced disease [table 1 ]. the most common histopathology type was adenocarcinoma (ad ; 71%), followed by squamous cell carcinoma (18%) and bronchoalveloar carcinoma (bac ; 11%). patients ' demographics complete remission was observed in 1 (1.6%) patient. there were 5 (7.9%) patients with partial remission. there were five patients who were lost to follow - up in our hospital and their response was unknown. the most common toxicity observed in the study patients was skin rash (32%). among the patients with this, one had severe skin rash, leading to discontinuation of treatment. the median duration of gefitinib treatment was 183 days (range 9 - 1094 days). the median duration of follow - up was 311 days (range 11 - 1544 days). the progression - free survival (pfs) was 161 days (95% ci : 124 - 200). the overall response rate (or) which included complete remission (cr) and partial remission (pr) was 11%, and disease control rate (cr + pr + stable disease (sd)) was 49%. the response was better among females (p = 0.028), nonsmokers (p = 0.065) : [figure 1 and 2 ] and previously untreated patients (p = 0.053). there was no statistically significant difference found with regard to pfs between patients who received gefitinib as first - line chemotherapy and those who received it as subsequent chemotherapy. on multivariate analysis, none of the variables showed statistically significant difference in relation to or or pfs [table 3 ]. survival according to sex of the patients (female, --- ; male,) survival according to smoking status of the patients (nonsmoker, --- ; smoker,) multivariate analysis sixty - three patients with nsclc, diagnosed from january 2006 to january 2010, were included in this study. the median age of patients was 63 years (range 39 - 86 years). majority of the patients (n = 55, 87%) had advanced disease [table 1 ]. the most common histopathology type was adenocarcinoma (ad ; 71%), followed by squamous cell carcinoma (18%) and bronchoalveloar carcinoma (bac ; 11%). complete remission was observed in 1 (1.6%) patient. there were 5 (7.9%) patients with partial remission. there were five patients who were lost to follow - up in our hospital and their response was unknown. the most common toxicity observed in the study patients was skin rash (32%). among the patients with this, one had severe skin rash, leading to discontinuation of treatment. the median duration of gefitinib treatment was 183 days (range 9 - 1094 days). the median duration of follow - up was 311 days (range 11 - 1544 days). the progression - free survival (pfs) was 161 days (95% ci : 124 - 200). the overall response rate (or) which included complete remission (cr) and partial remission (pr) was 11%, and disease control rate (cr + pr + stable disease (sd)) was 49%. the response was better among females (p = 0.028), nonsmokers (p = 0.065) : [figure 1 and 2 ] and previously untreated patients (p = 0.053). there was no statistically significant difference found with regard to pfs between patients who received gefitinib as first - line chemotherapy and those who received it as subsequent chemotherapy. on multivariate analysis, none of the variables showed statistically significant difference in relation to or or pfs [table 3 ]. survival according to sex of the patients (female, --- ; male,) survival according to smoking status of the patients (nonsmoker, --- ; smoker,) multivariate analysis this single - center study was an analysis of the patients with advanced disease nsclc, who received treatment with gefitinib either as first - line or as subsequent line of treatment. the drug was prescribed when conventional chemotherapy was felt to be not feasible or as too toxic for the individual in the first- and second - line setting. response rate of ideal 1 was 18.4, ideal 2 was 12%, single agent gefitinib (sign) study was 13.2%, and iressa survival evaluation in lung cancer (isel) india was 14%. thus, our studies or was comparable to that of previous studies which were conducted in an unselected group of patients. activating mutations in the egfr gene contribute to tumor progression, and also therefore confer hypersensitivity to the tkis, gefitinib and erlotinib, in patients with advanced nsclc. mutations in egfr and kras are the most commonly observed alterations in nsclc. as per previous studies response rates in egfr mutation - positive patients receiving gefitinib after previous chemotherapy have been reported to be between 76% and 91% in asian studies and between 58% and 70% in non - asian patients. this study was conducted in unselected patients, and response rate (rr) was 11%. it may have been better if egfr status was evaluated as shown in other studies in selected patients. there was no survival benefit noted in our study, which was similar to the results obtained in isel and iressa pan asia study (ipass). the median duration of treatment with gefitinib was 6.1 months (range 9 - 1094 days). improvement in symptoms was reported in 80% of the patients, the majority of such symptoms being dyspnea, cough, effort tolerance, and general well - being. patients of asian origin, never - smoking status, ad histology, and female sex have been reported to be the predictors of improved response in previous randomized trials of tkis. the nonsmoker status of lung cancer patients is the strongest indicator of activity of gefitinib in western populations. in our study, on univariate analysis, female sex and nonsmoker status were associated with an improved outcome. in previous studies, ad histology, especially the ad - bac subtype, is usually associated with higher likelihood of response. in our study, patients with ad or bronchoalveolar type had a higher or when compared with non - adenocarcinoma patients (44% vs. 19%). table 4 shows comparison of the present study data with the results of previously conducted clinical trials. our study showed median survival time of 10.4 months and 1-year survival of 44% (28/63). the response rate (11%) in the present study was lower than that obtained in the chinese study (27%) and taiwanese study (33.3%). this could be attributed to different frequency of mutation in egfr in indian patients compared to studied populations. summary of phase ii / iii gefitinib 250 mg / day efficacy data on unselected patients with advanced nsclc and comparison with the present study data the strength of the study is its adequately large sample size to say the results were statistically significant. this is the first indian study in which gefitinib was evaluated in unselected patients with nsclc. the data of the study may be helpful for planning future multicenter studies for more appropriate documentation of role of gefitinib in indian population. it will also be interesting to see the incidence of egfr mutation rate in tumors (nsclc). there are more improved drugs that inhibit egfr, in addition to erlotinib and gefitinib that are in trial. it is now possible to initiate treatment in a patient with an egfr inhibitor as primary chemotherapy if the tumor is positive for a mutation and has a wild k - ras. egfr mutation - positive status highly correlates with a favorable response, and in case of nsclc patients, -positive status highly correlates with a favorable response, and in case of nsclc patients, it should be done. but in resource - limited countries like india, where the test is not easily available, in case of advanced nsclc patients, treatment with gefitinib is still a viable option. female sex and nonsmoking status are the variables related to positive response in the present study. gefitinib therapy of 250 mg / day has a favorable toxicity profile and is well tolerated.	background : gefitinib, an epidermal growth factor receptor - tyrosine kinase inhibitor, represents a new treatment option for patients with advanced non - small - cell lung cancer (nsclc). we analyzed the data of patients who received gefitinib for nsclc in a tertiary care center in south india.materials and methods : sixty - three patients with advanced nsclc who had received gefitinib either after failure of conventional chemotherapy or were previously not treated as they were unfit or unwilling for conventional treatment were included in the analysis.results:the median follow - up for the cohort was 311 days (range 11 - 1544 days). median time to progression was 161 (range 9 - 883) days. complete and partial remission was seen in 1 (2%) and 6 (9%) patients, respectively, with overall response rate of 11%. twenty - four (38%) patients had stable disease. gefitinib was well tolerated with no significant side effects.conclusion:gefitinib shows anti - tumor activity in pretreated or previously untreated patients with advanced nsclc. it has a favorable toxicity profile and is well tolerated. gefitinib should be considered as a viable therapy in patients with nsclc.
the widespread use of pesticides contributes to population declines and mortality of birds in agroecosystems. among the various categories of pesticides, insecticides typically present a higher risk of acute effects [2, 3 ] because of their elevated inherent toxicities and high potential for exposure. documented cases of mass mortality by intoxication and the various studies reporting negative effects of insecticides on birds are clear evidence of the risk posed by insecticides on wild bird species (e.g., [211 ]). one case of mass mortality of birds that received a great deal of attention in argentina was the 1995 - 1996 mortality of swainson 's hawks caused by monocrotophos, an organophosphorus insecticide [12, 13 ]. after this event, monocrotophos registration was cancelled in argentina while the pyrethroid insecticides gained in importance and popularity. among insecticides, pyrethroids are a class of neurotoxic synthetic insecticides widely used due to their relative safety to mammals and birds, high insecticidal potency at low dosages, and fast biodegradation. insect axon sodium channels are 100-fold more sensitive to pyrethroid esters than mammalian channels. for these reasons, several studies about pyrethroids have been conducted on vertebrates (e.g., [1618 ]), the majority on rodents (e.g., [1923 ]). the neurotoxicity of pyrethroids to mammals depends on the stereochemical configuration and the pyrethroid structure [24, 25 ]. in contrast, little is known of the toxicity of pyrethroids to birds, probably because this class of insecticides is generally considered to have negligible toxicity to birds. beta - cyfluthrin (cyano-(4-fluoro-3-phenoxyphenil)-methyl-3-(2,2-dichloroethenyl)-2,2-dimethyl - cyclopropanecarboxylate) is the active ingredient of insecticide formulations used to control a wide variety of pests on cotton, corn, sunflower and soybean crops. like other pyrethroids, beta - cyfluthrin presents stereoselective interaction with a fraction of the sodium channels of the neuronal membranes, resulting in a prolongation of the inward sodium currents evoked in neurons by every incoming pulse of excitatory stimulation [2628 ]. beta - cyfluthrin is a mixture of four diastereomers, with diastereomers ii and iv predominating and determining chemical and physical properties of the substance [29, 30 ]. beta - cyfluthrin is a type ii pyrethroid, with a characteristic cyano group on the alpha carbon. type ii esters keep the sodium channel open for a more prolonged time period than type i esters. the main signs of intoxication of type ii pyrethroids in mammals include choreoathetosis and salivation (cs). the reported ld50 of beta - cyfluthrin to birds in the pesticide manual is > 2000 mg / kg in the japanese quail. database (http://cfpub.epa.gov/ecotox/) gives the same value, but associated with the northern bobwhite. yet, unpublished information cited in a report without details about vehicle or formulation gave an ld50 of ca. 100 mg / kg for beta - cyfluthrin in canaries as well as the more usual values for northern bobwhite and mallard duck of > 2000 mg / kg. this value for canaries, if real, casts doubts on the general wisdom that pyrethroids are non - toxic to birds. the present study had for objective independent corroboration of the ld50 of beta - cyfluthrin to canaries and determining the acute oral toxicity of commercially formulated beta - cyfluthrin to two novel and wild species, the shiny cowbird (molothrus bonariensis) and the eared dove (zenaida auriculata). the study was carried out in the research facilities of the inta (instituto nacional de tecnologa agropecuaria) at the paran agricultural experimental station (315053s, 603219w). the study was carried out in an aviary of 20 10 m, including an acclimation area with 6 groups of pens (each 3 2 3 m) and 24 individual test cages (each 0.5 0.5 0.5 m). the photoperiod and the average temperature of the testing room during the dosing were recorded (tables 2 and 3). the ventilation was controlled so as to maintain the indoor conditions of temperature and humidity within outdoor environmental ranges. the wild birds, shiny cowbirds, and eared doves were selected based on their large numbers in surrounding fields, which assured their availability, abundance, and capture success. healthy adult birds were weighted and grouped by sex before being acclimated to experimental conditions for at least 14 days. shiny cowbirds were fed insectivore certified commercial food, eared doves were offered a mix of wheat and sunflower seeds, and canaries, a commercial seed mix and ground egg. bottled water for human consumption was offered ad libitum to all species. because of the absence of a constituted animal care committee at inta or at the local university (universidad nacional del litoral) which provided academic supervision of this research, guidelines of the denver wildlife research center of the us department of agriculture were followed for the capturing, transportation, housing, care, euthanasia, and necropsy of the birds, in addition to other procedures of the study [3345 ]. to obtain the test doses (mg beta - cyfluthrin / kg body weight), we used a commercial formulation (bulldock of bayer cropscience), a suspension of 12.5 g a.i./100 ml of unreported inert ingredients. we assumed label concentration was correctly reported and administered to birds the necessary volume of formulated product corresponding to the required dose of beta - cyfluthrin. it is known that pyrethroid toxicity may be greatly influenced by the dosing vehicle. because wild birds are exposed to formulated products, we opted to test the formulation without an additional vehicle where possible and with distilled water as a diluent for several doses for canaries (see dilutions in the footnotes of table 3). doses were calculated according to standard equations for each stage of the approximate d - optimal design, in milligrams of a.i. per kilogram of body weight, as shown in table 3. the dosing volumes were calculated based on individual body weights measured within 12 hours of dosing (table 1). to prevent regurgitation, the higher dose volumes (> 0.17 ml for canaries, > 0.45 ml for shiny cowbirds, and > 1.0 ml for eared doves) were split and administered in up to four aliquots separated by 15 minutes. this split administration of doses took place for all species in the limit test, one canary in the first stage of the full test and all shiny cowbirds and eared doves in all stages of the full test (table 1). dose volumes never exceeded 16 ml / kg bw (body weight) in canaries, 27 ml / kg bw in shiny cowbirds, and 26 ml / kg bw in eared doves. individuals that regurgitated part or all of a dose and who survived the dose were substituted for others due to the fact that regurgitation modifies the dose and prevents the correct approximation of the ld50. forty - six percent of shiny cowbirds, 33% of eared doves and 16% of canaries regurgitated, despite being fasted before the dose. acute oral toxicity tests were carried out following draft guideline 223 of the organisation for economic cooperation and development. first, five individuals of each species were treated with a limit dose of 2000 mg / kg of test chemical. following any mortality at this limit dose, ld50 were estimated in sequential stages with the approximate d - optimal design (full test ; figure 1). in canaries, the first stage of the full test was carried out to confirm and improve the initial estimate of the canary ld50 (250 mg / kg, based on the aforementioned literature value and the result of a limit test). birds were randomly assigned to each test and were observed for 14 days after the dose. mortality, clinical symptoms, change in weight between the beginning and the end of the study, regurgitations, time to death (in hours), and recovery were recorded. also, livers and hearts were weighed and their relative weights calculated (1), in order to detect any pathology associated with any loss or increase in mass of these organs (hepatomegaly, necrosis, hypertrophy, etc.). we fit a probit model to the combined data from all stages (stat - sas 6.1) to obtain the ld50 estimates, confidence intervals confidences and slopes of dose - response curves. both the initial and final body weights and the relative weights of hearts and livers were compared by one - way anova using spss v.10 for windows. initial ld50 estimates obtained for the limit tests were 2247 mg / kg for both shiny cowbirds and eared doves because 40% of individuals died in both species. by contrast, all treated canaries died, and it was, therefore, impossible to obtain an initial estimate of ld50 with the limit test (table 2). with canaries, the ld50 values estimated at each sequential stage were 68 mg / kg, 110 mg / kg and 170 mg / kg, respectively. during the additional stage (similar to the third stage, performed in order to decrease the confidence intervals of the ld50), two of four individuals that received the highest dose regurgitated, and, for this reason, they were not included in the results. for shiny cowbirds and eared doves, although the doses administered in the second stage were the same because of similar results in the limit test, the mortality was different (table 3). the ld50 estimates after the second stage were 1589 mg / kg and 2338.6 mg / kg for shiny cowbirds and eared doves, respectively. the final ld50 estimates, obtained by fitting a probit model to the combined data of all stages for each species were 170 41 mg / kg for canaries, 2234 544 clinical signs included ruffled appearance, salivation (evidenced by constant deglutition movements and head shaking), decreased activity, prostration, panting, labored breathing, body tremor, balance loss and/or convulsions. signs appeared shortly after dosing and lasted from a few minutes to a few hours. there were doses that did not produce clinical signs and others that allowed recuperation of individuals with signs of intoxication, including convulsions (tables 2 and 3). there were no significant differences between the body weights of the survivors before dosing and 14 days after the dose, except for canaries in the third stage of the full test where 14-day postdose weights were higher than predose weights (p =.018). maximum time to death was 1.75 hours in canaries, 3 hours in eared doves, and 5 hours in shiny cowbirds. only canaries showed a tendency toward a shorter time to death with increasing dose (figure 3). relative weights of heart and liver (rl and rh) did not vary either (p >.05 in all cases). the ld50 value we obtained in canaries approximates the value presented in the report on beta - cyfluthrin of the european commission, confirming it as moderately toxic or class ii for this species. ld50 values for formulated beta - cyfluthrin obtained in shiny cowbirds (another passerine species) and eared doves, are close to the values reported for bobwhite quail and japanese quail [29, 30 ], confirming beta - cyfluthrin to be practically non - toxic or class iii for those species. the different sensitivity to beta - cyfluthrin between canaries and other tested birds may be due to differences in the characteristics of the sites of toxic action, the intestinal absorption, the metabolism, and/or the elimination of this substance. for instance, the low acute toxicity of cypermethrin to quail had been explained by a high resistance of the cns of quails to the lethal effects of cypermethrin, a greater extensive metabolism to a large number of products, and a rapid elimination in the excreta. it is possible that similar physiological mechanisms occur in shiny cowbirds and eared doves, explaining their low susceptibility to beta - cyfluthrin. in addition, both significant differences in sensitivity at the sites of toxic action and metabolic differences and different main detoxification routes and enzymatic activities [5159 ] may play a major role in the differential responses to these insecticides. on the other hand, differences in body size among the three bird species included in this study could have influenced the results., the small body size of the canary compared to the other two species might have contributed to the higher sensitivity. factors related to the experimental setting and our procedures could have influenced the results of this study. a relationship between temperature and the toxic effects of pyrethroids has been shown for several groups of animals, including insects, amphibians and mammals [51, 6164 ]. in our study, variations were noted in the ambient temperatures at the time of each stage of testing (table 3). however, even when variation was slightly higher in the case of shiny cowbirds, it is unlikely to have influenced results because we obtained the ld50 through combined data from sequential stages with different temperatures. additionally, the varying dose volumes and split administrations may have increased variability between experimental animals. in our work canaries were administered a volume well below those used for the other two species (table 1).. showed that increasing the amount of corn oil delivered with a dose of bifenthrin, another synthetic pyrethroid, changed the time course and potency of the pesticide in rats (a two - fold difference in potency was seen for a 5-fold increase in corn oil). the use of oil as dosing agent should be avoided when highly lipophilic pesticides are being tested. we hope to have avoided this problem by dosing with the neat formulation where possible, and therefore not changing the relative concentration of pesticide in the dosing solution. comparing the number of aliquots (table 1) to the mortality in the tests (table 3), suggests that the exact dosing regime probably did not affect our test results to any great extent. finally, because we tested formulated material (with unknown inerts) rather than the active ingredient, we can not ascertain definitively whether the canary is more sensitive to the active ingredient or to one of the formulants. however, the similarity to the value cited for the a.i. by the european union and the relative lack of toxicity of the formulants in the other two species (with ld50 values similar to those obtained in quail or mallard with the active ingredient alone) suggests differences in sensitivity to the pyrethroid and not to the inerts included in the formulated material. clinical symptoms observed in response to high doses in the three species of bird are in agreement with those described by sheets. in rats treated with beta - cyfluthrin and those observed by qadri., who tested permethrin and cypermethrin in chickens. these symptoms consisted of decreased activity, tremors in the whole body, salivation, agitated breathing, flattened posture, and choreoathetosis. nervous intoxication symptoms were observed a short time after intake and lasted up to a few hours, indicating that the removal of pyrethroids from the nervous system is rapid. the survivors from all species did not show loss of body weight, at least by the end of the 14-day observation period (table 4). moreover, the body weights of canaries increased significantly during the third stage of the full test. singh observed increased body weights after acute treatment with beta - cyfluthrin on albino rats. these authors postulate that the increase in body weight may be due to excessive food and water intake and increased food conversion efficiency of treated groups as compared to controlled ones. all deaths occurred within 24 hours after the dose, probably because peak levels in blood, liver, muscle and brain are reached the first day of treatment [25, 70 ]. times to death in shiny cowbirds and eared doves were quite similar to those obtained with fenvalerate by mumtaz and menzer in japanese quail (4 to 8 hours). rapid recovery is a characteristic of poisoning with pyrethroids in mammals. in the three bird species, regurgitations were not as frequent in canaries, suggesting differences in their physiological capacity to regurgitate. since the individuals that regurgitated were substituted for others, the results were not influenced by regurgitation in this study. acute and subacute studies have shown that pyrethroids at high doses cause liver hypertrophy, and, if death does not occur, these changes have been shown to be reversible [72, 73 ]. nevertheless and as in yavasoglu., who worked with cypermethrin in rats, relative liver weights (rl)as an indirect measure of changes in liver health or function did not show any effect of dose. although there are in vitro studies regarding the effects of pyrethroids on the cardiac muscle of rats and guinea pig [7577 ], effects associated with loss or increase of heart mass were not detected here. in conclusion, although there were factors that possibly exacerbated the differences of susceptibility within or between species (e.g., varying dose volumes, multiple dosing scheme, variable ambient temperatures, body size, etc.), the high sensitivity of canaries to beta - cyfluthrin was corroborated. on the other hand, formulated beta - cyfluthrin was found to be practically non - toxic to shiny cowbirds and eared doves. these results emphasize the need to test a broader range of species before generalizing about the toxicity of pyrethroids (and possibly other pesticides) to birds. in the case of beta - cyfluthrin specifically, although low application rates are generally used in the field (7.520 g a.i./ha according to tomlin), it is necessary to consider the potential variation in the toxicity of this pesticide in order to fully assess its safety to birds. in the present case, a species sensitivity approach would suggest that other species of birds, especially small bodied species, will show higher sensitivity to pyrethroids. future research is needed to explain why the canaries are more sensitive to beta - cyfluthrin, to determine whether canaries are similarly more sensitive to other pyrethroids, and more importantly, whether wildlife species related phylogenetically to canaries also present a high sensitivity to pyrethroids.	it is generally assumed that the toxicity of pyrethroid insecticides to birds is negligible, though few species have been tested. the oral acute toxicity of formulated beta - cyfluthrin was determined for canaries (serinus sp.), shiny cowbirds (molothrus bonariensis), and eared doves (zenaida auriculata). single doses were administered to adults by gavage. approximate lethal doses 50 (ld50) and their confidence intervals were determined by approximate d - optimal design. canaries were found to be substantially more sensitive to formulated beta - cyfluthrin (ld50 = (170 41) mg / kg) than the other two species tested (ld50 = (2234 544) mg / kg and ld50 = (2271 433) mg / kg, resp.). the ld50 obtained for canaries was also considerably lower than typical toxicity values available in the literature for pyrethroids. this study emphasizes the need for testing a broader range of species with potentially toxic insecticides, using modern up and down test designs with minimal numbers of birds.
factors contributing to the steady rise in prevalence of periodontal disease include poor oral health awareness. annual health budget is 2% of gross national product but no specific budget is earmarked for oral health. oral health knowledge is considered to be an essential prerequisite for health - related behavior. although only a weak association exists between knowledge and behavior in cross - sectional studies, there are studies that establish an association between knowledge and better oral health. we keep reading about studies done to judge the oral health and oral hygiene practices at so many places scattered all across the globe. according to the literature, no study has been done to assess the oral hygiene awareness in jodhpur till now, which, is the second largest city in rajasthan with an area of 22,850 square km and population of 3,685,681. this study was therefore initiated in vdch with a prime focus to determine the awareness levels among the patients visiting vdch, jodhpur, from jodhpur 's urban and rural localities. to assess the oral health awareness among people of jodhpur, rajasthanto learn about their oral hygiene practicesto provide insight into educational programs that have to be organized by health care professionals. to assess the oral health awareness among people of jodhpur, rajasthan to learn about their oral hygiene practices to provide insight into educational programs that have to be organized by health care professionals. to assess the oral health awareness among people of jodhpur, rajasthanto learn about their oral hygiene practicesto provide insight into educational programs that have to be organized by health care professionals. to assess the oral health awareness among people of jodhpur, rajasthan to learn about their oral hygiene practices to provide insight into educational programs that have to be organized by health care professionals. a cross - sectional study was conducted among the patients attending the outpatient department of vdch, jodhpur. a self - constructed 16-item close - ended questionnaire was distributed to all subjects above 18 years of age in english and was filled by a dental professional for illiterate persons [figure 1 ]. the questionnaire included information related to the patient 's name, age, gender, occupation, and residential area it was further categorized to evaluate the knowledge, practices, and behavior pattern related to oral health. the completed questionnaires were then analyzed statistically to obtain the results in terms of percentages. in the present study, questionnaire was distributed to 500 patients who were selected randomly. of the 500 participants, fifty - six percent of these participants were literate and the remaining 44% were illiterate. almost 58% of participants used tooth brush as a method to clean their teethin response to other methods of cleaning their teeth, 17% were using salt with finger, 18% indicated use of neem stick and 23% charcoal, 34% use tooth powder with finger, whereas 8% of the subjects even resort to using brick powder with finger [figure 2]forty - four percent people brush their teeth occasionally, 33% of them were brushing once a day, 23% were brushing twice a day, and none of them brushed more than 2 times a day [figure 3]approximately 50% of the participants were unaware about the type of tooth brush used by them and only 10% of the sample use soft tooth brush [figure 4]around 75% of the subjects brush their teeth in horizontal direction, which is the most dangerous method of brushing [figure 5]none of the patients use dental flossonly 10% uses mouth wash [figure 6]although 40% noticed bleeding from gums, only 10% among them want to get their teeth cleaned [figure 6]surprisingly enough, 50% of the population, visit dentist only in pain [figure 7]only 20% of the sample cleans their tongue [figure 8]twenty - nine percent of study sample rinse their mouth after eating a meal [figure 8 ]. almost 58% of participants used tooth brush as a method to clean their teeth in response to other methods of cleaning their teeth, 17% were using salt with finger, 18% indicated use of neem stick and 23% charcoal, 34% use tooth powder with finger, whereas 8% of the subjects even resort to using brick powder with finger [figure 2 ] forty - four percent people brush their teeth occasionally, 33% of them were brushing once a day, 23% were brushing twice a day, and none of them brushed more than 2 times a day [figure 3 ] approximately 50% of the participants were unaware about the type of tooth brush used by them and only 10% of the sample use soft tooth brush [figure 4 ] around 75% of the subjects brush their teeth in horizontal direction, which is the most dangerous method of brushing [figure 5 ] none of the patients use dental floss only 10% uses mouth wash [figure 6 ] although 40% noticed bleeding from gums, only 10% among them want to get their teeth cleaned [figure 6 ] surprisingly enough, 50% of the population, visit dentist only in pain [figure 7 ] only 20% of the sample cleans their tongue [figure 8 ] twenty - nine percent of study sample rinse their mouth after eating a meal [figure 8 ]. different types of cleaning methods other than tooth brushing types of tooth brush being used different types of brushing technique percentage of people noticed bleeding from gums and smell from mouth frequency of dental visit percentage of people cleaning their tongue and using mouth wash it has been observed that oral hygiene has mostly remained as an ignored and unrealized major social problem. majority of the people are unaware about the relationship between oral hygiene and systemic diseases or disorders. most diseases show their first appearance through oral signs and symptoms and they remain undiagnosed or untreated because of this missing awareness. according to the consumer usage and attitudes study done in 2010, among the most shocking of revelations is that nearly half of the indian population does not use a tooth brush and only 51% brushed their teeth using a tooth brush and toothpaste. over the past 20 years a significant amount of emphasis has been made on prevention of diseases rather than the treatment aspect. preventive oral health knowledge, behavior, and its practice are the important ways of keeping our teeth healthy. hence, in this study attempts were made to evaluate preventive oral health knowledge, practice, and behavior of the population of jodhpur. although brushing was the commonly used method of cleaning, the percentage of subjects brushing their teeth twice daily is 23% [figure 3 ], which is very less as compared with 58% of the police recruits in a study by dilip, 67% of the chinese urban adolescents in a study by jiang, 62% of the kuwaiti adults in a study by al - shammari, and 50% of the middle aged and 75% of the elderly chinese adults in urban areas in a study by zhu. it is noteworthy that 75% of the respondents brushed their teeth using traditional horizontal method, which will jeopardize the tooth structure [figure 5 ]. this finding is in agreement with that of the study done by zhu. only 10% of the subjects use soft brush, which is less than that observed among zhu. thirty percent change their toothbrush once in 3 months, and surprisingly 60% change their brush only when it is useless [figures 4 and 9 ]. percentage of people changing toothbrush there is generally a failure in the use of dental floss as a preventive tool. none of the subjects has used dental floss, which is similar to a study conducted by hanaa m. jamjoom in saudi arabia in 2001. in contrast, hamilton and couby found that a high percentage (44%) of the sample they studied in northeastern ontario used dental floss. reason for this may be the significant resource allocation to health education programs that are carried out in canada. this emphasizes the urgent need for educating and motivating the public to use this efficient method for oral health care. only 20% of the studied population showed that they clean their tongue either with tooth brush or tongue cleaner. this missing and very basic method of maintaining oral hygiene is a clear indication of lack of awareness. this study is in contrast with that of an epidemiologic survey of the general population of japan where 24% of the individuals examined complained about bad breath. buhlin. who showed that self - reported bleeding gums was high in percentage. this study is in contrast with the studies of nagarajan and pushpanjali in india, tervonen and knuttila, and kallio. who showed that most of the patients did not notice bleeding from gums. our study showed that 54% of the subjects visited a dentist when they were in pain, which is similar to the study done by nabil al - beiruti, in 1997, where 69.5% of the participants reported visiting a dentist only when they have pain. unsurprisingly, standards of oral health are very poor in india, with a large proportion of the population being affected due to poor socio economic conditions. missing awareness about the crucial role of regular dental checkups in preventing and detecting dental diseases is another gap in public education. as dentists, the indifferent results of this study in jodhpur are a critical indicator of the task on hand, the task to spread awareness among the masses about oral hygiene. the information on developments in vital combination of oral hygiene, oral diagnosis, and overall health needs to be spread by us, the dentists. establishing and demonstrating this connection will be critical to achieve this goal and this process will have to be taken at all levels including a definite beginning with our patients. we, as dentists, will have to keep reinforcing the importance of correcting all aspects related with brushing and flossing along with the importance of regular checkups. the task of spreading this awareness extends beyond our clinic to general masses and it will have to be achieved in a similar way by various outreach programs and relevant public health awareness measures through various mediums, such as print / press media, audio / radio / television, internet, and organizing social activities. all of these and more innovative methods of reaching the public will not only ensure a healthy individual but a healthy society as well.	background : according to world oral health report 2003, the prevalence of periodontitis is 86% in india. dental care can sometimes be a forgotten part of a healthy life style. while its importance is often underestimated, the need for regular dental care can not be overstated. oral health has been neglected for long in india. the scarce literature on dental health awareness, attitude, oral health - related habits and behavior among the adult population in rajasthan prompted us to assess the preventive oral health awareness and oral hygiene practices in patients attending outpatient department of vyas dental college and hospital (vdch), jodhpur through this study.materials and methods : a total of 500 patients in the age group 1550 years were selected using random sampling technique. a self - administered structured questionnaire including 16 multiple choice questions was given to them. the results were analyzed using percentage.results:the result of this study shows an acute lack of oral hygiene awareness and limited knowledge of oral hygiene practices. in jodhpur, few people use tooth brush.conclusions:hence, there is an urgent need for comprehensive educational programs to promote good oral health and impart education about correct oral hygiene practices.
breast cancer (bc) is the most common cancer among american women, with more than 200,000 new cases diagnosed each year. the lifetime risk for developing breast cancer has been reported to be as high as 12%, whereas the risk of death has been reported to be as high as 5%. in mexico in 2007, there were 16,340 reported hospitalizations for breast cancer and 4,872 breast cancer - related deaths. the introduction of mammography screening protocols in recent decades has facilitated the detection of breast cancer at increasingly earlier stages. for example, in the usa, stage 0 or i disease was found in 56% of cases in 1995 in comparison to only 45% in 1985. today, we are able to detect bc at earlier stages ; however, according to the literature, cases of locoregional and distant recurrences have been reported in 5 to 40% of cases. this wide range of reported results is probably due to inadequate axillary dissection, incomplete surgical technique, or suboptimal systemic treatment. the involvement of the axillary lymph nodes (lns) is the most important prognostic factor for recurrence in the early stages of bc according to the literature. patients with cancer - positive ln have been reported to have a four to eight times higher mortality rate in comparison to patients with negative lymph nodes. there is also a direct correlation of positive ln status with the risk of distant recurrence. in patients with negative ln, patients with tumors that were smaller than 1 cm in diameter had an overall 5-year survival rate of 99%, whereas patients with tumors of 35 cm in diameter had a survival rate of 86%. the scarff - bloom richardson (sbr) grading system takes into account the mitotic index and the differentiation and pleomorphism of the tumor. according to these characteristics, tumors can be well-, medium-, and poorly - differentiated (grades 1, 2, or 3, resp.). grade 3 tumors have a relative recurrence risk of 4.4 times higher compared to the reference group. this prognostic factor has a significant relevance in patients with small tumors and negative lns. in clinical stage i, some studies have reported a recurrence risk of 38% when lymphovascular invasion (lvi) is present in comparison to 22% in cases where lvi is absent ; however, some studies have not found any differences. histologically, tubular, mucinous, tubulolobular, and cribriform breast tumors have the best prognoses. ductal, lobular solid, and mixed - type (ductal and lobular) tumors have a 10-year os in only 50% of cases. the worst prognosis occurs with inflammatory carcinoma, which has a 10-year os in 30% of cases. hormone receptor status (hr) is both a prognostic and predictive factor in bc. a recent study has found that patients with positive hr had a higher percentage of 5-year dfs and os. other authors have found that hr are a prognostic factor, and hr are a strong predictive factor in relation to adjuvant treatment with tamoxifen or aromatase inhibitors [14, 15 ]. c - erb b-2 (her2/neu) is present in 2030% of bc patients. her2/neu is detected in tumors that are hr negative, have lymphatic infiltration, have high mitotic indices, and are bcl-2 negative. patients with positive lns and the presence or absence of her2/neu expression have a 10-year os in 50% and 65% of the cases, respectively. her2/neu expression can be used as a predictive factor of patient response to alkylating chemotherapy. tumors with her2 neu expression responded well and had a better survival rate in comparison to her2 neu negative tumors, when treated with anthracycline - based chemotherapy. the expression of her2/neu is a predictor of patient response to trastuzumab (herceptin), wherein 4% of patients have a complete response, 17% a partial response, and 30% stable disease. the grade of tumor proliferation can be measured in several different ways, and these different methodologies have been evaluated as prognostic factors. some of these different methodologies include ki-67, the mitotic index, and the s - phase fraction [2022 ]. genetic profiling (microarray) is typically used to identify gene expression profiles, which could help in the identification of prognostic or predictive factors in patients with small tumors and negative ln. studies have reported increased bc - related death rates among obese patients, in addition to a twofold increased risk of a contralateral breast cancer. the intent of this study was to analyze the prognostic and predictive factors that are responsible for locoregional or distant recurrences in a group of patients treated at the national cancer institute of mexico. this retrospective transversal study included all of the patients who presented with a recurrence of a treated breast cancer from january 1 to december 31, 2007, at the national cancer institute of mexico (incan). a total of 11,144 patients were treated from january 1, 1986, to december 31, 2006, and a total of 60 of these patients showed a recurrence in 2007. the control group consisted of 60 other patients who were matched by clinical stage, time, type of treatment, and age. all of the information used in this study was obtained from the clinical archive of the incan. the revised data used in this investigation consisted of pathology reports of true cut biopsies or the definitive operative material. in the majority of cases, disease recurrence was diagnosed using imaging techniques (ct of the lung and abdomen using a siemens somatom volumen zoom 2003 ; nuclear bone scanning using a siemens gama camera series signature e.cam ; abdominal ultrasound using a siemens antares acuson premium ; or x - ray of the chest wall using a philips super 80 cp diagnost 15). both groups received adequate surgical interventions and neoadjuvant or adjuvant therapy during their primary treatments according to the guidelines of the nccn. vascular invasion was defined as tumor penetration into the lumen of an artery or vein. vascular invasion should only be diagnosed when the tumor is demonstrated within one or both of a pair of vessels. extracapsular extension (ece) was determined by a rupture of the axillary node membrane or when tumor cells were found in perinodular tissue. if the lymph node capsule was infiltrated but not penetrated, this was considered as ece - absent. immunohistochemical staining for the her2 protein was performed using the dako herceptest immunocyto - chemical assay (dako, glostrup, denmark) on 4-m - thick sections of formalin - fixed, paraffin - embedded material by the pathology unit of our institute and according to the manufacturer 's instructions. this staining was scored as follows : 0 or 1 +, no staining or membrane staining in 10% of the tumor cells ; 2 +, weak to moderate complete membrane staining in > 10% of tumor cells ; 3 +, intense and complete membrane staining in > 10% of tumor cells. if ihc staining was indeterminate (2 +), then the fish method was conducted as a follow - up test. recurrence was defined as an occurrence of disease six or more months after the last treatment. statistical analyses were performed using the statistical package for the social sciences software (version 15.0 ; spss, chicago, il, usa). the chi - squared or fisher 's exact test was used to compare categorical variables. univariate and multivariate logistic regression were used to identify recurrence - associated factors. for categorical variables with multiple levels, the reference level was set to that with the lowest probability of the dependent variable. variables with a p < 0.2 in the univariate analysis were entered into the model using a forward stepwise procedure. the results were summarized as odds - ratios (or) and respective 95% confidence intervals (ci). the area under the receiver operating characteristic curve (aurocc) was used to evaluate the ability of the model to discriminate between patients in the recurrence and control groups. a goodness - of - fit (hosmer - lemeshow) in this series, the median age of the patients was 44.75 years (range 2573) in the recurrence group (rg) and 44.3 years (range 2574) in the control group (cg). the median tumor size was 5.08 cm (range 112 cm) in the rg and 4.35 cm (range 110 cm) in the cg. the median number of ln in the dissected material consisted of 16 ln in both groups (with a range from 235 in the rg and 238 in the cg). some of the patients from both groups, depending on their clinical stage, were treated with chemotherapy, radiotherapy, and chemoradio therapy as a neoadjuvant treatment. in 42 patients (70%) of the rg, we used different regimens of neoadjuvant chemotherapy, depending on the patient and tumor characteristics. the aforementioned treatment regimens have been applied in recent years for the breast cancer. after treatment with neoadjuvant chemotherapy, the clinical responses of patients to these treatments were analyzed. therein, 52% of patients in the rg, who presented with stable or progressive disease and were clinically or mammographically diagnosed following the neoadjuvant chemotherapy, were treated with chemoradio concomitant treatment or with radiotherapy alone. all of the patients were treated with surgery, and the type of surgery used in each case is presented in table 2. the number of positive lns and the tumor response therein in the group of patients who had been treated with neoadjuvant treatment were analyzed, and the results are summarized in table 3. all of the patients who had positive hr were treated with adjuvant hormone therapy for five years. approximately 20 mg tamoxifen was given to 43% of these patients (table 4). the median time of recurrence in our patients was 46.1 months (95% ci 34.857.4), as illustrated in figure 1. in the rg the results of the univariate analyses to identify the independent risk factors of early recurrence in the breast cancer are presents in table 6. on table 7, the multivariate analyses were used to identify the independent risk factors of early recurrence in the breast cancer. in this study, patients younger than 35 years of age (15% of the analyzed group) did not present with the usual tumor characteristics that have been previously reported such as big tumor size, positive ln, negative hr, and her2/neu expression). this subgroup exhibited a tumor size, negative hr, positive axillary ln, and her2/neu expression that were similar to those of the recurrence group (5.8 cm and 5.08 cm, 55.6% and 45.2%, 62.5% and 73.3%, and 25% and 23.8%, resp.). the clinical stage and skin involvement of patients in the analyzed group were used as criteria for comparisons to the cg, which is the reason why we did not include these attributes as prognostic factors. nevertheless, 55% of group had locoregional advanced clinical stage tumor, whereas 42% had skin involvement at the time of diagnosis. in a study involving 70 patients with locally advanced clinical stage disease presentation (iii - c) and who were treated with a combined modality treatment, a disease - free survival (dfs) and a median overall survival (os) of 1.9 years and 3.5 years, respectively, statistical analyses demonstrated a longer dfs (2.8 years) in group of locally advanced clinical stage disease presentation (iiia, iiib, iiic). this observation can be explained because of the aggressive locoregional treatment, which is standard treatment in our department for this group of patients. the group with advanced clinical stage disease presentation (iib, iiia, iiib, and iiic) were not statistically at higher risk for earlier recurrence in comparison to group with earlier stages of disease presentation (p = 0.084 ic 0.7119.14), and this observation has been confirmed by a multivariate analysis. this result is not similar to a report from montagna, who observed a 57% recurrence - free survival rate in the first two years of remission. in this series, it was not possible to confirm the findings of galea., who observed that tumor histology predicted the recurrence rate or influenced the 10-year survival rate. it has been reported in the literature that tumor histological grade could be a relevant recurrence prediction factor, especially in small tumors with negative ln. in this study, we did not confirm the predictive value of histological grading, probably because the 85% of patients in rg and cg had large tumors with positive ln. after performing a univariate analysis on the results, lvi was confirmed as a prognostic factor for tumor recurrence in this study, which agrees with the 60% higher mortality rate in patients when lvi is present, reported in the literature ; however, subsequent multivariate analyses exclude the role of lvi as prognostic factor for tumor recurrence. hr was observed not to be predictive of breast recurrence and disease - free interval when the two groups were compared after four years. this observation disagrees with the results reported by the nsabp b-06 study, which found that patients with positive hr had a higher percentage of 5-year dfs and os, compared to patients with negative hr. this has been explained by the presumption that the predictive power of hr is only relevant in the first three years, because these tumors are usually well differentiated and their growth is slower. when the positive hr was compared to the negative hormone receptors in the recurrence group, we found that 69% of patients with positive hr (estrogen, progesterone, or both) did not present with an early recurrence (up to 24 months from the termination of the initial therapy) (30.9 versus 69.1% p = 0.014). patients with negative hr did not show a statistically significant difference in terms of early or late recurrence. in this series, the histopathological reports could not distinguish gross from focal extracapsular extension (ece), which is most likely why ece was not identified as a prognostic factor. positive ln was identified as the most important prognostic factor for recurrence in this study. this observation agrees with those obtained by the nsabp study, which reported 5-year overall survival rates of 83%, 73%, 46%, and 28% for negative ln, 13 positive lns, 412 positive lns, and more than 13 positive lns, respectively. there were no differences in the expression of her2/neu in the recurrence and control groups, and we could not confirm her2/neu as a prognostic factor for recurrence in this study. these results are different from the findings reported in the literature [30, 31 ]. we believe that these results are because of the small number of patients with her2/neu expression in both groups analyzed. patients in the recurrence group exhibited a significantly lower complete pathological response in comparison to patients in the control group. we confirmed that patients with a complete pathological response had a lower risk of recurrence. in this study, the extent of nodal involvement and the complete pathological response to neoadjuvant chemotherapy were the most important predictors of breast cancer recurrence.	the purpose of this study was to analyze the prognostic and predictive factors that relate to locoregional or distant recurrences in breast cancer patients who have been treated at the national cancer institute of mexico. multivariate, time - dependent cox regression analyses indicate that the pn status (positive versus negative lymph node ; p = 0.003 ; hr (hazard ratio), 3.47 ; ci (confidence interval), 1.527.91) and the pathological complete response of the patient to neoadjuvant chemotherapy (yes versus no ; p = 0.061 ; hr, 0.38 ; ci, 0.141.04) were important prognostic factors for recurrence.
the temporal and spatial coupling between increased neuronal activity and cerebral blood flow (cbf), known as functional hyperemia or neurovascular coupling, is a highly regulated phenomenon that ensures adequate supply of oxygen and glucose to the neurons at work during a given task. although intuitively appealing, a direct link between energy state and blood flow is not universally accepted, and the physiological basis of neurovascular coupling still remains uncertain. indeed, neither the lack of glucose or oxygen appears to fully justify the hemodynamic response (powers., 1996 ; wolf. 2010), which may serve as a safety measure for substrate delivery during functional activation (leithner., 2010). despite these limitations, the changes in hemodynamic signals (bold response, cbf or cerebral blood volume, cbv) are commonly used as surrogate markers to map changes in neural activity in brain imaging procedures, such as functional magnetic resonance (fmri), positron emission tomography (pet), or diffuse optical imaging (doi) under both physiological and pathological conditions. accordingly, an adequate interpretation of imaging data imperatively requires understanding of the cellular basis of the activated neurocircuitry and its interaction with astrocytic and vascular targets. in contrast to the innervation of large cerebral arteries by ganglia from the peripheral nervous system, which is mainly involved in autoregulation (for a review, hamel, 2006), the neuronal circuitry at play here refers to pathways of the central nervous system that interact with the brain microcirculation. as such, it is known that the changes in activity are triggered by the incoming afferents, but that it is their local processing by the targeted cells that drives the perfusion changes (logothetis., 2001 ; these hemodynamic changes are mainly, if not exclusively, achieved by the control of the vasculature at the arteriolar level (hillman., 2007), from the pial surface down to the precapillary level where vascular pericytes stand (jones, 1970), the latter contractile elements being likely involved in a localized control of capillary tone (peppiatt., 2006). additionally, it has been shown that the hemodynamic response correlates with synchronized synaptic activity, a highly energy consuming process (arthurs., 2000), and that it is controlled by signaling molecules released during increased synaptic activity (devor., 2007). recently, using the neurovascular coupling response to various sensory stimuli (whisker, forepaw or hindpaw, visual), elegant experiments demonstrated that the spread of the hemodynamic activity accurately reflects the neural response (berwick., 2008), that it is driven by synaptic activity generated by intracortical processing (franceschini., 2008), and that the latter reflected the balance between excitatory and inhibitory signals (devor., 2005, 2007 ; shmuel., 2006). particularly, suppressed neuronal activity or functional neuronal inhibition has been associated with decrease in blood oxygenation and perfusion, which could explain the negative bold signals (shmuel., 2002, 2006) as it occurred concurrently with arteriolar constriction (devor., 2007). as an attempt to identify the underlying neuronal circuitry, an interesting study by lu. (2004)showed that laminar bold and cbv responses to rat whisker stimulation spatially correlated with increased neuronal activity evaluated by c - fos upregulation. in an unrelated study in rats treated with the serotonin releasing drug m - chlorophenylpiperazine, positive bold signals and c - fos immunoreactivity correlated in areas of increased activity, but not in those that displayed diminished bold signals, presumably due to decreased neuronal signaling (stark., 2006). recently, it was shown that stimulation of corticocortical and thalamocortical inputs to the same area of the somatosensory cortex induced completely distinct frequency - dependent changes in cbf and oxygen consumption, and evoked activity in different populations of cortical excitatory pyramidal cells and inhibitory gaba interneurons (enager., 2009). stimulation intensity increases occurred with the silencing or recruitment of distinct inhibitory interneurons, indicating that neural network activation is both stimulus- and frequency - dependent. particularly, the hemodynamic responses were attributed to activation of cyclooxygenase-2 (cox-2) pyramidal cells and somatostatin (som)/nitric oxide synthase (nos) inhibitory interneurons, consistent with pharmacological studies that implicated cox-2- and nos - derived vasodilator messengers in these neurovascular pathways (niwa., 2000 ; moreover, increased activity in inhibitory interneurons has been associated with the initiation of the hemodynamic response triggered by synchronized cortical activity, as induced by activation of the basal forebrain (niessing., 2005). stimulation of this basalocortical afferent input further indicated selective activation of cholinoceptive layers ii to vi som and neuropeptide y (npy) interneurons, as well as layer i gaba interneurons, with widespread activation of pyramidal cells, including those that contain cox-2 (kocharyan. although it was demonstrated that the cbf response was triggered by cholinergic afferents, its full expression required gabaa - mediated transmission on neuronal, vascular and/or astrocytic targets (kocharyan., 2008). together these anatomical, neurochemical and functional studies demonstrate the importance of identifying the cellular ensemble that underlies hemodynamic signals, highlighting that specific subsets of neurons are activated by a given stimulus, depending on the afferent input they receive and integrate. in addition to the difficulty in identifying the exact contribution of excitatory and inhibitory neurotransmissions in the evoked hemodynamic response, the effect of these neurotransmitter systems on perivascular astrocytes needs to be considered (for a detailed review, carmignoto and gomez - gonzalo, 2009). the enwrapping of synapses and blood vessels (kacem., 1998) by glial processes and, particularly, their intervening endfeet in multiple neurovascular appositions identified at the ultrastructural level led to the concept of a neuronal - astrocytic - vascular tripartite functional unit (vaucher and hamel, 1995 ; cohen., 1996 ; paspalas and papadopoulos, 1996 ; vaucher., 2000). the significance of these interactions in the regulation of cbf was first substantiated by the demonstration that astrocytes could synthesize vasodilatory messengers (table 1), particularly epoxyeicosatrienoic acids (eets) generated from p450 arachidonic acid epoxygenase activity (alkayed., 1996) that were involved in the cbf response to glutamate (alkayed., 1997 ; harder., soon after came the first in vitro (in cortical slices) and in vivo demonstrations for a role of astrocytes, through metabotropic glutamate receptor (mglurs)-induced ca transients, in microarteriolar dilation and the increase blood flow to forepaw stimulation, a response mediated by arachidonic acid products, possibly prostaglandin e2 (pge2) (zonta., a role for astrocytes in neurovascular coupling has been reaffirmed in various paradigms (carmignoto and gomez - gonzalo, 2009). neuronal and glial metabolism by - products (e.g., co2, h, adenosine) are not included. no, nitric oxide ; vip, vasoactive intestinal polypeptide ; pge2, prostaglandin e2 ; eets, epoxyeicosatrienoic acids ; nos, nitric oxide synthase ; npy, neuropeptide y ; cox, cyclooxygenase, nmda - r, n - methyl d aspartate receptors, iglurs ; ionotropic glutamate receptors, mglur, metabotropic glutamate receptors ; vsmc, vascular smooth muscle cell, sgc, soluble guanylate cyclase, vpac1, vip / pacap receptor type 1 ; ep, prostaglandin e2 receptors ; gpcr, g protein coupled receptor. kca, ca+-activated k channels ; kir, inward rectifier k channels ; g, conductance. another pathway activated in astrocytic endfeet following ca increases is the large - conductance, calcium - sensitive potassium (bk) channels that induce k release (table 1), activation of smooth muscle kir channels and relaxation (filosa., 2006). recently, it was shown that the extent of the ca increases in astrocytic endfeet determined the dilatory or contractile nature of the vascular response, both mediated by extracellular k (girouard., 2010). this novel mechanism would reunify previous apparently contradictory findings, in brain slices or in retina, of dilation and constriction being induced by increased ca signaling in astrocytes, and explained by the levels of no (mulligan and macvicar, 2004 ; metea and newman, 2006), oxygen (gordon., 2008), or the pre - existing tone of the vessels (blanco., 2008). (2010) showed that neuronal activation in vitro (electrical field stimulation) similarly acted through serial activation of astrocytic bk and smooth muscle kir channels. considering that gaba (nilsson. 2008) and peptides such as som that colocalize with gaba (somogyi., 1984 ; cauli., 2000) in interneurons also increase ca signaling in astrocytes, (straub., 2006), the latter could act as intermediaries to gaba in neurovascular coupling (table 1). whether or not the astrocytic / smooth muscle bk and kir channel activation cascade is involved still remains to be determined. similarly, it would be interesting to evaluate if the latter, or other contractile mechanisms of neuronal (cauli. 2006) or vascular origins (mulligan and macvicar, 2004) could explain the dilatory and constrictive phases seen in the central core of neuronal depolarization and surround region of hyperpolarization, respectively, after somatosensory stimulation (devor., 2007). to date the cellular and molecular mechanisms or even the functional significance of this response remains unknown in addition to the astrocyte - derived vasodilatory messengers pge2 (zonta., 2003), eets (alkayed., 1996), or k ions (filosa., 2006), iii pyramidal cells (yamagata., 1993 ; breder., 1995), nitric oxide (no) (gotoh., 2001) whose synthetic enzyme is expressed by discrete subpopulations of cortical gaba interneurones (kubota., 1994), vip (yaksh., 1987), acetylcholine (scremin., 1973) and corticotropin - releasing factor (de michele., 2005) synthesized by bipolar / bitufted gaba interneurones (morrison., 1984 ; chdotal., 1994a ; cauli., 1997 ; gallopin., 2006 for instance the highly ca permeable nmda receptors, expressed by cortical neurons (monyer., 1994 ; cauli., 2000), promote the release of pge2 (pepicelli., astrocytes, some gaba interneurons produce substances with vasocontractile properties, namely npy (abounader., 1995 ; cauli., 2004) and som (long., 1992 ; cauli., cortical neurons producing these vasoactive peptides are intimately associated with blood vessels through neuronal - astrocytic - vascular appositions described above (chdotal., 1994b ; abounader and hamel, 1997 ; estrada and defelipe, 1998 ; vaucher., 2000 ; wang., 2005), and their receptors are expressed by smooth muscle cells and astrocytes (chalmers., 1995 ; bao., 1997 ; abounader., 1999 ;, 2000 ; cauli., 2004 ; straub., 2006 ; cahoy., this raises the intriguing question of whether or not astrocytes are intermediaries for neuron - derived vasoactive messengers or, alternatively, if the latter exert direct effects on the microcirculation. it is widely admitted that an increase in intracellular ca (table 1) is a required early event for the production and/or release of vasoactive messengers from neurons (lauritzen, 2005) and astrocytes (straub and nelson, 2007). examination of ca dynamics in these cell types could provide a clue to decipher their relative and temporal contribution to functional hyperemia. the general view is that rapid ca events reflect an entry following fast (1012 ms) spiking response of neurons (petersen., 2003) and/or activation of ca permeable ionotropic receptors, whereas slower dynamics are mainly driven by activation of metabotropic receptors leading to the release of ca from intracellular stores (perea and araque, 2005). hence, cortical neurons, which express more frequently and abundantly ionotropic glutamate receptors (monyer., 1994 ; 2008), are likely to be responsible for the majority of fast ca responses. in contrast, group i mglurs ubiquitously expressed by cortical neurons (baude., 1993 ; cauli., 2000) and astrocytes (porter and mccarthy, 1996) would be responsible for slower ca dynamics in both cell types. consistent with this, in somatosensory or visual cortex, evoked ca events in neurons are virtually locked with sensory stimulations and precede those in astrocytes by a few seconds (stosiek. wang., 2006 ; schummers., 2008 ; murayama., 2009), although a small proportion (5%) of astrocytes can exhibit ca responses as fast as neurons (winship., 2007). calcium uncaging in astrocytic endfeet in vivo showed that arterioles start to dilate 500 ms after the onset of ca increase (takano., 2006) indicating that synthesis, release and effects of vasodilatory messengers must be achieved within this time window. since hemodynamic responses initiate 600 ms after the onset of sensory stimulations (kleinfeld., 1998 ; 2003), it appears that only cell types exhibiting fast evoked ca events (i.e., less than 100 ms) can account for the early phase of the hemodynamic response. therefore, vasoactive messengers produced by neurons and, possibly, also by astrocytes with fast ca events, could explain this response (figure 1). summary of the proposed regulation of cortical microvessels by pyramidal cells, gaba interneurons and astrocytes (a), and how their respective effects can temporally regulate cbf changes (b). (a) subcortical afferents from a variety of brain areas target distinct populations of neurons in the cerebral cortex. these activated neuronal networks can either directly act on local microvessels, which are endowed with receptors (geometric forms on the vessel wall) for most neurotransmitters / neuromediators, or indirectly via astrocytes that act as intermediaries to both pyramidal cells and interneurons. known direct vasoactive mediators released from pyramidal cells and interneurons correspond respectively to cox-2 derivatives like prostaglandin e2 (pge2) and no and, possibly, gaba, whereas astrocytes act chiefly by releasing dilatory eets, an effect comparatively slow as opposed to that of no and pge2 (or other neurally released vasoactive molecules or peptides). the possibility that sub - cortical afferents directly contact and act upon cortical astrocytes or microvessels also has to be taken into consideration. modified from figure 3 in hamel (2006). (b) schematic representation of the relative and temporal contributions of selected vasoactive mediators produced by pyramidal cells (pge2), interneurons (no) and astrocytes (eets), to the cbf response evoked by sensory stimulation (see table 1 for a more complete list). brief stimulations (1 s) are more likely to involve neurally - derived mediators whereas sustained stimulation (1 min) are more susceptible to recruit astrocyte - derived messengers. no being transiently released its contribution to cbf response during sustained stimulation is minor and could account for its permissive role. in the cerebral cortex, none of the vasoactive messengers implicated in neurovascular coupling (girouard and iadecola, 2006), whether of neuronal or astroglial origin, can individually account for the hemodynamic response, as demonstrated by genetic invalidation (ma. kitaura., 2007) or synthesis inhibition (lindauer., 1999 ; peng., 2002 ; hoffmeyer., 2007 ; leithner., 2010). when individually summed the inhibition of these messengers largely exceeds the expected value of 100% (iadecola, 2004), which suggests that their kinetics of action, temporal and spatial recruitment must be carefully considered to elucidate their relative contributions. alternatively, this may suggest that the activated pathways do not obligatorily operate independently from each other, and that, under certain circumstances, some may act like modulator rather than mediator of the perfusion responses, as documented for no in the somatosensory cortex (lindauer., 1999). no, one of the fastest diffusible (wood and garthwaite, 1994) vasodilator produced by a subset npy - expressing interneurons (dawson., correspondingly, shibuki 's group showed that neuronal no can account for up to 50% of the cbf response evoked by a brief (1 s) sensory stimulation (kitaura., 2007). in contrast, others only found a permissive role for no when long (1 min) stimulations were used (lindauer. these differences likely reflect the fact that no release is transient (buerk., 2003) which can be explained by the no scavenging effect of hemoglobin and/or by the adaptation of no producing interneurons (karagiannis., 2009). in contrast, vasodilatory prostanoids produced by cox-2, which is chiefly expressed by pyramidal cells (yamagata., 1993 ; breder., 1995), account for 50% of the cbf response evoked by both sustained (niwa., 2000) and brief stimulations (kitaura., 2007). consistent with an involvement of neuron - derived messengers in the early phase of neurovascular coupling, blockade of prostanoids and no synthesis almost completely abolished hemodynamic responses evoked by brief sensory stimulations (kitaura., 2007). during long lasting sensory stimulation, blockade of eets synthesis (peng., 2002) or their receptors (liu., 2008) blocked about 50% of the blood flow response, demonstrating that eets produced by astrocytes (alkayed., 1996) similarly, the local release of k from astrocytic endfeet (filosa., 2006) accounts for up to 50% of the cbf increase evoked by long lasting sensory stimulations (girouard., 2010 ; leithner., 2010) surprisingly, combined blockade of nos, coxs, p450 epoxygenase, bk channels and adenosine receptors (leithner., 2010) did not reach a total inhibition of the late phase of the hemodynamic response as expected from individual blockades (see above). this suggests that either multiple inhibition was incomplete or that other long lasting vasodilatory messengers are involved (cauli., 2004), possibly vip, which is contained within gaba interneurons targeted by thalamocortical afferents (staiger., 1996) and similarly vasodilatory messengers derived from the endothelium (rosenblum, 1986) such as no, prostacyclin (faraci and heistad, 1998) but also eets (campbell and fleming, 2010), might be recruited under certain circumstances as it was reported for endothelial no after muscarinic m5 receptor activation (elhusseiny and hamel, 2000 ; yamada., 2001). current evidence suggests that neuronal and astroglial signals that transduce changes in neuronal activity into an integrated vascular response are highly dependent upon the neurotransmitter released by the incoming afferents, and strictly determined by the target neurons within the activated area. particularly, depending on the nature of the afferent input (i) different neuronal or astroglial messengers, likely acting in sequence, mediate the hemodynamic changes, (ii) some recruited neurons release messengers that can directly alter blood vessel tone, (iii) others act by modulating neuronal and astroglial activity, and (iv) astrocytes may act as intermediaries for both excitatory and inhibitory neurotransmitters (figure 1). probably due to the large diversity of cortical neurons (ascoli., 2008), to our knowledge, no in vivo study has yet investigated ca dynamics in identified neurons producing vasoactive substances or the vascular effects of their stimulation. the growing development of transgenic mice expressing genetically encoded fluorescent reporters and/or optogenetic tools (cardin. 2009) in discrete subsets of cortical neurons (heintz, 2001) together with the emergence of ultrafast multispectral imaging systems (bouchard., 2009) that allow simultaneous monitoring of ca events and hemodynamics should help evaluate the contribution of specific neuronal types in neurovascular coupling. this should provide decisive conclusions on the temporal, spatial and extent of the neurally - driven hemodynamic alterations and how the latter can be interpreted in the context of brain imaging of normal or pathological physiology. indeed, since astrocytes appear as intermediary effectors in conveying signals for sustained hemodynamic responses, their alteration primarily expressed by a state of chronic activation in several chronic diseases of the central nervous system such as alzheimer 's disease or epilepsy, has to be seriously considered. altered perfusion signals detected by fmri, pet or doi may represent astroglial dysfunction and not necessary impaired neuronal activity. extending such thinking to the microcirculation itself, the functional endpoint in the intricate cascade of neuronal - astrocytic - vascular events evoked by increased brain activity, any diseases of the blood vessels themselves or alterations in their physical capacity to dilate or constrict, as seen in pathologies such as hypertension, diabetes, hypercholesterolemia and, even alzheimer 's disease (iadecola, 2004 ; zlokovic, 2008) would hinder the correct vascular response to totally normal neuronal activities. this further highlights that extreme caution should be applied to perfusion signals when making direct inference to altered neural activity (schleim and roiser, 2009 ; ekstrom, 2010). hence, a careful understanding of the neuronal circuitry at work will need to be interpreted in the context of a healthy or sick brain taking neuroinflammation and vascular diseases as possible confound factors. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.	in this article, we will review molecular, anatomical, physiological and pharmacological data in an attempt to better understand how excitatory and inhibitory neurons recruited by distinct afferent inputs to the cerebral cortex contribute to the coupled hemodynamic response, and how astrocytes can act as intermediaries to these neuronal populations. we aim at providing the pros and cons to the following statements that, depending on the nature of the afferent input to the neocortex, (i) different neuronal or astroglial messengers, likely acting in sequence, mediate the hemodynamic changes, (ii) some recruited neurons release messengers that directly alter blood vessel tone, (iii) others act by modulating neuronal and astroglial activity, and (iv) astrocytes act as intermediaries for both excitatory and inhibitory neurotransmitters. we will stress that a given afferent signal activates a precise neuronal circuitry that determines the mediators of the hemodynamic response as well as the level of interaction with surrounding astrocytes.
coronary artery spontaneous dissection is a rare and uncommon cause of sudden cardiac death and acute coronary syndrome. we report a case of a 31-year - old male admitted to our hospital with an acute coronary syndrome. a dissection of the left main coronary artery was diagnosed with a multidetector computed tomography (ct) scan and it was decided to perform an emergency coronary artery bypass grafting. a 31-year - old male, airline pilot, with no clinical history or cardiovascular risk factors, was admitted to the emergency room with 12 h history of retrosternal chest pain radiating to the jaw and upper extremities, during a trans - atlantic flight, which did not improve with analgesia. because of persistent pain, he came to our hospital. upon arrival to the emergency room, his vital signs were stable and he had a normal physical examination but complained of precordial pain. the cardiac enzymes were elevated and the ekg detected s - t changes consistent with mi (fig. 1). a multidetector ct coronary angiography was performed showing left main trunk dissection with 50% stenosis (fig. figure 2:multidetector coronary angio - tomography reveals dissection at the ostium of the left main coronary artery. multidetector coronary angio - tomography reveals dissection at the ostium of the left main coronary artery. the patient underwent emergent coronary revascularization with saphenous vein grafts to the first obtuse marginal and the left anterior descending artery (lad). control ct coronary angiography was performed 2 months later revealing patent aorto - coronary grafts (fig. figure 3:ct coronary angiography reconstruction showing patent grafts to the lad and obtuse marginal arteries. in 1931 harold pretty described the first case of spontaneous dissection of a right coronary artery during the autopsy of a woman who presented with precordial chest pain. this condition is a rare cause of ischemic heart disease and affects mainly young healthy women. in 1987, 85 cases were reported in the literature, currently more than 300 cases have been published [2, 3 ]. before the coronariography era, these cases were reported during autopsies of patients having sudden cardiac deaths and its incidence may have been under - estimated. with the advent of these studies, the incidence has been reported to range between 0.07 and 1.1%. dissection of the lad artery is most common in women, whereas in men it is the right coronary artery [2, 4 ]. the left main coronary artery involvement, like in our case, is rare, occurring in up to 12% of the cases, but is the most severe injury and presents as mi. hemodynamic and hormonal factors determine morphological changes in the arterial wall, and these may contribute to spontaneous dissection of the coronary arteries, with a peak incidence during the second week after birth [3, 4 ]. to date, the pathophysiology is unclear [24 ]. in a subgroup of patients it is not possible to identify a specific condition causing spontaneous coronary dissection and is therefore classified as idiopathic. in patients with connective tissue disorders such as marfan s and ehlers danlos syndromes, predisposing to the dissection arises from medial degeneration of the coronary arteries. certain vasculitis, including systemic lupus erythematosus and polyarteritis nodosa, has been associated with the occurrence of coronary artery dissection. other factors that can cause vascular spasm and coronary dissection include intense exercise, sneezing and prolonged cocaine abuse [2, 3 ]. it should show the presence of a double radiopaque lumen separated by a radiolucent intimal flap or a slow clearance of contrast from the false lumen. an intimal tear is present in only a minority of cases and a medial hematoma may not be recognized on coronary angiography as the medial hemorrhage may cause luminal narrowing or occlusion by pushing the inner media against the opposing wall. several cases in the literature have shown that ct coronary angiography provides additional information over invasive coronary angiography, with an accurate demonstration of the intimal flap and extent of the intramural hematoma. this study constitutes an emerging noninvasive alternative for diagnosis and also the follow - up of coronary artery spontaneous dissection. there are no specific guidelines for the management of spontaneous dissections of the coronary arteries [3, 5 ]. coronary artery bypass surgery is indicated in patients with multivessel dissection, failed angioplasty and dissection of the trunk of the left main coronary artery [35 ]. although spontaneous dissection of a coronary artery is an uncommon condition, treatment should be based on the clinical status and imaging studies, including conservative and surgical means depending on the clinical presentation, location and characteristics of the dissection, we elected to proceed with surgery based on the ct findings.	spontaneous dissection of the coronary arteries is a rare disease with a wide range of clinical presentations ranging from angina to myocardial infarction (mi) ; its pathophysiology has not yet been fully established. in this paper, we present the case of a 31-year - old male with an acute coronary syndrome. the initial results of the electrocardiogram and cardiac enzymes were consistent with mi. however, a coronary angio - tomography revealed a dissection of the left main coronary artery and the patient underwent emergent surgery with coronary artery bypass grafting. the treatment of spontaneous dissection of the coronary arteries depends on the anatomical location and the patient 's clinical presentation. coronary revascularization is associated with good results.
reducing under - five mortality is now a global concern. in 2001 as part of the millennium development goals (mdg) for health, nations pledged to ensure a two - thirds reduction in under - five mortality between 1990 and 2015 and at once a series of articles in lancet by the bellagio study group described various aspects of child survival[2, 3, 4, 5, 6 ]. although under - five mortality is declining worldwide as a result of socioeconomic development and implementation of child survival interventions, nearly 8.8 million children die every year before their fifth birthday. india alone accounted for 21% of the world s under - five deaths occurring in 2008 owing to its large population. in india, states such as assam, arunachal pradesh, bihar, chhattisgarh, jharkhand, madhya pradesh, orissa, rajasthan, uttar pradesh, have higher under - five mortality than the rest of india. the national average for under - five mortality is 74 per 1000. the ministry of health and family welfare, india, established empowered action group (eag) in 2001 to have special focus by monitoring and facilitating the attainment of national health goals on some of these states which are demographically lagging behind. the eag states constitute 45% of the total population of india and also have higher neonatal and infant mortality rates. in developing countries, efforts have been made during the past three decades to reduce child mortality. despite socioeconomic development and implementation of child survival interventions, studies on determinants of child mortality have mainly used either logistic regression or cox proportional hazards model assuming that the outcomes are independent. to find more accurate estimates for the determinants of child mortality that has critical implications for resource allocation for improving child survival, sibling structures in child mortality data from demographic surveys have been treated as multivariate failure time data[10, 11, 12, 13 ]. as failure time data, many attempts have been made to extend the cox proportional hazards model. in this context, the variance - corrected cox model has received much attention[14, 15 ]. in the variance - corrected cox model, regression parameters of the determinants are estimated by ignoring intra - family correlation but adjusted for in the inference procedure ; however, it ignores the variation of underlying risk among families. to overcome this, these frailties are common to observations from the same cluster and assumed to follow a given statistical distribution, known as multivariate random effects model or cox frailty model. in india, studies on child mortality have mainly addressed the role of maternal, socioeconomic and health - related determinants[9, 17, 18 ]. these studies were restricted to the analysis of mortality risks in children at individual level and not considered the correlation among children of the same family. we also want to emphasize those determinants which are nearer in time to the outcome and can be modified by program than those which are remote or far apart in time to the outcome of concern. the former covariates are referred to as programmable determinants and the latter as background variables. therefore, we aimed to identify the programmable determinants of under - five mortality using cox frailty model to account for sibling - level correlation for providing valid estimates needed for policy - decision making. in order to appreciate the influence of sibling - level correlation over the estimates of the determinants of under - five mortality, the results of cox frailty model were compared with the cox proportional hazards model and variance - corrected cox model. the third round of national family health survey-3 (nfhs-3) in india was completed during 2005 - 06 covering a nationally representative sample of ever married women aged 15 - 49 years. this survey collected data on fertility, family planning, infant and child mortality, maternal and child health, etc. using a two - stage sample design in rural areas for each state of india. the first stage involved selection of primary sampling units, i.e., villages, with probability proportional to population size and the second stage involved systematic selection of households within each selected village. the response rates for household and eligible women identified in the household were 98.5% and 95.5% respectively. the rural data of nfhs-3 for eight eag states, viz., bihar, chhattisgarh, jharkhand, madhya pradesh, orissa, rajasthan, uttar pradesh and uttarakhand were combined and analyzed to identify the determinants of under - five mortality. in rural eag states, a total of 14,184 live births occurred within five years preceding the survey and mortality experience of 13,785 children were analyzed in this study. in 399 cases the information was missing on some of the variables used in the analysis. the primary outcome, under - five mortality, was defined as time to death of a live born baby before his / her fifth birthday. available potential predictors of child survival as summarized in the conceptual framework of mosley and chen was considered and grouped into programmable (proximate) determinants and background variables. programmable determinants included mother s age at birth, delivery assistance, place of delivery, 62 mode of delivery, combined variable of birth order and birth interval, survival status of previous child, maternal subjective assessment of baby s size at birth, sex of the baby and ever breastfeeding ; and the background variables included region (eight states), religion, caste, mother s education, mother s occupation, household wealth index, number of children in the family and desired time for pregnancy. the variance - corrected and frailty hazard models are multivariate not only in the usual sense of having multiple predictors, but also in the sense of having multiple responses, that is, responses from more than one child in the family. mathematically, it is written as h(t) = h0(t) exp(zk), t > 0,. (1) where, h0(t) is an unspecified baseline hazard function and denote the vector of the true regression coefficients for covariates zk, (k=1, 2,, we could obtain an estimator of based on the working assumption that the under - five deaths in each family were independent of one another. we supposed that conditional on covariate vector (zik), the marginal hazard function hik(t) for failure time of the kth child in the ith family, (k = 1,2,3,., ki ; i = 1,2,3,,n) with the usual proportional hazards form and is given by hik(t) = h0(t) exp(zik), t > 0,.. (2) we could obtain an estimator of based on the working assumption that the under - five deaths in each family were independent of one another. but the equation (2) assumes that the births are related and hence adjusts for it in the inference, that is, the standard error by means of sandwich - type estimators and so it is called as variance corrected models. for the frailty model, we supposed that conditional on the frailty, vi the hazard function hik(t) for the failure time of the kth children in the ith family (k = 1,2,3,.,ki ; i = 1,2,3,,n) follows the usual proportional hazards form and is given by : hik (t) = h0 (t) vi exp(zik), t > 0,.. (3) where, vi, group - level (family) frailty. these frailties are unobservable, assumed to be independent and identically distributed with unit mean and unknown variance. each family could have different values of random effects and the variability in the vis reflect heterogeneity of risks between families. if the variance of the random effect (frailty) is 0, then children from the same family are independent. the variance of the random effect lies between 0 and a. a larger variance implies greater heterogeneity in frailty across families and greater correlation among children belonging to the same family. the frailty (family) often assumed to follow gamma distribution for the sake of computational convenience and convergence[21, 22, 23 ] and this model is expected to yield correct z - ratios, on which researchers rely heavily for their conclusions. equations (2) and (3) reduce to the traditional cox proportional hazards model, if the responses from each child in the family are assumed to be independent. the complete data for all the eag states were downloaded from demographic health survey data distribution system website : http://www.measuredhs.com. all the variables were read and coded using stata 9.0 (college station, texas, usa). the under - five mortality rate (u5mr) and its 95% ci with respect to potential determinants influencing under - five mortality was calculated. we identified potential determinants of under - five mortality using three models : the traditional cox proportional hazards model, the variance - corrected cox proportional hazards model, and the cox frailty model. programmable determinants were adjusted for background factors in the multivariate analysis in all the three models. the r - software (version 2.11.1, 2010, the r foundation for statistical computing) was used to fit all the models. the third round of national family health survey-3 (nfhs-3) in india was completed during 2005 - 06 covering a nationally representative sample of ever married women aged 15 - 49 years. this survey collected data on fertility, family planning, infant and child mortality, maternal and child health, etc. using a two - stage sample design in rural areas for each state of india. the first stage involved selection of primary sampling units, i.e., villages, with probability proportional to population size and the second stage involved systematic selection of households within each selected village. the response rates for household and eligible women identified in the household were 98.5% and 95.5% respectively. the rural data of nfhs-3 for eight eag states, viz., bihar, chhattisgarh, jharkhand, madhya pradesh, orissa, rajasthan, uttar pradesh and uttarakhand were combined and analyzed to identify the determinants of under - five mortality. in rural eag states, a total of 14,184 live births occurred within five years preceding the survey and mortality experience of 13,785 children were analyzed in this study. in 399 cases the information was missing on some of the variables used in the analysis. the primary outcome, under - five mortality, was defined as time to death of a live born baby before his / her fifth birthday. available potential predictors of child survival as summarized in the conceptual framework of mosley and chen was considered and grouped into programmable (proximate) determinants and background variables. programmable determinants included mother s age at birth, delivery assistance, place of delivery, 62 mode of delivery, combined variable of birth order and birth interval, survival status of previous child, maternal subjective assessment of baby s size at birth, sex of the baby and ever breastfeeding ; and the background variables included region (eight states), religion, caste, mother s education, mother s occupation, household wealth index, number of children in the family and desired time for pregnancy. the variance - corrected and frailty hazard models are multivariate not only in the usual sense of having multiple predictors, but also in the sense of having multiple responses, that is, responses from more than one child in the family. mathematically, it is written as h(t) = h0(t) exp(zk), t > 0,. (1) where, h0(t) is an unspecified baseline hazard function and denote the vector of the true regression coefficients for covariates zk, (k=1, 2,, we could obtain an estimator of based on the working assumption that the under - five deaths in each family were independent of one another. we supposed that conditional on covariate vector (zik), the marginal hazard function hik(t) for failure time of the kth child in the ith family, (k = 1,2,3,., ki ; i = 1,2,3,,n) with the usual proportional hazards form and is given by hik(t) = h0(t) exp(zik), t > 0,.. (2) we could obtain an estimator of based on the working assumption that the under - five deaths in each family were independent of one another. but the equation (2) assumes that the births are related and hence adjusts for it in the inference, that is, the standard error by means of sandwich - type estimators and so it is called as variance corrected models. for the frailty model, we supposed that conditional on the frailty, vi the hazard function hik(t) for the failure time of the kth children in the ith family (k = 1,2,3,.,ki ; i = 1,2,3,,n) follows the usual proportional hazards form and is given by : hik (t) = h0 (t) vi exp(zik), t > 0,.. (3) where, vi, group - level (family) frailty. these frailties are unobservable, assumed to be independent and identically distributed with unit mean and unknown variance. each family could have different values of random effects and the variability in the vis reflect heterogeneity of risks between families. if the variance of the random effect (frailty) is 0, then children from the same family are independent. the variance of the random effect lies between 0 and a. a larger variance implies greater heterogeneity in frailty across families and greater correlation among children belonging to the same family. the frailty (family) often assumed to follow gamma distribution for the sake of computational convenience and convergence[21, 22, 23 ] and this model is expected to yield correct z - ratios, on which researchers rely heavily for their conclusions. equations (2) and (3) reduce to the traditional cox proportional hazards model, if the responses from each child in the family are assumed to be independent. the complete data for all the eag states were downloaded from demographic health survey data distribution system website : http://www.measuredhs.com. all the variables were read and coded using stata 9.0 (college station, texas, usa). the under - five mortality rate (u5mr) and its 95% ci with respect to potential determinants influencing under - five mortality was calculated. we identified potential determinants of under - five mortality using three models : the traditional cox proportional hazards model, the variance - corrected cox proportional hazards model, and the cox frailty model. programmable determinants were adjusted for background factors in the multivariate analysis in all the three models. the r - software (version 2.11.1, 2010, the r foundation for statistical computing) was used to fit all the models. the trends in under - five mortality rates by major states in rural india for five years preceding the nfhs-1 (1992 - 92) and nfhs-3 (2005 - 06) are given in table 1. among the eag states, no change in under - five mortality was found in chhattisgarh and the highest decline in mortality (38.1%) was found in bihar between the two surveys. the percentage decline in under - five mortality in rural india was 31.3 between the two surveys. trends in under - five mortality rates by major states in rural india for five years preceding the nfhs-1 (1992 - 93) and nfhs-3 (2005 - 06) data represent under - five mortality rates for the complete states the distribution of live births by family is shown in table 2. more than one third (41%) of the families contributed two or more children to the sample. a total of 1,068 under - five deaths occurred to 969 (10%) families. distribution of live births by family the number of live births and under - five mortality with respect to the background factors and programmable determinants are shown in table 3. one third of the total live births were from uttar pradesh ; only 36.4 percent live births belong to scheduled caste (21.3%) and scheduled tribes (15.1 %) and mothers of two - thirds of the live births were illiterate. the under - five mortality (per 1,000 live births) was 86.9 in uttar pradesh, 89.9 among scheduled caste mothers, 86.7 among illiterate mothers, 81.7 among families with more than two children, 87.9 in the poorest wealth quintile, 104.2 among children born to mothers aged 12 - 19 years, 139.3 in mothers having previous birth interval of less than two years and parity more than three, 107 among small sized babies, and 143 among children with history of dead sibling, which were having very high under - five mortality than their counterparts. the distribution of live births and under - five mortality rates across categories of the background variables and programmable determinants in rural eag states for five years preceding the nfhs-3 (2005 - 06) survey of india (n = 13,785) the results of programmable determinants of under - five deaths adjusting for the background variables using all the three models are given in table 4. the estimates are exactly the same in models 1 and 2 ; only standard errors are corrected in model 2, and in model 3, both estimates and standard errors are corrected. the determinants found to be significant in model 1 were also significant in model 3 except death of a previous child and in model 2 except mother s age at birth. in the frailty model, the mortality hazards for children born to mothers aged 12 - 19 years at birth were 1.41 (95% ci : 1.14, 1.75) times higher than children born to mothers aged 20 - 30 years at birth and in the variance - corrected model, the hazard ratio (1.19) for the same variable was not statistically significant. the mortality hazard for the female child has increased from 17% to 22% when unobserved familial effect is taken into account. small size babies at birth had 42% excess hazard than the average size babies at birth. the mortality hazards for first - born children and fourth - or - higher birth order children with preceding birth interval of less than two years were 2.04 (95% ci : 1.52, 2.73) and 2.42 (95% ci : 1.84, 3.18) times the hazard for second or third birth order children with a longer birth interval (p < 0.001). infants who were not breastfed had significantly higher hazard of death (hr = 102 ; 95% ci : 81, 128) than those who were breastfed. programmable determinants and background variables of under - five mortality using traditional cox proportional hazards, cox variance - corrected and cox frailty models in rural eag states for five years preceding the nfhs-3 (2005 - 06) survey of india (n = 13,785) reference categories : mother s age at birth 20 - 30 y, institutional delivery, delivery assistance by doctors / anm / hp, male children, 2 - 3 birth order & birth interval 2y, very large & large size of the baby at birth, surviving previous sibling, breastfed children, uttarakhand state, other caste, hindu religion, literate mothers, professional / clerical / sales, 2 children, poorest household wealth index and then for desire for pregnancy ; adjusted for background factors such as eag states, religion, caste, mother s education, mother s occupation, number of children, wealth index, desire time for pregnancy and other determinants ; p- p - value for multivariate analysis and p<0.05, statistically significant. the state, uttarakhand, was selected as the reference category due to low under - five mortality rates among the eag states. the hazard ratios were increased in all the eag states except jharkhand after adjustment for programmable determinants and other background variables. however, the adjusted hazard ratios were statistically significant for only madhya pradesh, chhattisgarh, rajasthan, orissa and uttar pradesh. the other background variables such as caste, mother s education and household wealth index were significantly associated with under - five mortality as shown in table 4. most hazard ratios for the proximate determinants are similar across the three types of models but the most notable finding is the change in the effect of the death of a previous child variable. the multiplicative effect of this variable changes from an 86% excess risk to an 18% reduction in risk (albeit not statistically significant) when unobserved familial effect is taken into account as a gamma frailty. the gamma frailty is 2.86 which means that larger unmeasured familial effect is present and is statistically significant (p<0.001). in general, the z - statistics (not shown here) are found to be smaller in the random - effects / frailty model than in the traditional cox and variance - corrected cox models except for some of the covariates. the r2 and log likelihood / i - likelihood are preferred for comparing the three models. the cox frailty model was considered the best model as it had the highest r2 and lowest log likelihood compared to the other two models. the primary goal of the study was to assess the determinants of under - five mortality by applying an appropriate model to account for sibling - level correlation and thus provide valid estimates for correct statistical inference needed for policy - decision making. we found that children born in chhattisgarh had higher risk of dying before age five, followed by children born in uttar pradesh and madhya pradesh. these states require health interventions that target under - five mortality reduction, particularly in rural areas. next, mother s education and wealth index emerge as powerful background covariates of under - five mortality in the eag states, for the reason that both are known to be associated with better child care practices. thus, the study urges the policy makers to focus on educating illiterate mothers about the child care ; however, policy aiming at improving maternal education and poverty reduction is needed for sustainability. we know that changes in the z statistics depend on the size of the parameter estimates along with the magnitude of the standard error. in general the z - statistics are smaller in magnitude in frailty model as compared to other models which we also observed in our results, clearly indicating that the sample of correlated observations contains less information than the independent sample. we also observed higher z - statistics for some covariates as observed by sastry, for example, mother s age at birth of 12 - 19 y (zmodel 3 = 3.13 vs. zmodel 1 = 1.98) in the cox frailty model than the traditional cox model. the assumption of the cox proportional hazards model is likely to be incorrect if we suspect that siblings share environmental or genetic influences beyond explicit covariates included in the model. to account for this correlation, if we correct the standard error alone, it might lead to the biased inference, casting doubt especially on the more marginally significant results. the covariate, maternal age 12 - 19 years at child birth, was found to be marginally significant in the traditional cox model and was statistically not significant in the variance - corrected cox model. however, this variable was highly significant in the cox frailty model which reiterates the importance of simultaneous correction of both parameter estimates and the standard error when analyzing correlated observations. there were remarkably stable in all the three models except survival status of previous child variable. this has been already noted in previous studies[10, 11, 12 ] that the positive effect of this variable indeed acts as a proxy in the traditional cox model. as pointed out by guo and rodrguez g, the hazard ratio of less than one in frailty model suggests that the death of a previous child lowers the risk of the surviving siblings through less competition for family resources or inducing changes in the parental behavior since death is a traumatic event. a non - protective role of institutional deliveries in the present study was found as pointed out by titaley and this might be complicated deliveries brought to the institution with three delays. estimation of family influences is difficult in that familial effects other than general socioeconomic status are very difficult to observe. clustering of deaths in families was explained in rural punjab and in guatemalan families by household s economic status and mother s education. we found high variance of unobserved familial effect of 2.86 in the rural area of eag states even after taking into account all possible cultural and socio - economic variables. this large unobserved heterogeneity at family level could be a result of greater variability in child care practices, health care and mother s personal abilities. also the female child is more likely to die before reaching age five than the male child which might be related to behavioral and environmental factors[5, 27 ]. thus, parental competence, genetic and other factors like nutritional deficiency, personal illness of the child etc which were not included in the present study might be the explanation for the family frailty in these rural eag states. the strengths of this study are the use of nationally representative survey of nfhs-3 (2005 - 06) data and the application of the cox frailty model to estimate unbiased parameter estimates for determinants after accounting for familial effect. the variable, breastfeeding, was not considered as a time - dependent covariate due to methodological difficulty of the frailty model. in conclusion, this paper confirms the hypothesis that the risk of under - five death among families is heterogeneous and identifies determinants associated with under - five deaths. many determinants can be modified by child survival programs to enhance child survival, such as intensive antenatal and delivery care to young pregnant women and women having parity of more than two with preceding birth interval of less than two years ; providing ideal nutritional supplement to infants who are small and or very small at the time of birth ; improving mother s child care practices by health education if mother has lost previous child ; and reemphasizing exclusive breastfeeding for six months with introduction of complementary feeding at appropriate time. in the setting of correlated observations, the cox frailty models are recommended for providing statistically valid estimates of the effects of proximate determinants after adjusting for the background variables and unobserved random effects.	objectivesin india there has been a decline in overall under - five mortality, with some states still showing very high mortality rates. it is argued that there is family clustering in mortality among children aged < 5 years. we explored the effects of programmable (proximate) determinants on under - five mortality by accounting for family - level clustering and adjusting for background variables using cox frailty model in rural empowered action group states (eag) in india and compared results with standard models.methodsanalysis included 13,785 live births that occurred five years preceding the national family health survey-3 (2005 - 06). the cox frailty model and the traditional cox proportional hazards models were used.resultsthe cox frailty model showed that mother s age at birth, place of delivery, sex of the baby, composite variable of birth order and birth interval, baby size at birth, and breastfeeding were significant determinants of under - five mortality, after adjusting for the familial frailty effect. the hazard ratio was 1.41 (95% ci=1.141.75) for children born to mothers aged 12 - 19 years compared to mothers aged 20 - 30 years, 1.42 (95% ci=1.121.79) for small - sized than average - sized babies at birth, and 102 (95% ci=81128) for non - breastfed than breastfed babies. children had significantly lower mortality risks in the richest than poorest wealth quintile. the familial frailty effect was 2.86 in the rural eag states. the hazard ratios for the determinants in all the three models were similar except the death of a previous child variable in the cox frailty model, which had the highest r2 and lowest log-likelihood.conclusions and public health implicationswhile planning for the child survival program in rural eag states, parental competence which explains the unobserved familial effect needs to be considered along with significant programmable determinants. the frailty models that provide statistically valid estimates of the covariate effects are recommended, when observations are correlated.
thirty two consecutive patients with jme admitted at the epilepsy clinic of sina hospital, and 32 healthy controls in tehran, iran were enrolled in the study. jme was diagnosed using international league against epilepsy (ilae) classification (6). entry criteria for patients included normal brain mri, age between 16 - 60 years, completion of primary school as the minimum level of education, juvenile onset of myoclonic jerks with or without generalized tonic - clonic seizure, generalized discharges of an irregular mixture of 3 - 6 hz spike / polyspikes - slow waves on electroencephalography (eeg). the exclusion criteria were the presence of an illness other than jme that may affect cognitive functions including neurological or psychiatric disorders, occurrence of a generalized seizure within the previous month or myoclonic jerks or absence seizures within the previous 24 hours, mental retardation (iq score below 85 based on raven s progressive matrices test), progressive psychiatric and neurological disorders, alcohol or drug abuse, ongoing use of any medications affecting central nervous system. age-, sex- and education - matched healthy control subjects collected from patient s accompanies or other healthy subjects who were admitted to sina hospital and had no medical, neurological or psychiatric illnesses, neither had family history of seizures and history of medication use which may affect cognitive function. subjects in both groups signed their written informed consents prior to being included in this study. jme diagnosis and eeg recording were done by an expert neurologist in the field of epilepsy. subjects completed a questionnaire including clinical and socio - demographic information as gender, age, level of education, duration of epilepsy, type and frequency of seizures, and drug treatment. neuropsychological tests were administered in a sound - attenuated, temperature - controlled room by a single trained neurologist. general information, orientation, mental control, logical memory, forward and backward digit spans, visual memory, associative learning, and memory quotient (mq) from the persian version of wechsler memory scale (wms)-revised were evaluated (17). the wisconsin card sorting test (wcst) perseverative errors was selected for evaluating executive function (18). attention and control of inhibition evaluated using the stroop test (color and word) (19). iq scores were obtained from raven s progressive matrices test (20) and depression was evaluated using the persian version of beck depression inventory (bdi - ii) (cronbach s alpha = 0.87 and r = 0.73) (21). cognitive functions and depression between the jme and control groups were compared. in the jme group, we evaluated correlation between cognitive performance and age, sex, level of education, history of febrile convulsion, familial history of epilepsy, disease and medication history. spss 11.0 (spss inc, chicago, illinois, usa) software was used for statistical analysis. student s t - test and the mann - whitney u - test were used for independent normally and non - normally distributed continuous variables, respectively. correlation between neuropsychological scores, patient characteristics, and epilepsy variables was evaluated by pearson s correlation analysis. the characteristics of 32 patients with jme and 32 controls has been compared in table 1. there were no significant differences in age, gender, and level of education between the jme and control groups. twenty one patients (65.6%) had a family history of epilepsy and six patients (18.8%) had experienced febrile convulsion. the proportions of patients who received valproate or topiramate were 21 (65.6%) and 1 (3.1%), respectively, and other patients received polytherapy (different combinations of valproate, topiramate, carbamazepine, phenytoein, phenobarbital, lamotrigine, and sodium valproate). as shown in table 2, patients with jme exhibit worse performances in more domains of cognitive functions compared to controls. however, significant differences were observed between jme and control groups with respect to scores of mental control (7.21.5 vs. 8.11.2, p=0.015), forward digit span (5.70.7 vs. 6.2 0.7, p=0.004), total digit span (9.81.1 vs. 10.6 1.2, p=0.008), and iq (106.711.8 vs. 115.811.8, p=0.003). also, difference in memory quotient (mq) value was significant between two groups in p<0.06. the patients with jme who graduated from the university had higher values of logical memory (11.03.4 vs. 8.54.3, p=0.015), visual memory (11.13.5 vs. 9.23.0, p=0.048), forward digit span (6.00.7 vs. 5.30.5, p=0.013), mq (110.314.3 vs. 102.814.1, p=0.044), iq (113.612.5 vs. 106.412.8, p=0.036), and lower value of stroop color (1.81.8 vs. 3.82.1, p<0.001) compared to other patients. however, there were not significant association between the scores of cognitive function tests and sex, history of febrile convulsion and familial history in the patients with jme. there were significant correlation between age of patients with jme and scores of associative learning (r=-0.663, p<0.001), perseverative errors (r=0.394, p=0.028), stroop color (r=0.616, p<0.001), and iq (r=-0.530, p=0.002). duration of treatment and epilepsy had significant correlation with values of mental control (r=-0.574, p=0.001 ; r=-0.373, p=0.035), backward digit span (r=-0.475, p=0.006 ; r=-0.370, p=0.037), total digit span (r=-0.517, p=0.002 ; r=-0.359, p=0.044), associative learning (r=-0.395, p=0.025 ; r=-0.532, p=0.002), perseverative errors (r=0.446, p=0.012 ; r=0.437, p=0.014), stroop color (r=0.782, p<0.001 ; r=0.608, p<0.001), and iq (r=-0.603, p<0.001 ; r=-0.535, p=0.002). moreover, duration of treatment was correlated with scores of logical memory (r=-0.373, p=0.043) and forward digit span (r=-0.424, p=0.016). however, the poor performances in cognitive function tests in the jme group were not correlated with dose of antiepileptic medications. the negative impact of jme is reflected in several cognitive domains including iq, mental control, forward and total digit span, compared to the healthy controls, whereas mood and education level were similar in two groups. moreover, age, level of education, duration of epilepsy and medication had an impact on several cognitive functions in the patients with jme. neuropsychological studies have shown that the deficits in the area of behavior, social adjustment and executive functions, which are observed in jme, point to frontal lobe involvement in this disorder (3). dysfunction of these regions, especially the prefrontal cortex, results in disturbances in the organization and self - regulation of behavior. these functions involve in the elaboration of cognitive and behavioral responses and strategies for the attainment of immediate or future goals (3, 22). in addition, significant executive dysfunction in children with jme has been associated with significantly smaller thalamus and increased frontal cerebrospinal fluid (14). thus, frontal and thalamic volumes appear to mediate the relationship between executive functioning and brain structure in patients with jme (4). the present study confirmed previous evidences for frontal lobe dysfunction in jme (10, 11, 23 - 27). intelligence quotient (iq) : some investigations have shown a decline in the intellectual function of ige patients (6 - 8, 28, 29). in agreement with our results, pascalicchio (3) found significantly lower iq score in the patients with jme compared to controls, whereas some previous studies reported no significant differences in iq score between these patients and control groups (11, 23, 30). lower iq scores were observed in the patients with a younger age at onset, more frequent seizures, more frequent episodes of status epilepticus, longer duration of seizure attacks, structural abnormalities in the brain and atypical absence seizures (6, 22 and 31). however, our results showed that the iq scores of patients with jme had positive correlation with educational level and had negative correlation with age, disease duration and medication. in agreement with several studies such as pascalicchio (12), we could find a positive relationship between the level of education and higher scores in several cognitive domains in the patients with jme. the neuropsychological assessment of the patients with higher level of education (more than 16 years) showed significant preference in the scores of logical memory, visual memory, forward digit span, mq and iq compared to patients with lower level of education. the implication of this difference might be an important finding for modification of cognitive deficits and its negative consequences on their quality of life. considering the side effects of antiepileptic drugs on cognitive function, the basic neuropathology of seizure disorders including jme, it may not be possible to prevent cognitive deficits in all of the patients, therefore education could be a modifiable variable for more resistance against cognitive deficits in these patients. we found significant differences between patients with jme and controls in respect to forward and total digit span score which was in consistent with two previous studies (7, 30). however, sonmez (23) reported no signiffcant difference in the digit span test between patients with jme and control groups. they found the digit span scores of patients with a familial history of seizures were lower than patients without this. our results showed that this test could be influenced by the level of education, disease duration and medication. one of the justifications of our results may be explained by the known side effects of antiepileptic medications on concentration and memory. visual memory and visuospatial function : sonmez. (23) found impaired visual memory and visuospatial function in the patient with jme, which were in contrast to our results and kim study (30). also, they reported a significant difference between two groups on stroop test. these differences might be attributable to the lack of assessment of depression in the study of sonmez ; hence their study does not exclude the effect of depressed mood on global cognitive performance. on the other hand, we found deficit in mental control in the patients with jme which was not reported in the previous studies. duration of the epilepsy was correlated with cognitive performance, which confirmed in two previous studies (7, 30). evidence suggests that prolonged exposure to abnormal neural activity during a critical period of cerebral maturation may disrupt the structural and functional changes in the brain that contribute to the establishment of the neural substrate for language development, learning and lateralization of functions (3). however, the results of our study are not compatible with somnez (23) which did not find such correlation in their series of jme patients. in addition, there was significant correlation between duration of medication use and cognitive function or alternatively, it may be due to duration of disease. most antiepileptic drugs (aeds), such as topiramate, have the potential to exert detrimental effects on cognitive function. however, the small percentage of our patients (3.1%) has been on topiramate and 21 patients (65.6%) received valproate that has little detrimental impact on cognitive function (32, 33). therefore, we did not find significant correlation between dose of medication and cognitive performance. although depression and anxiety were reported as common psychiatric disorders in the patients with epilepsy (34), the results of bdi scores did not differ between the case and controls, which was in consistent with kim study (30). neuropsychological studies have shown that the deficits in the area of behavior, social adjustment and executive functions, which are observed in jme, point to frontal lobe involvement in this disorder (3). dysfunction of these regions, especially the prefrontal cortex, results in disturbances in the organization and self - regulation of behavior. these functions involve in the elaboration of cognitive and behavioral responses and strategies for the attainment of immediate or future goals (3, 22). in addition, significant executive dysfunction in children with jme has been associated with significantly smaller thalamus and increased frontal cerebrospinal fluid (14). thus, frontal and thalamic volumes appear to mediate the relationship between executive functioning and brain structure in patients with jme (4). the present study confirmed previous evidences for frontal lobe dysfunction in jme (10, 11, 23 - 27). intelligence quotient (iq) : some investigations have shown a decline in the intellectual function of ige patients (6 - 8, 28, 29). in agreement with our results, pascalicchio (3) found significantly lower iq score in the patients with jme compared to controls, whereas some previous studies reported no significant differences in iq score between these patients and control groups (11, 23, 30). lower iq scores were observed in the patients with a younger age at onset, more frequent seizures, more frequent episodes of status epilepticus, longer duration of seizure attacks, structural abnormalities in the brain and atypical absence seizures (6, 22 and 31). however, our results showed that the iq scores of patients with jme had positive correlation with educational level and had negative correlation with age, disease duration and medication. in agreement with several studies such as pascalicchio (12), we could find a positive relationship between the level of education and higher scores in several cognitive domains in the patients with jme. the neuropsychological assessment of the patients with higher level of education (more than 16 years) showed significant preference in the scores of logical memory, visual memory, forward digit span, mq and iq compared to patients with lower level of education. the implication of this difference might be an important finding for modification of cognitive deficits and its negative consequences on their quality of life. considering the side effects of antiepileptic drugs on cognitive function, the basic neuropathology of seizure disorders including jme, it may not be possible to prevent cognitive deficits in all of the patients, therefore education could be a modifiable variable for more resistance against cognitive deficits in these patients. we found significant differences between patients with jme and controls in respect to forward and total digit span score which was in consistent with two previous studies (7, 30). however, sonmez (23) reported no signiffcant difference in the digit span test between patients with jme and control groups. they found the digit span scores of patients with a familial history of seizures were lower than patients without this. our results showed that this test could be influenced by the level of education, disease duration and medication. one of the justifications of our results may be explained by the known side effects of antiepileptic medications on concentration and memory (23) found impaired visual memory and visuospatial function in the patient with jme, which were in contrast to our results and kim study (30). also, they reported a significant difference between two groups on stroop test. these differences might be attributable to the lack of assessment of depression in the study of sonmez ; hence their study does not exclude the effect of depressed mood on global cognitive performance. on the other hand, we found deficit in mental control in the patients with jme which was not reported in the previous studies. duration of the epilepsy was correlated with cognitive performance, which confirmed in two previous studies (7, 30). evidence suggests that prolonged exposure to abnormal neural activity during a critical period of cerebral maturation may disrupt the structural and functional changes in the brain that contribute to the establishment of the neural substrate for language development, learning and lateralization of functions (3). however, the results of our study are not compatible with somnez (23) which did not find such correlation in their series of jme patients. in addition, there was significant correlation between duration of medication use and cognitive function or alternatively, it may be due to duration of disease. most antiepileptic drugs (aeds), such as topiramate, have the potential to exert detrimental effects on cognitive function. however, the small percentage of our patients (3.1%) has been on topiramate and 21 patients (65.6%) received valproate that has little detrimental impact on cognitive function (32, 33). therefore, we did not find significant correlation between dose of medication and cognitive performance. although depression and anxiety were reported as common psychiatric disorders in the patients with epilepsy (34), the results of bdi scores did not differ between the case and controls, which was in consistent with kim study (30). one of the limitations of this study was the lack of functional mri data of patients which hindered our ability to demonstrate any relationship between localization of epilepsy and cognitive functions. moreover, it is preferable to investigate patients with epilepsy who are not receiving aed medications, because of adverse effects of aeds which potentially contribute to cognitive impairment. finally and in practice, it was not possible to consider a blind examiner for cognitive assessments. in conclusion, this study indicates that jme is associated with impairment in specific cognitive domains and more specifically in the frontal, prefrontal and memory domains. in addition age, education level, duration of disease and medication had an impact on cognitive performance. however more investigation should be performed in the patients with jme to understand the depth and extension of neuropsychological deficits to provide a better clinical care and neuropsychological rehabilitation in the management of this group of patients. another important finding of this study is the presence of a relationship between the level of education and the scores of cognitive tests in the patients with jme. it may be claimed that higher level of education may play a role - as a reserve- against cognitive deficits which seemed to be an inevitable consequence in the patients who have to struggle with both epilepsy and the negative impacts of antiepileptic medications on their cognitive functions.	objectivethe aim of present study was to verify possible cognitive dysfunction in the patients with juvenile myoclonic epilepsy (jme) and its correlation to factors related to epilepsy and patients demographic variables.material and methodsthirty two consecutive patients with jme and 32 healthy controls were evaluated in neuropsychological domains including orientation, mental control, logical memory, forward and backward digit spans, visual memory, associative learning, and memory quotient (using persian version of wechsler memory scale (wms)-revised), preservative errors (using wisconsin card sorting test (wcst)), stroop test (color and word), iq score (using raven s progressive matrices test), and depression (using the persian version of beck depression inventory (bdi)). spss 11.0 (spss inc., chicago, illinois, usa) software was used for statistical analysis. student s t - test and the mann - whitney u - test were used for independent normally and non - normally distributed continuous variables, respectively.resultsour study showed significant differences between patients with jme and control group with respect to scores of mental control (p=0.015), forward digit span (p=0.004), total digit span (p=0.008) and iq (p=0.003). in addition, age, education level, duration of epilepsy and medication showed an impact on several cognitive functions in the patients with jme.conclusionit is indicated that jme is associated with impairment in specific cognitive domains, despite any evidence in favor of depression.
type 2 diabetes is characterized by chronic hyperglycemia resulting in microvascular and macrovascular complications which may contribute to an increased risk of early cardiovascular morbidity and early mortality. now - a - days, the primary approach for type 2 diabetes management is steadily and continuously blood glucose control, with the aim of decreasing the risk of diabetic complications. a diabetes outcome progression trial (adopt), which compared three antidiabetic agents currently available for the treatment of type 2 diabetes, showed that in terms of glycemic control, rosiglitazone showed a long - term glycemic control. since metformin and rosiglitazone act through different mechanisms, their combined use may be indicated in patients whose disease is poorly controlled with a maintenance dose of metformin. studies have shown that rosiglitazone in combination with metformin can decrease glycated hemoglobin (hba1c) while increase insulin sensitivity and improve -cell function. at present, china has listed the compound tablet containing a fixed dose of rosiglitazone and metformin, avandamet, which may improve patient compliance. data showed that, due to the increased number of patients with medication, it was very difficult to long - term adherence to their drug regimen. a compound tablet containing two different fixed dose drugs due to a different mechanism can reduce the number of medication, maintaining a more strict glycemic control, improving the compliance of patients with general. the aim of this randomized open study was to compare the efficacy and safety of avandamet and up titrated metformin in patients uncontrolled with metformin alone. patients aged 1865 years who have been diagnosed with type 2 diabetes (defined according to world health organization criteria) inadequately controlled with metformin were considered for this 48-week, multicenter, randomized, open - label, parallel group study. the study was conducted in accordance with good clinical practice guidelines and the 1996 version of the declaration of helsinki. written informed consent the study protocol and informed consent were approved by the ethics committee of peking university people 's hospital according to local requirements. participants with body mass index (bmi) 24 kg / m, 7.5% hba1c 9.5%, 7 mmol / l fasting plasma glucose (fpg) 15 mmol / l at screening, receiving 0.751.0 g metformin alone at constant dosage for at least 6 weeks prior to entry were included in this trial. exclusion criteria included : (1) treatment with glucose - lowing agents other than metformin within 12 weeks before screening ; (2) allergic to thiazolidinedione or their ingredients ; (3) treatment with exogenous insulin or receiving insulin within 12 weeks before screening ; (4) history of diabetic ketoacidosis ; (5) hemoglobin disease or chronic anemia with the level of hemoglobin 2.5 times the upper limit of normal, suffering from hepatic disease ; (8) type 1 diabetes, gestational diabetes, or other type of diabetes (mody,.) ; (9) a history of ischemic heart disease or heart failure according to the new york heart association classification class i iv ; (10) patients taking nitrates ; (11) ongoing edema requiring pharmacological treatment ; (12) systemic blood pressure > 170 mmhg or diastolic blood pressure > 100 mmhg while on anti - hypertensive treatment ; (13) any chronic disease requiring continuous intermittent treatment with corticosteroids ; (14) breastfeeding, pregnant or planning pregnancy women (women of childbearing age before interview one to use effective contraception for at least 1 month) ; (15) patients with abnormality during screening by the investigator judgement ; (16) active drug or alcohol abuse within the last 6-month or any associated condition that could preclude completion of the study ; (17) patients probably accept the iodinated contrast agent during study duration ; (18) electrocardiogram with qtc > 500 ms or unadjusted qt > 600 ms ; or patients with bundle branch block with qtc > 530 ms. this was a 48-week, multicenter, randomized, open - label, parallel group study, consisting of a screening visit and 48-week of follow - up after randomization. in an open regimen, patients were randomized into two groups receiving avandamet 4 mg/1000 mg or metformin 1500 mg as the initial daily dose. four weeks later, for each treatment group, drug dosage was increased to the maximum dosage (8 mg/2000 mg of avandamet or 2500 mg of metformin daily) according to the protocol. if fpg level > 7.0 mmol / l (126 mg / dl) and the maximum dosage was not reached, a dose increase was required at each visit ; while a dose reduction was permitted if adverse events (aes) occurred. the first patient was recruited on november 19, 2009 and the last patient completed follow - up visit on march 15, 2011. the authors confirm that all ongoing and related trials for this drug / intervention are registered. the primary efficacy outcome is the percentage of patients who reached the target hba1c (hba1c 7%) at week 48 in avandamet treatment group and metformin treatment group. hypothesis was made as that the between group difference more than 15% of the percentage of patients who reached hba1c 7% at week 48 suggested that superiority was reached. the secondary outcomes included : (1) changes in hba1c from baseline ; (2) changes in fpg and prandial plasma glucose (ppg) from baseline ; (3) percentage of patients reached hba1c 6.5% at week 48 ; (4) percentage of patients with the changes from baseline in hba1c 0.7% in avandamet group and metformin group ; (5) changes in fasting insulin from baseline. all aes were recorded and judged by an investigator as the severity and possible relationship to study medication. safety laboratory assessments (hematology and biochemistry) and vital signs were assessed at each study visit. mild - to - moderate hypoglycemia was defined if the patient had symptoms or a self - measured capillary glucose level (cg) 2.5 times the upper limit of normal, suffering from hepatic disease ; (8) type 1 diabetes, gestational diabetes, or other type of diabetes (mody,.) ; (9) a history of ischemic heart disease or heart failure according to the new york heart association classification class i iv ; (10) patients taking nitrates ; (11) ongoing edema requiring pharmacological treatment ; (12) systemic blood pressure > 170 mmhg or diastolic blood pressure > 100 mmhg while on anti - hypertensive treatment ; (13) any chronic disease requiring continuous intermittent treatment with corticosteroids ; (14) breastfeeding, pregnant or planning pregnancy women (women of childbearing age before interview one to use effective contraception for at least 1 month) ; (15) patients with abnormality during screening by the investigator judgement ; (16) active drug or alcohol abuse within the last 6-month or any associated condition that could preclude completion of the study ; (17) patients probably accept the iodinated contrast agent during study duration ; (18) electrocardiogram with qtc > 500 ms or unadjusted qt > 600 ms ; or patients with bundle branch block with qtc > 530 ms. this was a 48-week, multicenter, randomized, open - label, parallel group study, consisting of a screening visit and 48-week of follow - up after randomization. in an open regimen, patients were randomized into two groups receiving avandamet 4 mg/1000 mg or metformin 1500 mg as the initial daily dose. four weeks later, for each treatment group, drug dosage was increased to the maximum dosage (8 mg/2000 mg of avandamet or 2500 mg of metformin daily) according to the protocol. if fpg level > 7.0 mmol / l (126 mg / dl) and the maximum dosage was not reached, a dose increase was required at each visit ; while a dose reduction was permitted if adverse events (aes) occurred. the first patient was recruited on november 19, 2009 and the last patient completed follow - up visit on march 15, 2011. the authors confirm that all ongoing and related trials for this drug / intervention are registered. the primary efficacy outcome is the percentage of patients who reached the target hba1c (hba1c 7%) at week 48 in avandamet treatment group and metformin treatment group. hypothesis was made as that the between group difference more than 15% of the percentage of patients who reached hba1c 7% at week 48 suggested that superiority was reached. the secondary outcomes included : (1) changes in hba1c from baseline ; (2) changes in fpg and prandial plasma glucose (ppg) from baseline ; (3) percentage of patients reached hba1c 6.5% at week 48 ; (4) percentage of patients with the changes from baseline in hba1c 0.7% in avandamet group and metformin group ; (5) changes in fasting insulin from baseline. all aes were recorded and judged by an investigator as the severity and possible relationship to study medication. safety laboratory assessments (hematology and biochemistry) and vital signs were assessed at each study visit. mild - to - moderate hypoglycemia was defined if the patient had symptoms or a self - measured capillary glucose level (cg) 0.05). baseline characteristics of the lost - to - follow - up patients did not differ from those who completed the study. baseline characteristics of the lost - to - follow - up patients between avandamet group and metformin group did not differ either. the primary efficacy outcome is the percentage of patients reached hba1c 7% at week 48 in avandamet group and metformin group. at the end of week 48, the percentage of patients who reached hba1c 7% was 83.33% in avandamet group and 70.00% in metformin group (p 0.05) [table 4 ]. the most commonly reported aes (occurring in > 5% of avandamet - treated patients vs. metformin - treated patients) were abdominal discomfort (7.1% vs. 9.5%, p > 0.05), dyslipidemia (7.6% vs. 8.5%, p > 0.05), upper respiratory tract infection (6.6% vs. 6.5%, p > 0.05), edema (6.6% vs. 0.5%, p 0.05). there were no cases of heart failure in either treatment group, but two occurrence of angina pectoris in avandamet treated patients. one is a 61-year - old female patient who was incharged into hospital for the treatment of angina pectoris with the investigator judgement as probably correlated with study drug and discontinued avandamet treatment. another is a 51-year - old female patient who was incharged into hospital with the investigator judgement as probably not correlated with study drug and continued avandamet treatment. both patients recovered and discharged from hospital soon. aes, hypoglycemia between the two groups (n) aes : adverse events ; sae : serious adverse event. five hundred and eighty - eight patients who met the inclusion criteria were recruited for the study. at the end of run - in period, 398 patients who still met the inclusion criteria were randomly assigned into avandamet treatment group and metformin treatment group, while 156 participants in avandamet treatment group and 160 participants in metformin treatment group were included in the fas [figure 1 ]. baseline characteristics of patients between avandamet treatment group and metformin treatment group were comparable except that the average duration of diabetes in avandamet group was significantly longer than that in metformin group (3.56 3.49 years vs. 2.73 3.10 years, p = 0.01) [table 2 ]. baseline characteristics of the patients baseline characteristics were comparable between the two groups except for diabetic duration (p = 0.01). bmi : body mass index ; sbp : systolic blood pressure ; dbp : diastolic blood pressure. the percentage of patients who did not complete the study did not differ significantly between avandamet group and metformin group (23.23% vs. 24.00%, p > 0.05). baseline characteristics of the lost - to - follow - up patients did not differ from those who completed the study. baseline characteristics of the lost - to - follow - up patients between avandamet group and metformin group did not differ either. the primary efficacy outcome is the percentage of patients reached hba1c 7% at week 48 in avandamet group and metformin group. at the end of week 48, the percentage of patients who reached hba1c 7% was 83.33% in avandamet group and 70.00% in metformin group (p 0.05) [table 4 ]. the most commonly reported aes (occurring in > 5% of avandamet - treated patients vs. metformin - treated patients) were abdominal discomfort (7.1% vs. 9.5%, p > 0.05), dyslipidemia (7.6% vs. 8.5%, p > 0.05), upper respiratory tract infection (6.6% vs. 6.5%, p > 0.05), edema (6.6% vs. 0.5%, p 0.05). there were no cases of heart failure in either treatment group, but two occurrence of angina pectoris in avandamet treated patients. one is a 61-year - old female patient who was incharged into hospital for the treatment of angina pectoris with the investigator judgement as probably correlated with study drug and discontinued avandamet treatment. another is a 51-year - old female patient who was incharged into hospital with the investigator judgement as probably not correlated with study drug and continued avandamet treatment. both patients recovered and discharged from hospital soon. aes, hypoglycemia between the two groups (n) aes : adverse events ; sae : serious adverse event. the aim of this randomized open study was to compare the efficacy of avandamet and up titrated metformin in patients uncontrolled with metformin alone in a chinese population. avandamet treatment for 48-week in patients poorly controlled with metformin alone led to statistically significant and clinically relevant reductions in hba1c, fpg and ppg. compared with metformin up titreated treatment, treatment with showed trends toward a greater proportion of patients achieving hba1c < 7% as well as achieving hba1c < 6.5%. results concluded from this study were comparable with that of the previous published studies in caucasians. fonseca. reported in a study that after 26 weeks, compared with metformin - placebo treatment, rosiglitazone - metformin treatment led to a 1% in hba1c decreased and 2.2 mmol / l in fpg decreased. rosenstock. reported from a randomized open - label trial that after 24 weeks of avandamet treatment, the average hba1c decreased 4.0% 2.2% and the average fpg decreased 7.7 4.4 mmol / l, as well as 33% patients reached the target of hba1c 6.5%. in another randomized double - blinded trial, after 32 weeks of avandamet treatment, the average hba1c decreased 2.3% and the average fpg decreased 4.1 mmol / l with significance, and about 60% and 77% patients with avandamet treatment reached the target of hba1c 6.5% and hba1c 7% respectively, which was higher than the proportion of patient receiving metformin treatment. borges. in an 80-week randomized double - blinded trial reported that, the avandamet treatment was superior to metformin treatment both in hba1c and fpg. in terms of fasting insulin, results from this study indicated that avandamet treatment led to a decrease of fasting insulin more than metformin treatment, which was also in adherence with previous reports. kahn. concluded from the results of adopt that compared with sulfonylurea, both rosiglitazone and metformin can decrease fasting insulin level, improving insulin sensitivity. combinated with rosiglitazone or pioglitazone showed that, except for glucose improving, metformin combinated with rosiglitazone decreased fasting and postprandial insulin level significantly. a 6-month small study in chinese also showed that metformin combination with rosiglitazone could decrease fasting and postprandial insulin level, which indicated that insulin resistance improving. according to results from this study, compared with up titrated metformin treatment, avandamet treatment was associated with less reduction in body weight as well as less decrease in lipid profile. in fonseca. 's study, total cholesterol, hdl - c and ldl - c were significantly higher in rosiglitazone - metformin treatment and body mass was increased compared with metformin - placebo control group. reasons for the increasing of lipid profile may be associated with the mechanism of glucotoxicity improvement or different percentage of usage of lipid lowering therapy between groups, or other reasons we still no know. the weight gain in avandamet group might be associated with fluid retention or increased appetite which was explained in other studies previously. the more reduction of crp in avandamet group might be associated with the efficacy in lowering free fatty acid or other inflammation factors. there was no significant difference of the number of aes and saes between the two groups. the incidence rate of diarrhea in patients with avandamet treatment was significantly lower than that of patients in metformin group, while the incidence rate of edema with avandamet treatment was significantly higher. the types of aes as well as the incidence rate of total aes of this trial were similar with those results of other clinical trials. in addition, no case of heart failure in either treatment group was reported during the 48-week of follow - up, but two occurrences of angina pectoris in avandamet treated patients were reported. both patients recovered and discharged from hospital soon, one continued the study drug while the other one discontinued. of course, longer duration and with the primary endpoint of study is needed to evaluate the cardiovascular outcome of avandamet treatment in the future. although some of our study design was similar with that of previous studies carried out in caucasians, there are some different points. first, this study was carried out in chinese type 2 diabetes patients, which is a totally different ethnic population. ethnicity difference might be associated with the incidence of type 2 diabetes as well as the treatment efficacy on glucose control or -cell function or body weight changes, therefore, results from this study will give us the answer whether the superior efficacy of avandamet treatment to metformin treatment alone could be shown in a chinese population. secondly, baseline characteristics of patients were different from previous studies in caucasian population such as bmi, hba1c, fpg, duration of diabetes and so on. results from different kinds of patients may result in a different conclusion, therefore, we just want to see whether the efficacy of rosiglitazone combination with metformin treatment was superior to that of up titrated metformin treatment. third, different dosage forms between our study and other studies. in most previous study, they prescribed patients with metformin plus placebo or metformin plus rosiglitazone. in our study, we prescribed patients only one tablet with avandamet or metformin. what 's more, study duration in this trial was 48-week, which is a longer duration for us to evaluate the efficacy of avandamet treatment. according to the result concluded from this study in chinese type 2 diabetes patients, avandamet treatment led to a higher proportion of patients achieving hba1c < 7% compared with metformin although the superiority was not reached. clinical significance for this result might indicate that for poorly controlled chinese type 2 diabetes patients with metformin alone, avandamet treatment instead of up titrated metformin may be a good choice for glycemic control and insulin resistance improvement. reasons for that might be the combination mechanisms of rosiglitazone and metformin, the possible better adherence of avandamet treatment than up titrated metformin. comparable results concluded from this study in chinese type 2 diabetes patients also indicated that there may be no ethnicity difference in avandamet treatment. first, patients who lost follow - up in avandamet group and metformin group were 23.23% and 24.00% respectively, the rate of which exceeded the expected lose rate (abscission rate up to not more than 15%), which may be the reasons for this study that failed to demonstrate the superiority of avandamet treatment with respect to the higher dose of metformin monotherapy. but there was no significant difference in the abscission rate between groups with the p value of 0.9063, and the baseline characteristics of patients completed the trial as well as those of patients withdrawn did not show any significant difference between the avandamet group and metformin group either (data was shown in supplement material), which might indicate that the higher loss rate was not resulted from different treatment group. secondly, baseline characteristics of patients between avandamet treatment group and metformin treatment group were comparable except that the average duration of diabetes in avandamet group was significantly longer than that in metformin group (3.56 3.49 years vs. 2.73 3.10 years, p = 0.01). however, this difference at baseline did not seem to be associated with the efficacy difference between groups, just because patients in avandamet group had a longer duration of diabetes which may indicate poorer insulin secretion and therefore poorer effect in hypoglycemic treatment. controversely, at the end of the study, patients in avandamet group had a higher decrease in hba1c and a better control of glycemic treatment, which indicated that the baseline difference did not affect the final results. in conclusion, results from this randomized, open label, parallel group study indicated that compared with up titrated metformin treatment, the efficacy of avandamet treatment in patients uncontrolled with metformin alone was better, the proportion of patients who reached the target hba1c was higher in chinese type 2 diabetes patients.	background : at present, china has listed the compound tablet containing a fixed dose of rosiglitazone and metformin, avandamet, which may improve patient compliance. the aim of this study was to evaluate the efficacy and safety of avandamet or uptitrated metformin treatment in patients with type 2 diabetes inadequately controlled with metformin alone.methods:this study was a 48-week, multicenter, randomized, open - labeled, active - controlled trial. patients with inadequate glycaemic control (glycated hemoglobin [hba1c ] 7.59.5%) receiving a stable dose of metformin (1500 mg) were recruited from 21 centers in china (from 19 november, 2009 to 15 march, 2011). the primary objective was to compare the proportion of patients who reached the target of hba1c 7% between avandamet and metformin treatment.results:at week 48, 83.33% of patients reached the target of hba1c 7% in avandamet treatment and 70.00% in uptitrated metformin treatment, with significantly difference between groups. the target of hba1c 6.5% was reached in 66.03% of patients in avandamet treatment and 46.88% in uptitrated metformin treatment. the target of fasting plasma glucose (fpg) 6.1 mmol / l was reached in 26.97% of patients in avandamet treatment and 19.33% in uptitrated metformin treatment. the target of fpg 7.0 mmol / l was reached in 63.16% of patients in avandamet treatment and 43.33% in uptitrated metformin treatment. fasting insulin decreased 3.24 0.98 u / ml from baseline in avandamet treatment and 0.72 1.10 u / ml in uptitrated metformin treatment. overall adverse event (ae) rates and serious ae rates were similar between groups. hypoglycaemia occurred rarely in both groups.conclusions:compared with uptitrated metformin, avandamet treatment provided significant improvements in key parameters of glycemic control and was generally well tolerated. registration number : chictr - trc-13003776.
family planning is done for future pregnancies with the goal of prevention from unplanned pregnancies, which plays a major role in the improvement of maternal health, especially in developing countries. the negative outcome of not using a reliable contraceptive method is an unexpected pregnancy, which has physical, mental, and socioeconomic destructive effects on the maternal health as well as negative consequences including mothers death, illegal abortions, delayed antenatal care, neonatal low birth weight, and reduction in breast feeding. half of the pregnancies are unplanned, resulting from not using a contraceptive method, application of an inefficient contraceptive method, fear of complications, lack of health services, lack of education, lack of a constant method, and finally, failure of contraceptive methods. health problems and modern methods have been reported as the most common reason for following the traditional methods in turkey. research shows that 70% of women have no sexual desire for a pregnancy, while 49% face unexpected pregnancies and half of them are involved in pregnancy termination method (abortion). although about 120 million women in the world have no desire for pregnancy, they do not use any contraception method. the related reasons have been reported as low availability of services and facilities, and spouse 's or relative 's disagreement. in iran, although 5.9% of married women have no desire for pregnancy, they use no contraceptive method. if a method is appropriately and constantly applied, it can have the highest efficiency. the most important factor to be considered in the selection of a method by the individuals is its efficiency. planning of better and high - quality family planning services needs actual participation of the society and is associated with the level of social recognition and knowledge of the necessity to conduct such programs and apply appropriate contraceptive methods. family planning helps the couples regulate their number of children based on their economic and physical and mental power through knowledge, insight, and accountability, with the help of one of the contraceptive methods. women 's knowledge concerning appropriate contraception is a key element in the prevention of mortality and in conducting family planning programs. selection of an appropriate method plays a key role in the level of learning and desire for behavior change in relation with individuals health and practice. the educator should primarily consider the efficiency of various educational methods and learner 's educational and socioeconomic level before planning and select the most efficient educational methods accordingly. various studies concerning family planning have been conducted globally and in iran, but fewer systematic studies using educational methods have been carried out in this issue. in fact, the value of health educational programs depends on their efficiency and proper use of health education theories and models. educational theories and models are responsible for defining and detecting the obstacles in behavioral change and for their adjustment with existing socio - cultural contexts. basnef is a comprehensive and complete model which is adopted to study behaviors and plans to change them and to define the factors effective on individuals decision making. based on this model, individuals develop a new behavior when they believe it is beneficial for them. then, their evaluation leads to formation of their attitude toward their behavior. parallel to this process, key persons in one 's life can influence his / her decision for a new behavior and act either as a facilitator or as an inhibitor. individuals subjective norms are determined by their beliefs. in other words, how the new behavior is confirmed or rejected by the people is important in one 's mind. combination of attitude and subjective norms leads to formation of individuals decision making to attain a new behavior. on the other hand, factors such as skill, money, costs, etc.. these factors should exist prior to occurrence of a behavior. among the already conducted studies on basnef model in iran, sharifirad 's study on breast feeding, study on the effective factors on dormitory students smoking, taghdisi 's study on safety practice of coke workers in steel mill corporation, and ebadiazar 's study on prevention behavior concerning adolescents mental health can be pointed out. what prompted the researchers to adopt basnef model was the fact that existing problems in other behavior change models (health beliefs, behavior intention), such as lack of consideration of subjective norms and enabling factors, have been modified in this model, and almost all factors of health belief model, behavior intention, and precede behavioral factors have been included in it. important elements in the selection and change of a behavior in iran are among the important scales of basnef model which is specified for developing countries. literature review showed no study conducted in iran based on basnef model ; therefore, this study aimed to define the impact of an educational program based on basnef model in choosing contraceptive methods in women referring to health care centers in minoodasht in 2012. the study population comprised women not using any contraceptive methods or using traditional contraceptive methods, meeting the inclusion criteria, and referring to the health care centers (number 1 and number 2 of minoodasht) during aug.- sept. 2012. based on existing studies and ethnical and cultural structures and due to lack of precise studies, the sample size was finally calculated as 100 subjects with consideration of possible number of subjects dropping out in each of study and control groups (total of 200), with a confidence level of 95% (= 0.05, z1-/2 = 1.96) and a power of 80 (= 0.20, z1- = 0.84). health care centers with number 1 and number 2 were randomly assigned to the study and control groups, respectively. due to possible contamination of the subjects, it was decided to select the subjects in the study and control groups from separate centers. the subjects were left out of the study in case of spouse 's death or divorce. minoodasht has two health care centers (number 1 and number 2) which were randomly assigned to the study and control groups. as the town is not so big and there was possibility of subjects contamination, the population was divided into two parts and each one of it was associated with one center. the subjects were selected based on the inclusion criteria which were as follows : married women, muslim, iranian, fertility age of 10 - 49 years, healthy with no history of a known disease (contraindication for contraceptive methods), not using any contraceptive methods or on natural contraception (traditional), no desire for pregnancy, and literate enough to read and write. the exclusion criteria were : spouse 's death, divorcee, and unwilling to participate in the study. data collection tool was a researcher - made questionnaire including demographic characteristics and a section including age, length of marriage, education, occupation, number of pregnancies, deliveries and abortions, history of former deliveries and unexpected pregnancies, history of use of contraceptive method, and economic status, as well as a basnef questionnaire including six subscales of knowledge (7 questions), attitude (11 questions), subjective norms (5 questions), enabling factors (7 questions), behavior intention (2 questions), and practice (1 question). since practice and behavior refer to selection of a contraceptive method in the present study, the behavior intention is the intention to select a method, which was measured by three questions. practice was also determined concerning the method selection and the type of method by one question. content validity was adopted for testing the validity of the questionnaire. to confirm reliability, the questionnaire was filled by 20 women from a target group (not attending the study), and then was modified and checked with a confidence level of 95% (cronbach 's alpha = 84%). knowledge scores included seven questions and measured women 's information in a multiple choice answer format. attitude questions were 11 in number of which the first 7 were on personal beliefs and were scored 1 - 5 by likert 's scale as absolutely agree (score 1) to absolutely disagree (score 5), so that a positive attitude obtained a score of 1. subjective value comprised five questions of which three were of yes / no type and two were multiple choice questions in relation to the individuals and groups effective on women 's decision making. questions on enabling factors were seven in number of which three were yes / no questions and four were multiple choice questions on the available resources and facilities, education, family support, and skills. questions on behavior intention were three in number and were yes / no questions with the responses which showed women 's intention after the intervention and were answered during follow - up of practice at 3 months after the intervention. practice was measured by one yes / no question which was answered 3 months after the intervention during a follow - up and was considered positive if a contraceptive method was used by the w omen. all questions were scored, so that questions with correct answers were given a score of 1 and those with negative responses were given score. questions of attitude were scored in likert 's scale, wherein the respondents chose answers of absolutely agree, agree, no idea, disagree, and absolutely disagree and the answers were scored 1 - 5 with a different value for each item. as negative and positive directions of questions should be considered in likert 's scale, an answer key was prepared to give the highest score in a positive phrase to the option of absolutely agree and vice versa. after obtaining the approval of ethics committee and on confirmation of questionnaire validity and reliability, the researcher referred to the selected centers. after obtaining authorities permission and consent from the women qualified for the study primary data were analyzed, and educational intervention and its content were designed through valid scientific references accordingly. after obtaining the sample size, basnef - based education was conducted in five intervention groups (each containing 20 subjects). the period of the intervention was 3 months in the form of four educational sessions. firstly, group educational sessions were held face to face with women for 1 month. finally, an educational session was held for the families (individuals effective on women 's decision making). the participants were informed the gooya telephone number 149 (for spreading health messages) and it was emphasized that they obtain most of their needed information through this source. the persons who used a contraceptive method were invited during sessions and most of the subjects questions were answered through group discussion. to increase subjects motivation, conducting course exam and quiz tests and giving gifts were considered. with regard to use of condoms, practical education also (with the help of a moulage) was conducted. during the sessions, women 's knowledge of family planning programs and beliefs in relation with contraceptive methods was discussed in group discussion. basnef - based education improved the subjects knowledge through lecture method, group discussion, film show, health quizzes, practical education, and poster panel. the educational booklet and pamphlet were prepared and distributed to subjects in the intervention group. at the end of the fourth session, researchers phone numbers were given to the women in case they needed to contact the researchers. after women 's education (four sessions), an educational class was held for the group that was already determined in pre - test (subjects spouses, their mothers, their mothers in law, etc.). unexpected pregnancy complications and high - risk pregnancy were shown in a film, and the subjects were given examples of those involved in such complications and, through which the researcher tried to change subjects attitude. for those absent in the sessions, face - to - face education was provided at home visits. one and two months after the educational program (four educational sessions for women and one for families), two follow - up sessions were held. three months after, post - test was conducted and all subscales of basnef questionnaire (knowledge, attitude, subjective norms, enabling factors, behavior intention, and practice) were filled for both groups. the obtained results were analyzed by independent t - test, chi - square, paired t - test, mcnemar, and regression tests through spss version 14. the ethics committee of tehran university of medical sciences faculty of nursing andmidwifery approved the study. the ethics committee of tehran university of medical sciences faculty of nursing andmidwifery approved the study. independent t - test showed no significant difference in mean age of control and study groups [27.23 (6.22) and 27.72 (6.37) years, respectively ] and mean length of marriage in the control [7.30 (5.94) years ] and study groups [7.62 (6.23) years ]. findings showed that most of the subjects in the control (85%) and study (90%) groups were homemakers and their level of education was high school diploma (36% and 29%, respectively). most of the subjects in the control and study groups (68% and 70%, respectively) had one to five pregnancies, and 27% of women in the control group and 19% in the study group had no history of pregnancy. mean numbers of abortion in the control and study groups were 26 and 31 cases, respectively. history of unexpected pregnancy in the control and study groups was 19% and 23%, respectively. chi - square test showed no significant difference in the control and study groups concerning occupation, education, type of former delivery, history of unexpected pregnancy, and use of a contraceptive method. number 1and number 2 shows that there was no significant difference in the score of knowledge before education in the study and control groups (p = 0.291), but there was a significant difference after education (p = 0.003). comparison of mean scores of women 's attitude showed a significant difference in the study and control groups after intervention (p < 0.0001). analysis of covariance (ancova) showed a significant difference in mean scores of attitude before and after intervention in the study group (p < 0.0001). meanwhile, this difference in the control group was not significant (p = 0.271). with regard to effect of education on enabling factors, independent t - test showed a significant difference in the study and control groups before and after educational intervention (p < 0.0001). paired t - test showed no significant difference before and after educational intervention in the control group (p = 0.099). meanwhile, this test showed a significant difference in the study group before and after educational intervention (p < 0.0001). independent t - test showed a significant difference in subjective norms in the study and control groups before and after educational intervention (p < 0.0001). paired t - test showed no significant difference before and after educational intervention in the control group (p = 0.106). meanwhile, this test showed a significant difference in the study group before and after educational intervention (p < 0.0001) [table 1 ]. mcnemar test showed a significant difference in relation to spouse and health care personnel in the study group before and after educational intervention (p < 0.0001). paired t - test showed no significant difference before and after educational intervention in the control group (p = 0.106). meanwhile, this test showed a significant difference in the study group before and after educational intervention (p < 0.0001) [table 2 ]. mcnemar test showed a significant difference in relation to spouse and health care personnel in the study group before and after educational intervention (p < 0.0001), but no significant difference in relation to spouse 's family, wife, and friends. this test showed no significant difference in relation to spouse, wife 's family, friends, and health care personnel in the study group before and after intervention, but the difference in relation to spouse 's family was significant (p < 0.0001). chi - square test showed a significant difference in women 's practice in the study and control groups after intervention (p < 0.0001) and it was the same before education in both groups (they had no contraception). through logistic regression test and step - by - step analysis, the best subscale of basnef model in prediction of women 's contraceptive method selection was considered. the best introduced model and its enabling factors in prediction of women 's contraceptive method selection have been presented in table 3. the mean scores of different structures of basnef model in the two groups after intervention the mean scores of different structures of basnef model in two groups after intervention step - by - step regression model of knowledge component effect on women 's practice change nowadays, family planning is considered as the main part of life as it has a reciprocal association with health. provision of appropriate family planning services necessitates existence of trained forces, the required facilities and equipments, and appropriate planning. the main problem in use of contraceptive methods is low knowledge and an indifferent or negative attitude toward contraceptive methods which act as the main reasons for no referrals to health care centers. in a similar study conducted in 1985 on the effect of an educational model and women 's contraceptive behavior, robertson reported that use of health belief model directly influences the understanding, attitudes, and mental beliefs of contraception users as well as those not using any contraceptive method (25). the latter study showed that basnef - based educational intervention is effective on women 's knowledge, attitude, and practice. our findings showed that despite conducting education in health centers, women had low knowledge, which is consistent with the reports of sharifirad. bogale observed that the knowledge concerning contraceptive methods was low in most of the women, which is consistent with the result of the present study. mean scores of knowledge significantly increased 3 months after intervention, which reveals the effect of basnef - based educational intervention on increase of women 's knowledge, which is in line with the studies conducted on basnef model. the obtained results show that the mean score of women 's attitude notably increased after educational intervention in the study group, revealing the efficiency of basnef - based educational intervention in the domains of belief, continuity, and preservation and improvement of women 's attitude toward selection of a contraceptive method, which is in line with other studies. bani aghil and khoddam showed that education through increase of knowledge leads to motivation, and attaining more knowledge and a better attitude due to educational session results in practice of using contraceptive methods. three months after intervention, the attitude showed a little increase in the control group, which can possibly be due to increase of knowledge in this group leading to a more positive attitude among them. the enabling factors showed a significant difference before and after educational intervention in the study group (p = 0.0001), which reveals the positive role of family in supporting women in using a contraceptive method. in this regard, the results of sharifirad. 37 concerning breast feeding behavior of pregnant women and pirzadeh. on students nutritional education, as well as those of alizadeh concerning workers safety and mohamaei on the prevention of cardiac risk factors are consistent with the results of the present study. meanwhile, taghdisi., in a study on evaluation of basnef model efficiency in health promotion of cancer patients, observed significant difference in enabling factors. in the present study, enabling factors such as efficient education, time of giving service, educational facilities, and trained person had an active role in transformation of individuals intention to change behavior to select a safe contraceptive method, which is consistent with the report of jeihouni. laveist (1995) showed that enabling factors are much more important than cultural and behavioral differences to develop a behavior. in the present study, mean score of subjective norms significantly increased after educational intervention in the study group. this increase resulted from basnef - based educational intervention conducted in the study group as well as involvement of families, especially mothers and mothers in law. in this regard, studies showed that efficient education of mothers and mothers in law through basnef - based education leads to increase in the participation trend of women and their behavioral change, but some influencing persons (spouse and health care personnel) showed no significant difference (p < 0.0001). present study is consistent with pirzadeh 's study on students nutritional education in this regard. one of the key factors in developing a behavior is the subjective nor ms. in the present study, subjective norms effective on women 's behavior were detected as spouse, health care personnel, and wife 's and spouse 's family, respectively, which is consistent with the report of cherkazi. concerning health care personnel, while jeihouni. found that the most influencing persons were health care personnel, physicians, and the family. in a study in north ireland, emphasized that improper value worsens mothers correct behavior ; therefore, there should be a social approach in educational programs. conducted a study on theory of planned behavior applied to condom use among african american adolescents and found that health education models including theory of planned behavior are effective on attitude, subjective norms, and individuals intention either to develop or change a behavior, which is consistent with the present study. the most frequent source of information was health care personnel in both groups, which reveals that these personnel play an efficient role in the selection of contraceptive method among women. therefore, staffs education and their promotion of counseling skills can be effective in this regard. in some studies, mass media was reported as the most common source of information, but it had a lean role in this regard in the present study. in the present study, improvement of model components ultimately led to promotion of subjects practice in the study group, so that it raised from zero (no contraception) to 54% after intervention, which reflects the effect of basnef - based intervention on women 's practice and is consistent with the results of other studies on basnef model. in order to investigate the effect of basnef model subscales on the change in practice, the effect of each change on practice was, along with other factors, investigated in a regression analysis (ancova), and it showed that enabling factors were the most effective factors in the components of the model. with regard to subjective norms in iranian culture and enabling factors, use of an educational model which contains these components, like basnef, is recommended. with respect to the important goal of family planning program (prevention from unexpected pregnancies) and importance of mothers mortality (as an important index for development), it seems that if the model is precisely used, efficient steps could be taken toward prevention of unexpected pregna ncy and, consequently, improve maternal health. there may be problems and obstacles for behavioral change, such as cultural values and norms. educational models and patterns play a role in their definition and diagnosis, as well as their adaption to socio - cultural structures. it seems that through application of basnef model, an association was made between women 's knowledge, attitude, intention and behavior, and selection of a contraceptive method. this is a principle of basnef model. with regard to the subjective norms in iranian culture and enabling factors, the fallowing issues are suggested for promotion of women 's health : holding basnef - based educational sessions in health centers and hospitals for women, concerning appropriate pregnancy spacingapplication of models in educationapplication of basnef model in selection of delivery typeas the present study showed that basnef model is effective on women 's selection and behavior concerning use of a contraceptive method, this model is suggested to be used in selection of delivery type (natural or cesarean section). holding basnef - based educational sessions in health centers and hospitals for women, concerning appropriate pregnancy spacing application of models in education application of basnef model in selection of delivery type as the present study showed that basnef model is effective on women 's selection and behavior concerning use of a contraceptive method, this model is suggested to be used in selection of delivery type (natural or cesarean section).	background : quality of services, making communications with target groups, and educating them are among the most important success factors in implementation of family planning programs. provision of public access to contraception and related methods, counseling services, and paying attention to social specification and cultures are important in promotion of service quality. with regard to applicability of health education theories and models, the present study aimed to find the impact of an educational program based on basnef model to choose contraceptive methods in women referring to health care centers in minoodasht in 2012.materials and methods : this is a quasi - experimental study. data were collected using basnef questionnaire by the researcher from women referring to health care centers in two groups of study and control (n = 100 in each group). educational intervention (in the form of four educational 1-h sessions once a week during 1 month and two additional review sessions) was conducted in the form of group and face - to - face discussions and educational booklets were distributed. data were analyzed by chi - square, analysis of covariance (ancova), paired t - test, and t - test through spss version 14.results:after intervention, mean score of knowledge was significantly higher in the study group compared to control (p < 0.003). intervention led to a better attitude in the study group (p < 0.0001). mean score of subjective norms and enabling factors after intervention was significantly higher in the study group compared to control (p < 0.0001). the change in practice had a significant increase in the study group (54%) compared to control (9%).conclusions : basnef - based educational intervention was effective in increasing women 's knowledge, attitude, and practice. as family and educational facilities are among the influencing factors on contraceptive method selection, interventional planning is hoped to be based on educational model.
epidemiology of malocclusion and assessment of orthodontic treatment needs are of national importance in many countries and were thus included in numerous national level surveys [116 ]. malocclusion features the third highest prevalence among oral pathologies, secondarily to dental caries and periodontal disease and therefore ranks third among worldwide public health dental disease priorities. according to world health organization the main oral diseases should be subjected to periodic epidemiologic surveys. these assessments are necessary to plan sufficient treatment facilities and develop adequate training programs for respective specialists. malocclusions can be assessed with various methods [1823 ] but not one has gained universal acceptance. the index of orthodontic treatment need (iotn) was developed to grade malocclusion on the basis of the significance of various occlusal traits for dental health and esthetic impairment. the iotn incorporates a dental health component (dhc) based on the recommendations of the swedish medical board and an esthetic component. several prevalence studies have been conducted on children in mixed or permanent dentitions [2533 ]. however, prevalence of malocclusion and orthodontic treatment needs for nepalese children had not yet been reported in the indexed literature. therefore, the present study aims to assess the epidemiology of malocclusion and orthodontic treatment needs in nepalese schoolchildren aged 1215 years in eastern nepal using iotn index and compare these data with that of other population. this study was conducted after ethical clearance from institutional review board, bp koirala institute of health sciences and permission from concerned school authorities. the sample comprised two thousand seventy - four children (1149 males and 925 females) from twenty high schools consisting of both public and private schools. the subjects from the selected schools were included only if their chronological age was 1215 years and if they were permanent inhabitants of nepal. those who were undergoing orthodontic treatment or who had completed orthodontic treatment earlier or suffering from any other systemic diseases were excluded from the study. the examiner and recording assistant were trained prior to the commencement of the study to ensure reliability. a validation exercise was conducted during the study and subsamples of 10% were reexamined to check intraexaminer variability, which was found to be satisfactory (kappa value = 0.8). the examination for malocclusion was made according to the molar relationship (angle) and the criteria laid down by dhc of iotn. in addition the presence of anterior spacing, a feature overlooked by the dhc was evaluated. all the data was analyzed with spss software (version 16.0 for windows @ 2007 spss inc. out of 2074 children selected for the study, 14 did not return the signed parental consent document, 45 were not present at school on the assessment day, and 5 had already started orthodontic treatment. the age distribution of the remaining 2010 children, according to the gender, is presented in table 1. angle 's class i malocclusion was found in 48.50% of all children, class ii div. 1 in 29.35%, class ii div. 2 in 3.33%, class iii in 4.32%, and normal occlusion in 14.42% (table 2). table 3 shows the percentage scores of individual malocclusion traits according to the dhc of the iotn. crowding was the most common type of malocclusion presented by the study group (19.75%) followed by increased overjet (17.51%) and deep overbite (13.23%). features like scissor bite (0.89%), reverse overjet (1.79%), and open bite (2.03%) were least noticed in the study group. the iotn (dhc) showed the following distribution : grade 115.02% (n = 302) ; grade 214.7% (n = 296) ; grade 324.07% (n = 484) ; grade 424.67% (n = 496) ; grade 521.59% (n = 432). grades 3 and 4 were more commonly observed in nepalese children followed by grades 5 and 1 (table 4). the present study was designed to provide information about the prevalence of malocclusion and orthodontic treatment needs among 12- to 15-year - old school going children. although assessment of malocclusion in nongrowing population is more reliable, this range was chosen because it represents the majority of schoolchildren with developing malocclusion who require orthodontic treatment. the distribution of malocclusion and treatment needs among schoolchildren in nepal had not yet been reported in the literature. in our study 14.42% had a normal occlusion, 48.50% had class i, 29.35 had class ii div. the frequency of angle 's class i malocclusion in this study was similar to that reported by proffit. in united states but less than chinese and turkish populations. the frequency of angle 's class ii malocclusion was, however, higher than that being reported in united states, turkish, and chinese populations. in case of class iii malocclusion, the frequency was higher than that in united states population but less than that of chinese and turkish populations. the difference in the frequency of the various angle 's classes in all these studies can mainly be explained by differences in sample size and ethnicity. in this study crowding was the most common individual malocclusion trait as in accordance with studies in maltese and brazilian populations. the high prevalence of crowding can partially be explained by the great incidence of carious lesions and extractions of deciduous molars which favours migration of the first permanent molars as well as inclinations and rotations. however, scissor bite was the least common malocclusion in this study as in maltese population while in brazilian population ankylosed deciduous teeth were the least common trait. table 5 compares the finding of dental health component of iotn in various studies in different populations. the present investigation showed a greater frequency of grades 4 and 5 which is severe and extreme treatment needs. this is due to a greater frequency of impacted, submerged deciduous teeth and hypodontia ; further differences in sample size, study design, and ethnicities of the sample may account for differences in result. one can note that the same type of malocclusion falls into different levels of orthodontic treatment need according to its severity. therefore, the degree and priority of orthodontic treatment need among populations, which are important factors in public health planning, can not be fully known by just evaluating the malocclusion prevalence. if no specific index is used, determination of who really needs treatment becomes difficult and arbitrary, particularly among dentists and pediatric dentists, who end up inappropriately referring their patients to orthodontic treatment. in the present study, however, the normative evaluation based on the index of orthodontic treatment need may not be enough because of the often inherent elective nature of this treatment. as a result, other factors such as perceptual, functional, and social needs may interfere with treatment demand and service planning since those factors therefore, further studies investigating the patient 's perception and his or her concern regarding orthodontic treatment should be carried out in order to enhance the iotn efficacy [40, 41 ]. angle 's class i malocclusion is the most common while angle 's class iii is the least prevalent malocclusion in eastern nepalese schoolchildren. the pattern of malocclusion in this population sample is in general similar to that published in the international literature. it differs in the fact that this population have a high number of grade 1 but a lower number of grade 2 treatment needs of dhc of iotn. further this population exhibits a higher number of grade 5 treatment needs emphasizing an extreme treatment need.	objective. to evaluate the prevalence of malocclusion and orthodontic treatment needs among 12- to 15-year - old schoolchildren in eastern nepal and compare the findings with those of other populations. methods. two thousand seventy - four children (1149 males and 925 females) aged between 12 and 15 years were evaluated. their orthodontic treatment need was assessed using the index of orthodontic treatment needs (iotn) (dental health component (dhc)). angle 's classes of malocclusion were also evaluated. results. the prevalence of classes i, ii, and iii was 48.50%, 32.68%, and 4.32%, respectively. the iotn showed that 21.59% had an extreme treatment need, 24.67% had severe treatment need, 24.07% had moderate treatment need, 14.7% had mild treatment need, and 15.02% had no treatment need. conclusion. class i malocclusion is the most common, while class iii is the least prevalent in eastern nepal. the majority of the children need orthodontic treatment.
we analyzed the sample of older adults (65 years of age or older, n = 3,810) participating in nhanes 19992004, a continuing statistical survey of the noninstitutionalized civilian u.s. all study participants have blood pressure measurements taken, whereas those in the nhanes morning subset (n = 1,556) also have measurements taken of fasting plasma glucose and lipids. in accordance with american diabetes association criteria for epidemiological studies (20), study participants were considered to have diabetes if they met one or more of the following conditions : their fasting plasma glucose level was 126 mg / dl, or they reported being told by a physician that they had diabetes, or they were taking glucose - lowering medications. a person was considered aware if he / she responded positively to the question, have you ever been told by a doctor that you have diabetes or sugar diabetes ? study participants who reported taking insulin or oral hypoglycemic agents were classified as treated. a treated person was considered controlled if hba1c was less than 7%. hypertension was defined in accordance with the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure (jnc 7) (7). a person was deemed to have hypertension if the nhanes examination indicated that, based on the average of three measurements, the systolic blood pressure (sbp) was 140 mm hg, or the diastolic blood pressure (dbp) was 90 mm hg, or if he / she reported current use of antihypertensive medication. consequently, a person who may have been informed that he / she had hypertension, but who was not on medication and who tested normotensive on the day of the examination was not classified as a prevalent case of hypertension. a person with hypertension according to the jnc 7 definition was considered aware if he / she gave a positive response to the question, have you ever been told by a doctor or other health professional that you had hypertension, also called high blood pressure ? a person with hypertension was classified as treated if he / she reported taking antihypertensive medication at the time of the survey. a treated person was considered controlled if his / her average sbp was < 140 mm hg and average dbp was < 90 mm hg. study participants were classified as having dyslipidemia if their low - density lipoprotein (ldl) cholesterol exceeded the appropriate risk - based threshold established by the national cholesterol education panel (ncep) adult treatment panel iii (atp iii) (4) or if they reported taking an antihyperlipidemic drug at the time of the examination. similar to hypertension, this definition excludes persons who report having previously been told that they had high cholesterol but who test negative in nhanes and are not on medication. a person with dyslipidemia according to the atp iii definition was considered aware if he / she gave a positive response to the question, have you ever been told by a doctor or other health professional that your blood cholesterol level was high ? those who reported taking prescribed medicine to lower their blood cholesterol were considered treated. controlled if his / her ldl was lower than the appropriate ncep - atp iii goal. we applied survey weights to the nhanes 19992004 data to derive population estimates of the prevalence of diabetes, hypertension, and dyslipidemia, as well as awareness, treatment, and control rates of these conditions among persons aged 65 or older. calculations were performed using sas version 9.1 (sas institute inc., cary, nc, 2004), and standard errors and p values computed using the procedure surveyfreq, which takes into account the effect on estimator variance attributable to the complex nhanes multistage stratified cluster sample design. to determine independent risk factors, multivariate logistic regression models were estimated incorporating covariates for sex, age, usual place of care, doctor visits in past year, household income, living with spouse / partner, and education level. we analyzed the sample of older adults (65 years of age or older, n = 3,810) participating in nhanes 19992004, a continuing statistical survey of the noninstitutionalized civilian u.s. all study participants have blood pressure measurements taken, whereas those in the nhanes morning subset (n = 1,556) also have measurements taken of fasting plasma glucose and lipids. in accordance with american diabetes association criteria for epidemiological studies (20), study participants were considered to have diabetes if they met one or more of the following conditions : their fasting plasma glucose level was 126 mg / dl, or they reported being told by a physician that they had diabetes, or they were taking glucose - lowering medications. a person was considered if he / she responded positively to the question, have you ever been told by a doctor that you have diabetes or sugar diabetes ? study participants who reported taking insulin or oral hypoglycemic agents were classified as treated. a treated person was considered controlled if hba1c was less than 7%. hypertension was defined in accordance with the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure (jnc 7) (7). a person was deemed to have hypertension if the nhanes examination indicated that, based on the average of three measurements, the systolic blood pressure (sbp) was 140 mm hg, or the diastolic blood pressure (dbp) was 90 mm hg, or if he / she reported current use of antihypertensive medication. consequently, a person who may have been informed that he / she had hypertension, but who was not on medication and who tested normotensive on the day of the examination was not classified as a prevalent case of hypertension. if he / she gave a positive response to the question, have you ever been told by a doctor or other health professional that you had hypertension, also called high blood pressure ? a person with hypertension was classified as treated if he / she reported taking antihypertensive medication at the time of the survey. a treated person was considered controlled if his / her average sbp was < 140 mm hg and average dbp was < 90 mm hg. study participants were classified as having dyslipidemia if their low - density lipoprotein (ldl) cholesterol exceeded the appropriate risk - based threshold established by the national cholesterol education panel (ncep) adult treatment panel iii (atp iii) (4) or if they reported taking an antihyperlipidemic drug at the time of the examination. similar to hypertension, this definition excludes persons who report having previously been told that they had high cholesterol but who test negative in nhanes and are not on medication if he / she gave a positive response to the question, have you ever been told by a doctor or other health professional that your blood cholesterol level was high ? those who reported taking prescribed medicine to lower their blood cholesterol were considered treated. controlled if his / her ldl was lower than the appropriate ncep - atp iii goal. we applied survey weights to the nhanes 19992004 data to derive population estimates of the prevalence of diabetes, hypertension, and dyslipidemia, as well as awareness, treatment, and control rates of these conditions among persons aged 65 or older. calculations were performed using sas version 9.1 (sas institute inc., cary, nc, 2004), and standard errors and p values computed using the procedure surveyfreq, which takes into account the effect on estimator variance attributable to the complex nhanes multistage stratified cluster sample design. to determine independent risk factors, multivariate logistic regression models were estimated incorporating covariates for sex, age, usual place of care, doctor visits in past year, household income, living with spouse / partner, and education level. table 1 shows the characteristics of adults aged 65 and older in the united states. based on nhanes 19992004, 55.2% are 6574 years old, more than half of all elders are married or living with a partner, and virtually all older adults are insured. adults 65 years of age and older, national health and nutrition examination survey 19992004 the overall prevalence of hypertension (table 2) among older adults is 70.8%, with prevalence increasing as age increases. women have a significantly higher prevalence of hypertension than men (76.6% vs 63.0%) and a significantly lower rate of control on treatment (42.9% vs 57.9%). at 54.0%, blood pressure control is higher among the youngest old (age 6574) compared with 41.7% and 44.6% in the two older age groups (ages 7584, 85), respectively. overall, 42% of older adults have uncontrolled isolated systolic hypertension (sbp 140 mm hg and dbp < 90 mm hg [data not shown ]). women have significantly higher levels than men of isolated sbp 140 mm hg and < 160 mm hg (28.6% vs 22.1%, p <.0002) and of isolated sbp of 160 mm hg or greater (21.0% vs 10.1%, p <.0001). isolated systolic hypertension accounts for 88.4% of undiagnosed hypertension among older adults. hypertension, dyslipidemia, and diabetes prevalence, awareness, treatment, and control among u.s. adults 65 years of age and older, national health and nutrition examination survey 19992004 notes : p.05 difference between male and female, or between the age group and age 6574. p.01 difference between male and female, or between the age group and age 6574. p.001 difference between male and female, or between the age group and age 6574. men are significantly less likely to be aware of their condition than women (59.1% vs 71.1%), although both genders are equally likely to be treated (40.9% vs 45.1%). the oldest old have a significantly lower prevalence, are less aware, and have a lower treatment rate for dyslipidemia than the youngest old. two thirds of all older adults medically treated for dyslipidemia reach their ldl cholesterol goal. diabetes (table 2) affects 21.2% of adults 65 years of age and older. the awareness rate for diabetes (71.4%) is driven by a significantly higher awareness among women than among men (78.5% vs 63.6%). half of all prevalent diabetes cases are treated pharmacologically, and only half of all treated cases reach hba1c goal attainment of < 7%. tables 3, 4, and 5 present the results of estimating separate logistic regression models for the rates of awareness, pharmacologic treatment, and control of the three conditions, adjusting for the effects of age, sex, education, number of doctor visits, living arrangement, having a usual health care provider and annual household income. logistic regression models of predictors of awareness, treatment, and control of hypertension among u.s. adults 65 years of age and older, national health and nutrition examination survey 19992004 notes : or = odds ratio ; ci = confidence interval. logistic regression models of predictors of awareness, treatment, and control of dyslipidemia among u.s. adults aged 65 and older, national health and nutrition examination survey 19992004 notes : or = odds ratio ; ci = confidence interval. logistic regression models of predictors of awareness, treatment, and control of diabetes among u.s. adults aged 65 and older, national health and nutrition examination survey 19992004 notes : or = odds ratio ; ci = confidence interval. for elders with hypertension, higher age is associated with lower rates of awareness (odds ratio [or ] = 0.71 [age 7584 ] and 0.54 [age 85 ] vs age 6574), treatment (or = 0.70 [age 7584 ] and 0.60 [age 85 ] vs age 6574), and control (or = 0.59 [age 7584 ] vs age 6574). among those with dyslipidemia, the oldest (age 85) have significantly lower rates of awareness and treatment (or = 0.29 and 0.38 vs age 6574, respectively). although there are no significant differences between the sexes in awareness or treatment for hypertension, women on treatment are only half as likely as men to have their blood pressure controlled (or = 0.51). women are more likely than men to be aware and treated for dyslipidemia (or = 2.60 and 1.56, respectively) and more likely to be aware of their diabetes (or = 3.14). there are no significant associations between education (high school or more) and awareness, treatment, or control of hypertension and diabetes, however, education is positively associated with awareness (or = 1.39) and treatment (or = 1.62) of dyslipidemia. the number of doctor visits in the past year is positively and strongly associated with awareness and treatment of all three conditions. living arrangement (married / living with partner) is not associated with awareness, treatment, or control of any of the conditions. elders who have a usual health care provider are far more likely than those who do not have a usual provider to be aware (or = 2.87) and treated (or = 5.10) for their hypertension. elders with low annual household income (less than $ 15,000 in 2002 dollars) are much less likely than those with income more than $ 45,000 to be aware or controlled on treatment for their dyslipidemia (or = 0.36 and 0.39, respectively). those in the middle - income range of $ 15,000$45,000 are less likely than persons with household income more than $ 45,000 to be aware of their diabetes (or = 0.36). tables 3, 4, and 5 present the results of estimating separate logistic regression models for the rates of awareness, pharmacologic treatment, and control of the three conditions, adjusting for the effects of age, sex, education, number of doctor visits, living arrangement, having a usual health care provider and annual household income. logistic regression models of predictors of awareness, treatment, and control of hypertension among u.s. adults 65 years of age and older, national health and nutrition examination survey 19992004 notes : or = odds ratio ; ci = confidence interval. logistic regression models of predictors of awareness, treatment, and control of dyslipidemia among u.s. adults aged 65 and older, national health and nutrition examination survey 19992004 notes : or = odds ratio ; ci = confidence interval. logistic regression models of predictors of awareness, treatment, and control of diabetes among u.s. adults aged 65 and older, national health and nutrition examination survey 19992004 notes : or = odds ratio ; ci = confidence interval. for elders with hypertension, higher age is associated with lower rates of awareness (odds ratio [or ] = 0.71 [age 7584 ] and 0.54 [age 85 ] vs age 6574), treatment (or = 0.70 [age 7584 ] and 0.60 [age 85 ] vs age 6574), and control (or = 0.59 [age 7584 ] vs age 6574). among those with dyslipidemia, the oldest (age 85) have significantly lower rates of awareness and treatment (or = 0.29 and 0.38 vs age 6574, respectively). although there are no significant differences between the sexes in awareness or treatment for hypertension, women on treatment are only half as likely as men to have their blood pressure controlled (or = 0.51). women are more likely than men to be aware and treated for dyslipidemia (or = 2.60 and 1.56, respectively) and more likely to be aware of their diabetes (or = 3.14). there are no significant associations between education (high school or more) and awareness, treatment, or control of hypertension and diabetes, however, education is positively associated with awareness (or = 1.39) and treatment (or = 1.62) of dyslipidemia. the number of doctor visits in the past year is positively and strongly associated with awareness and treatment of all three conditions. living arrangement (married / living with partner) is not associated with awareness, treatment, or control of any of the conditions. elders who have a usual health care provider are far more likely than those who do not have a usual provider to be aware (or = 2.87) and treated (or = 5.10) for their hypertension. elders with low annual household income (less than $ 15,000 in 2002 dollars) are much less likely than those with income more than $ 45,000 to be aware or controlled on treatment for their dyslipidemia (or = 0.36 and 0.39, respectively). those in the middle - income range of $ 15,000$45,000 are less likely than persons with household income more than $ 45,000 to be aware of their diabetes (or = 0.36). using the combined nhanes 19992004 data, these analyses provide up - to - date estimates of prevalence, awareness, treatment, and control of hypertension, dyslipidemia, and diabetes among adults aged 65 and older in the united states. our results are consistent with previous studies of prevalence of these three cardiovascular risk factors in older adults (810,16,17) ; expand the body of knowledge regarding awareness, treatment, and control rates ; and identify gender disparities among the rates. we found that older women are more likely than older men to have hypertension, equally likely to be aware and treated, but less likely to have their blood pressure controlled on pharmacotherapy. this finding is consistent with recent studies showing a positive trend of increasing blood pressure control among older adults (age 60) in the nhanes study population (18,21). the improvement in control may be due, in part, to physician education and changing practice patterns based on the jnc 7 guidelines (7). notwithstanding this success, in the present study, women are only half as likely as men (after adjusting for covariates) to attain blood pressure goal. a similar finding for women (age 20) was reported in a previous nhanes 19992002 study of racial disparities in hypertension, after adjusting for race (22), suggesting that additional blood pressure control programs specifically targeted toward women are warranted. the paradigm shift of the jnc emphasizing the importance of diagnosing and treating systolic hypertension among older adults (23,24) further merits the need for such gender - specific programs. we found the prevalence of dyslipidemia to be similar by gender, with women more likely to be aware and treated. although overall treatment is only 43%, there has been a suggestive upward trend of pharmacological treatment for dyslipidemia among older adults (age 60), based on estimates from the nhanes data (16). this trend may be attributed to some degree, to increasing adoption of the ncep guidelines (4). control rates for dyslipidemia in this study are similar by gender and by age groups. our study found that women with diabetes are more likely than men to be aware of their condition and that only 50% of patients on pharmacotherapy are controlled (hba1c < 7%). other studies in various clinical settings have documented poor diabetes control among the elderly adults (25,26). this study provides newer evidence that confirms the need for improved quality of care. in our study, continued physician efforts to improve treatment rates and successfully treat patients to goal are needed. although our study shows that increasing numbers of doctor visits are associated with awareness and treatment for all three conditions, and with goal attainment for dyslipidemia, the interpretation of this finding is ambiguous. it may be that more frequent visits provide added opportunity for physicians to detect and treat undiagnosed disease, or the causality may be reversed, and those who already are aware and/or treated make more visits for the purpose of refilling prescriptions or monitoring treatment effectiveness. an important national public health effort to reduce cvd risk factor burden through the healthy people 2010 has had ongoing successes (16,27), and knowledge of our study 's findings on gender differences in awareness and control may be useful to clinicians in the effort to improve outcomes. the strengths of this study include its population - based sampling frame and the availability of clinical measurements to determine undiagnosed and diagnosed disease prevalence and to determine goal attainment. additionally, using 6 years of nhanes data provides a large sample and precise estimates of conditions affecting older adults. to our knowledge, this is the first study to use nhanes 19992004 to report on the prevalence and management of cvd risk factors among adults 65 years and older a limitation in our study is that physician - specific variables, such as practitioner age, group practice size and setting (eg, university medical center), and practice patterns are not available in nhanes. we used a blood pressure goal of < 140/90 mm hg for adults with hypertension with and without diabetes, a method that has been applied in other studies (8,9). the current blood pressure goal for adults with hypertension and with diabetes is < 130/80 mm hg (7). applying this threshold to adults with hypertension and with diabetes would result in a lower estimate of blood pressure control (7,9). women with hypertension are disproportionately affected by higher prevalence and lower control rates than men. women with dyslipidemia or diabetes are more likely than men to be aware of their condition. for all three conditions, improvements in treatment and control rates are needed. in light of the expected growth in this population,	backgroundadults aged 65 and older are disproportionately affected by hypertension, dyslipidemia, and diabetes, which are established risk factors for cardiovascular disease (cvd). although risk reduction strategies among older adults, including control of cvd risk factors, can lead to a decline in premature cvd morbidity and mortality, the prevalence of these risk factors has generally increased in the past decade among elders and risk factor control rates have been suboptimal. we assess prevalence, awareness, treatment, and control rates among u.s. adults aged 65 and older with respect to hypertension, dyslipidemia, and diabetes and describe predictors associated with awareness and management of these factors.methodsanalysis of nationally representative data collected from adults aged 65 and older (n = 3,810) participating in the national health and nutrition examination survey 19992004.resultswomen have a significantly higher prevalence of hypertension than men (76.6% vs 63.0%) and a significantly lower rate of control when treated pharmacologically (42.9% vs 57.9%). dyslipidemia prevalence is 60.3% overall, and women are significantly more likely to be aware of their condition than men (71.1% vs 59.1%). diabetes affects 21.2% of older adults, and 50.9% of prevalent cases are treated pharmacologically. goal attainment among those treated is problematic for all three conditions hypertension (48.8%), dyslipidemia (64.9%), and diabetes (50.4%). having two or more doctor visits annually is associated with goal attainment for dyslipidemia.conclusionsknowledge of cardiovascular health in older adults and understanding gender gaps in awareness can help physicians and policymakers improve disease management and patient education programs.
acinetobacter baumannii was recently recognized as a successful nosocomial pathogen, with an increasing morbidity and mortality due the rise in multi- and pan - drug - resistant strains (perez. its clinical importance led to extensive studies on different aspects of the biology and pathogenesis (mcconnell., the genomes of 16 a. baumannii strains have been sequenced and those of more than 200 other isolates are as draft stage in genbank. genome comparisons showed high variability, which could be the result of the combination of natural competency and the presence of active recombination system(s) (smith. these processes could also explain the unusual tendency of a. baumannii to acquire multiple antibiotic resistance determinants and to survive in the hospital environment (hornsey. a recent study showed that three strains belonging to the international clonal lineage 2 (icl2) have an elevated number of single nucleotide polymorphisms (snps) when compared with the icl2 prototype acicu strain. recombinant regions, which was interpreted as the product of homologous recombination - mediated dna swapping (snitkin. taking into account these results, a comparison of the genomes of a tigecycline resistant with a susceptible strain isolated from a patient before and after 1-week treatment showed significant differences in addition to a single nucleotide mutation in the ades gene that accounts for resistance to the antibiotic (hornsey. conversely, another comparative study between two a. baumannii strains, one of them an extreme - drug resistant and the other a pan - drug resistant, showed only 61 snps between them (tan. however, despite the relatively small number of snps observed between the two strains, the authors concluded that the changes are indicative of fast evolution because the mutations occurred within a 1-month period (tan. we recently initiated the characterization of the first laboratory confirmed naturally competent a. baumannii strain, ab a118, an isolate that unlike other clinical isolates is susceptible to numerous antibiotics, supports replication and stable maintenance of different plasmid replicons and took up fluorophore labeled oligonucleotides (ramirez. ab a118 was isolated in the year 1995 in buenos aires city, argentina, from a blood culture of an intensive care unit patient and did not belong to any of the predominant clonal complexes widespread in our region and in the world. interestingly, although the comm gene is interrupted by the insertion of the abar resistance island in numerous strains, it is intact in ab a118, a characteristic that could account for the susceptible phenotype and natural competency exhibited by this isolate (ramirez., a comparative study to gain insight into the nature and extent of the genomic differences found in ab a118 strain when its genome is compared with those of strains atcc 17978, aye, and acicu is described. the available scaffolds (accession number aeow01000000) were ordered and oriented with the mauve contig mover (darling. genomes were aligned with the open - source mauve aligner version 2.3.1 using the progressive algorithm. the alignments were generated using the default settings (http://gel.ahabs.wisc.edu/mauve/, last accessed august 29, 2014). coding sequence predictions and annotations were made by rast version 4.0 and glimmer2 software (delcher. snps were displayed using the circos software (http://circos.ca, last accessed august 29, 2014). 2011) has an average gc content of 38.4%, and 88 trna genes and 3,520 coding sequences were identified, of which 93.64% could be annotated with high confidence and also manually curated using fasta and blast results. figure 1, obtained using circos software, shows a diagram of the ab a118 genome with the annotated genes in color codes according to clusters of orthologous group (cog) category. ring 2 : open reading frame (orf) distribution, plus strand. ring 3 : orf distribution, negative strand. ring 5 : gc content, blue represents the above average content and red the bellow average content. ring 6 : gc skew, calculated in artemis. ring 5 : gc content, blue represents the above average content and red the bellow average content. ring 6 : gc skew, calculated in artemis. the phylogenetic tree diagram obtained using mauve and splitstree4 showed that atcc 17978 strain is the most closely related strain to ab a118 (supplementary fig. the ab a118 genome showed 98% average nucleotide identity with that of atcc 17978 (accession number cp000521), and 96% with those of aye (accession number nc_010410) and acicu (accession number nc_010611). these latter strains are representatives of the most widespread clonal complexes 1 and 2. gene order comparison using mauve alignment showed multiple changes and 19 regions with high homology named local colinear blocks (lcbs), which encompassed an average of 81.73% of each genome (fig. table 1 shows the numbers of inversions, insertions, and deletions identified when comparing the ab a118 genome with those of strains atcc 17978, aye, and acicu. the results show that the acicu strain has the small number of arrangements compared with ab a118. the nucleotide sequences of ab a118 genome (accession number aeow01000000), atcc 17978 (accession number cp000521), aye (accession number nc_010410), and acicu (accession number nc_010611) were compared using the mauve aligner version 2.3.1 (darling. areas that are completely white within an lcb are not aligned and probably contain sequence elements specific to a particular genome. table 1genome rearrangements and snps obtained from the comparison of ab 118 genome to those of strains atcc 17978, aye, and acicustraininversionsinsertionsdeletionssnps (%) atcc 1797812206626 + 43,784 (1.1496)aye627535044,130 (1.158)acicu520160143,914 (1.153) multiple alignment of ab a118, atcc 17978, aye, and acicu genomes. the nucleotide sequences of ab a118 genome (accession number aeow01000000), atcc 17978 (accession number cp000521), aye (accession number nc_010410), and acicu (accession number nc_010611) were compared using the mauve aligner version 2.3.1 (darling. areas that are completely white within an lcb are not aligned and probably contain sequence elements specific to a particular genome. genome rearrangements and snps obtained from the comparison of ab 118 genome to those of strains atcc 17978, aye, and acicu these genomic variations support the existing idea that a. baumannii has a distinct ability to exchange genetic material and rearrange its genome, a property that must impact its ability to adapt to hostile environments. the number of snps presented by the ab a118 genome in comparison with all three other strains is shown in table 1 and figure 3. the number of snps identified between ab a118 and atcc 17978, aye, and acicu strains was 43,784 (1.1496%), 44,130 (1.158%), and 43,914 (1.153%), respectively (fig. 3 and table 1). the snps were located in 2,571 genes (73%), 2,791 genes (79%), and 2,882 genes (82%) with respect to the genomes of atcc 17978, aye, and acicu, respectively. these genes are predicted to be involved in core biological functions, such as metabolism, replication, transcription, natural competence, antibiotic resistance, cytoskeleton, and others. 3.distribution of snps among ab 118, atcc 17978, aye, and acicu genomes. distribution of snps among ab 118, atcc 17978, aye, and acicu genomes. apart from the remarkable difference with other strains for having an intact comm gene, all other presumptive competent - related genes in strain ab a118 shared between 94% and 100% of amino acidic identity with those of all the complete annotated a. baumannii genomes. analysis of the presence of snps in 12 presumptive competent - related genes when comparing strains ab a118 and atcc 17978 showed 186 snps with an average of 16 per gene (table 2). however, the snps found in the genes annotated as pily1 and comec / rec2, which was recently reported as an important gene with a role in natural competence (wilharm. the high variability among these genes and the presence of an intact comm gene may play a role in the natural competency. functional and comparative studies are needed to determine the role of these genes in the mechanisms and efficiency of natural transformation in these strains. table 2snps and amino acid variation found presumptive competence genesfeature namecoordinate in ab a118snpsnonsynonymous substitution / amino acidpilin - like competence31408073141067160/0pilin - like competence tfp pilus assembly protein pile3141224314165244/4possible pilus assembly protein tip - associated adhesin pily1(comc)314171931455644830/30possible pilus assembly protein pilw3146528314734321/1pild - dependent protein pddc26355602635940120/0pild - dependent protein pddd2636016263661290/0putative competence protein (coml)1992614199340260/0competence factor involved in dna uptake comec / rec2104101110434434725/25putative membrane protein comm3618207361921780/0putative membrane protein comn3617566361820742/2putative membrane protein (como)3616829361756981/1putative outer membrane protein (comq)36140763616241221/1feature name found in the acinetobacter baumannii atcc 17978 strain. snps and amino acid variation found presumptive competence genes feature name found in the acinetobacter baumannii atcc 17978 strain. acinetobacter baumannii presents some remarkable survival features as its capacity to develop resistance to all antibiotics currently in use as well as its ability to withstand desiccation and resist most disinfectants. these traits, together with other characteristics such as the ability to form biofilms, provide a. baumannii strains the genetic armory to survive for long periods of time in the hospital environment. its intrinsic genetic plasticity must be a key factor in the rapid adaptation necessary to survive the hostile environments in hospitals (smith. the ab a118 genome includes a variety of genomic arrays with respect to others and includes a high number of snps consistent with high genomic variability. in particular, considering the previous finding that the comm gene is uninterrupted by an abar insertion (ramirez. 2011), there is also a high variability in at least two other genes potentially involved in natural competency. this opens questions about the potential of this strain to behave differently than others with respect to acquisition of dna from the environment. comparative studies of the competency of several strains as well as mutagenesis analysis will contribute to the clarification of the mechanisms, efficiency, and roles of natural competency in a. baumannii. the phylogenetic tree diagram obtained using mauve and splitstree4 showed that atcc 17978 strain is the most closely related strain to ab a118 (supplementary fig. the ab a118 genome showed 98% average nucleotide identity with that of atcc 17978 (accession number cp000521), and 96% with those of aye (accession number nc_010410) and acicu (accession number nc_010611). gene order comparison using mauve alignment showed multiple changes and 19 regions with high homology named local colinear blocks (lcbs), which encompassed an average of 81.73% of each genome (fig. 2). table 1 shows the numbers of inversions, insertions, and deletions identified when comparing the ab a118 genome with those of strains atcc 17978, aye, and acicu. the results show that the acicu strain has the small number of arrangements compared with ab a118. the nucleotide sequences of ab a118 genome (accession number aeow01000000), atcc 17978 (accession number cp000521), aye (accession number nc_010410), and acicu (accession number nc_010611) were compared using the mauve aligner version 2.3.1 (darling. areas that are completely white within an lcb are not aligned and probably contain sequence elements specific to a particular genome. table 1genome rearrangements and snps obtained from the comparison of ab 118 genome to those of strains atcc 17978, aye, and acicustraininversionsinsertionsdeletionssnps (%) atcc 1797812206626 + 43,784 (1.1496)aye627535044,130 (1.158)acicu520160143,914 (1.153) multiple alignment of ab a118, atcc 17978, aye, and acicu genomes. the nucleotide sequences of ab a118 genome (accession number aeow01000000), atcc 17978 (accession number cp000521), aye (accession number nc_010410), and acicu (accession number nc_010611) were compared using the mauve aligner version 2.3.1 (darling. areas that are completely white within an lcb are not aligned and probably contain sequence elements specific to a particular genome. genome rearrangements and snps obtained from the comparison of ab 118 genome to those of strains atcc 17978, aye, and acicu these genomic variations support the existing idea that a. baumannii has a distinct ability to exchange genetic material and rearrange its genome, a property that must impact its ability to adapt to hostile environments. the number of snps presented by the ab a118 genome in comparison with all three other strains is shown in table 1 and figure 3. the number of snps identified between ab a118 and atcc 17978, aye, and acicu strains was 43,784 (1.1496%), 44,130 (1.158%), and 43,914 (1.153%), respectively (fig. 3 and table 1). the snps were located in 2,571 genes (73%), 2,791 genes (79%), and 2,882 genes (82%) with respect to the genomes of atcc 17978, aye, and acicu, respectively. these genes are predicted to be involved in core biological functions, such as metabolism, replication, transcription, natural competence, antibiotic resistance, cytoskeleton, and others. 3.distribution of snps among ab 118, atcc 17978, aye, and acicu genomes. distribution of snps among ab 118, atcc 17978, aye, and acicu genomes. apart from the remarkable difference with other strains for having an intact comm gene, all other presumptive competent - related genes in strain ab a118 shared between 94% and 100% of amino acidic identity with those of all the complete annotated a. baumannii genomes. analysis of the presence of snps in 12 presumptive competent - related genes when comparing strains ab a118 and atcc 17978 showed 186 snps with an average of 16 per gene (table 2). however, the snps found in the genes annotated as pily1 and comec / rec2, which was recently reported as an important gene with a role in natural competence (wilharm. the high variability among these genes and the presence of an intact comm gene may play a role in the natural competency. functional and comparative studies are needed to determine the role of these genes in the mechanisms and efficiency of natural transformation in these strains. table 2snps and amino acid variation found presumptive competence genesfeature namecoordinate in ab a118snpsnonsynonymous substitution / amino acidpilin - like competence31408073141067160/0pilin - like competence tfp pilus assembly protein pile3141224314165244/4possible pilus assembly protein tip - associated adhesin pily1(comc)314171931455644830/30possible pilus assembly protein pilw3146528314734321/1pild - dependent protein pddc26355602635940120/0pild - dependent protein pddd2636016263661290/0putative competence protein (coml)1992614199340260/0competence factor involved in dna uptake comec / rec2104101110434434725/25putative membrane protein comm3618207361921780/0putative membrane protein comn3617566361820742/2putative membrane protein (como)3616829361756981/1putative outer membrane protein (comq)36140763616241221/1feature name found in the acinetobacter baumannii atcc 17978 strain. snps and amino acid variation found presumptive competence genes feature name found in the acinetobacter baumannii atcc 17978 strain. acinetobacter baumannii presents some remarkable survival features as its capacity to develop resistance to all antibiotics currently in use as well as its ability to withstand desiccation and resist most disinfectants. these traits, together with other characteristics such as the ability to form biofilms, provide a. baumannii strains the genetic armory to survive for long periods of time in the hospital environment. its intrinsic genetic plasticity must be a key factor in the rapid adaptation necessary to survive the hostile environments in hospitals (smith. 2007 ; iacono. 2008 ; peleg. 2008 ; adams. 2009 the ab a118 genome includes a variety of genomic arrays with respect to others and includes a high number of snps consistent with high genomic variability. in particular, considering the previous finding that the comm gene is uninterrupted by an abar insertion (ramirez. 2011), there is also a high variability in at least two other genes potentially involved in natural competency. this opens questions about the potential of this strain to behave differently than others with respect to acquisition of dna from the environment. comparative studies of the competency of several strains as well as mutagenesis analysis will contribute to the clarification of the mechanisms, efficiency, and roles of natural competency in a. baumannii.	recent studies have demonstrated a high genomic plasticity in acinetobacter baumannii, which may explain its high capacity to acquire multiple antibiotic resistance determinants and to survive in the hospital environment. acinetobacter baumannii strain a118 (ab a118) was isolated in the year 1995 from a blood culture of an intensive care unit patient. as this particular strain showed some peculiar characteristic such as being naturally competent and susceptible to numerous antibiotics, we performed whole - genome comparison (wgc) studies to gain insights into the nature and extent of the genomic differences. the ab a118 genome is approximately 3,824 kb long with a 38.4% gc content and contains 3,520 coding sequences. wgc studies showed that the ab a118 genome has 98% average nucleotide identity with that of a. baumannii atcc 17978, and 96% average nucleotide identity with that of strains aye and acicu. at least 12 inversions, 275 insertions, and 626 deletions were identified when the ab a118 genome was compared with those of strains atcc 17978, aye, and acicu using mauve wgc. multiple gene order arrangements were observed among the analyzed strains. mauve wgc analysis identified 19 conserved segments, known as locally colinear blocks. the number of single nucleotide polymorphisms found when comparing the ab a118 genome with that of strains atcc 17978, aye, and acicu was 43,784 (1.1496%), 44,130 (1.158%), and 43,914 (1.153%), respectively. genes comea, pilq, pild, pilf, coml, pila, comec, pili, pilh, pilo, piln, pily1(comc), pile, pilr, and comm, potentially involved in natural competence were found in the ab a118 genome. in particular, unlike in most strains where comm is interrupted by an insertion of a resistance island (abar), in strain ab a118 it is uninterrupted.
among smokers who are aware of the ill effects of smoking, 3 out of 4 individuals are interested in quitting. one of the most important responsibilities of a countries health care system is to treat nicotine dependence. this effort consists of different methods such as simple clinical counseling, medications and quit lines. the cost of these methods differs, however and these methods are not equally effective. it also is important to match the treatment method to the local and cultural context as well as to clients current needs. repeated consultation is important in reinforcing the necessity of quitting smoking at every clinical visit. this intervention is relatively cost - effective, because it is part of the existing health care services which are used by the majority of smokers. these interventions are effective because they are offered by health care providers, who are respected by the public and smokers also have a good relationship with them. moreover, it is important to provide the quit lines services free- of- charge from any telephone. even old telephone line systems that responded only to incoming calls showed very significant results. in addition to clinical counseling and quit lines for consultation, an effective treatment can include medication. medication includes nicotine replacement therapy (nrt) (patches, gum, sublingual tablets, lozenges, inhalers and nasal spray) and prescribed drugs such as bupropion and varenicline. in general, medication is more expensive than medical counseling and quit lines. now after 9 years from the first educational interventions for quitting smoking and one of two complementary integrated programs in the primary health care network, the situation for counseling, human resources and medication is not suitable at the country level and there is insufficient documentation and studies in this regard. thus, it was considered beneficial to identify the best current evidence and incorporate the latest outcomes. this would allow for development of an appropriate framework necessary to assess different methods in order to select the best approaches for essential interventions at the country level. this study specifically targeted decision makers in iran to enable a quantitative systematic review based on the two following two main questions : which smoking cessation methods are most published?which one is the most effective method ? based on a cross - sectional study, english publications from 2000 to 2012 in pubmed were selected. to find articles related to quitting smoking, we considered one of the following keywords from a title : quit or cessation, nrt, champix or varenicline or chantix, zyban or bupropion sustained - release, combination therapy, training, self - learning material, behavior therapy, quit and win, hypnosis, telephone consulting, acupuncture, interactive voice response (ivr), e - cigarettes, pharmacological treatments, non - pharmacological treatments, personal methods, non - nicotine medications. in an expert panel meeting it was proposed to include relevant articles (at a minimum, these must include the abstracts). unpublished and duplicated articles and opinion based papers were excluded as they were not classified as relevant. articles were reviewed separately by two qualified assistants, who were trained and calibrated to achieve desirable agreement in the selecting of articles. in order to reach check reliability, a pilot inter - rater reliability assessment was conducted by the author (gh). the inter - rater reliability was 67% before discussion ; any discrepancies in answers were discussed and a consensus was achieved on review. after the training and pilot assessment, the author (gh) determined that the assistants were ready to do the search. the reports of the assistants were observed again by the author (gh), who determined when the assistants were ready to work alone. finally, the assistants selected and categorized the articles according to the following criteria for each method : total number of articles, the number of articles that supported quit smoking efforts, the number of articles that did not support quit smoking efforts and the number of articles without any firm conclusions. again in order to assess the reliability of each assistant, one method was selected randomly and the recorded results were compared by the author (gh). if there were discrepancies in the figures, these were corrected by author (gh). different methods were used for rating articles and the strengths and weakness of each were discussed through an expert panel meeting. finally, the best scoring model was accepted for each stop - smoking method, which included a maximum of 40 scores of which 20 scores were for articles that supported methods (positive articles minus negative articles) and 20 points for the highest proportion of supported to the total number of articles. for scoring of each article, one point was possible, since we did not consider differences between articles which were published in pubmed. however, a point was not given for articles that did not have a clear result. because newer treatment methods had less chance for having more published articles ; an index of positive articles to total articles this search resulted in a folder with 3081 titles of articles ; of which 1436 were inaccessible (at a minimum an abstract). out of 1645 remaining articles, 713 were not about treatment and its effect on quitting smoking (they were about history, smoking cessation programs in countries, relevant policies, rules and regulations for stopping smoking, article 14 of world health organization [who ] framework convention on tobacco control, etc.) of the 932 remaining articles, 62 articles did not support quit smoking methods (with no good results or side - effects). of 870 remaining manuscript, 90 cases did not demonstrate a clear result (even whether the method is usable) [figure 1 ]. study selection flow diagram nrt, champix and zyban respectively with 352, 117 and 71 cases were the most advised method and e - cigarette and non - nicotine medication with one case were the least advised methods. for each method the number of advising articles to the total was 31 out of 32 and for nrt was 376 of 407 (which was considered the maximum score). willpower with two out of 9 was the lowest score and finally nrt with a score of 39 and champix and training with a score of 36 were the best methods. the number of selected articles and the results are shown in tables 1 and 2. prevalence of smoking cessation methods in included articles (n=932) and the results obtained by number of articles and based on classification of the studies prevalence of quit smoking methods in the included articles (n=932 and their scores) unfortunately, after a decade of offering quit smoking services, the situation is not so positive in iran and is still based on older methods such as 15 mg nicotine patches and group therapy. these services have not been updated on the basis of more recent evidence. for determining which quit smoking methods are maximally effective, a comprehensive review of all recent articles was a necessity in order to obtain suitable qualitative indicators for selecting effective quit smoking methods. in this study, it was found that three methods nrt, champix and education for quitting smoking are advised most frequently based on the articles referenced in pubmed between the years 2000 and 2012. these findings are informative in updating the health care services at the country level and a combination of these methods might be provided. many countries that offer cessation services follow better methods through evaluation and incorporating necessary modifications to these methods. as displayed in the results, nrt, champix, training, combination treatment, zyban and self - help materials received the highest scores with over 30 points ; multi - drug treatment, acupuncture, some methods without medication such as nurse counseling, non - nicotine drugs, will power and e - cigarettes had the lowest scores with less than 20 and the remaining methods including behavioral methods, quit and win, telephone counseling and ivr and hypnotism were in the middle. the results of this study are consistent with the findings of high impact articles in who, centers for disease control and prevention and cochrane, and these results also were consistent with the writers experience. however, it was necessary to prioritize quitting methods based on a new quantitative analysis and simple qualitative methods in order to gain the support of decision makers for further necessary interventions. that is because the systematic reviews, such as cochrane, do not provide guidance on which of the effective methods are best for a particular environment. in the systematic reviews articles finding the relevant articles was challenging in the current study because we selected a huge number of articles for a particular selected method (based on the accessibility of the full text or abstract). boyle. and linder. in their studies have reported that use of electronic medical registration and continuous use of nicotine patches can increase success of smoking cessation attempts. this study was the first quantitative study, which provided us with a wide perspective about all tobacco cessation methods. scores were based in part on number of publications, thereby creating a bias in favor of older methods with more opportunity for publication. some of the newer methods are quite recent and therefore not surprisingly have few published assessments. this study could be a useful indicator of effective quit smoking methods based on updated scientific documents. the results of this study could be used as a reference for decision makers who are charged with selecting cessation methods at the country level that will produce good outcomes. methods that obtained the higher scores have been applied most frequently in interventions that have had positive effects on quit rates. according to the updated scientific and updated evidence based on a simple and quantitative approach, quit smoking methods with high and better scores including nrt, champix and training have been recognized as effective and the other methods have received lower scores. further studies are needed to implement a comparative study on qualitative and quantitative smoking cessation methods that could lead to increased quit rates at the country level.	background : during recent years, there have been many advances in different types of pharmacological and non - pharmacological tobacco control treatments. in this study, we aimed to identify the most effective smoking cessation methods used in quit based upon a review of the literature.methods:we did a search of pubmed, limited to english publications from 2000 to 2012. two trained reviewers independently assessed titles, abstracts and full texts of articles after a pilot inter - rater reliability assessment which was conducted by the author (gh). the total number of papers and their conclusions including recommendation of that method (positive) or not supporting (negative) was computed for each method. the number of negative papers was subtracted from the number of positive ones for each method. in cases of inconsistency between the two reviewers, these were adjudicated by author.results:of the 932 articles that were critically assessed, 780 studies supported quit smoking methods. in 90 studies, the methods were not supported or rejected and in 62 cases the methods were not supported. nicotine replacement therapy (nrt), champix and zyban with 352, 117 and 71 studies respectively were the most supported methods and e - cigarettes and non - nicotine medications with one case were the least supported methods. finally, nrt with 39 and champix and education with 36 scores were the most supported methods.conclusions:results of this review indicate that the scientific papers in the most recent decade recommend the use of nrt and champix in combination with educational interventions. additional research is needed to compare qualitative and quantitative studies for smoking cessation.
the overall incidence of ovarian tumors in pregnancy is 2.4 - 5.7% (1). of these tumors, currently, an ultrasound is routinely used early in pregnancy, and this has led to an early diagnosis and management of asymptomatic ovarian tumors. in contrast to previous guidelines for managing gynecological malignancies in pregnancy, today, most women with ovarian cancer complicating pregnancy hope to maintain the pregnancy and preserve fertility, which has been encouraged by late maternity and a low birthrate. in the absence of large prospective randomized trials and cohort studies, it is difficult to know how to manage patients best and make standard guidelines for management of these tumors during pregnancy. to establish standard a guideline for management of ovarian cancer complicating pregnancy, it is necessary to identify surgical strategies with or without adjuvant intrapartum chemotherapy that results in safe oncologic and fetal outcomes. this study was undertaken to help establish a guideline for management of ovarian malignancy complicating pregnancy. we retrospectively analyzed the medical records of patients (n=27) with ovarian cancer complicating pregnancy who were treated at cheil general hospital and women 's healthcare center between january 1996 and november 2006. in these patients, twenty - five of these patients were diagnosed in our institute, and two were transferred from other hospitals. conservative surgical management was defined as cystectomy, or unilateral salpingo - oophorectomy (uso), or uso with omentectomy and multiple biopsies. specialized pathologists at our institution reported findings of intra - operative frozen biopsies and post - operatively confirmed pathology specimens for all 27 patients. the 1988 figo staging system was retrospectively used for each case after reviewing the operative reports and pathologic findings. follow - up information was recorded up to the date of last contact or death. mean age of the patients was 29.1 yr (range 23 - 40). of 27 patients with ovarian tumor complicating pregnancy, 25 were diagnosed during pregnancy and the others were incidentally diagnosed at cesarean section. mean time of diagnosis of ovarian tumor was 12.5 weeks of gestational age (ga) (range 5.0 - 41.0 weeks of ga). in the patients diagnosed during pregnancy, 6 had the presenting symptoms such as abnormally distended abdomen due to huge ovarian tumor. most patients without symptoms (n=21) were diagnosed incidentally at early ga by a routine ultrasound examination (table 1). histopathologic characteristics included ovarian borderline malignancy (n=15), epithelial ovarian cancers (eoc) (n=7) and malignant germ cell tumors (gct) (n=5). the mean size of the ovarian tumors measured by ultrasound sonogram was 11.4 cm (range, 4 - 22 cm) and three of 27 patients had an ovarian tumor less than 6 cm in diameter. the initial operation included a conservative operation (n=26) and a cytoreductive operation with hysterectomy (n=1). the conservative surgical management included cystectomy (n=10), uso (n=13) and uso with omentectomy and multiple biopsy (n=3). patients with ovarian borderline malignancies underwent cystectomy (n=7), uso (n=6) or uso with omentectomy and multiple biopsy (n=2). the patients with germ cell tumor underwent cystectomy (n=2), uso (n=2) or uso with omentectomy and multiple biopsy (n=1), with no recurrence in any patient with malignant germ cell tumor. in the patients with eoc, the surgical approaches were cystectomy (n=1), uso (n=5) or cytoreductive operation with hysterectomy (n=1). in this group, of the 22 patients who underwent surgical interventions during pregnancy, there were no definite feto - maternal complications after surgical management. of these 22 patients, nineteen (86.4%) received surgical intervention during second trimester and three (13.6%) during the first trimester. a total of 8 patients received adjuvant chemotherapy ; 3 patients received chemotherapy with fetus in utero, and 5 patients received chemotherapy just after delivery. one patient with eoc complicating ectopic pregnancy received postoperative chemotherapy. of five patients with germ cell tumor, 3 patients received it with the fetus in utero (range, 22.8 - 30.6 weeks of ga) and two patients at 2 weeks after cesarean delivery with 4 courses of bleomycin, etoposide and cisplatin (bep). three patients with eoc received 6 courses of paclitaxel plus carboplatin at 2 weeks after cesarean delivery. all 26 patients with ovarian cancer complicating pregnancy were successful in having a full term delivery. of the 26 healthy full - term infants, 15 were delivered by normal vaginal delivery and eleven were by cesarean section. an ultrasound sonogram is a routine method for evaluating fetal status in women with pregnancy and it can also be used for early detection of an incidental ovarian tumor. with the wide use of routine prenatal ultrasound, finding of an adnexal mass in pregnancy is increasing (4). in contrast to past delivery modes, cesarean delivery is now more frequent, and this can also contribute to the early incidental diagnosis of ovarian tumor complicating pregnancy. the incidence of ovarian tumor detected during pregnancy is 1/300 to 1/556 pregnancies (5 - 7). of the ovarian tumors detected during pregnancy, the incidence of ovarian malignancy is 1/15,000 to 1/32,000 in the most reports (4, 8). in our series, ovarian cancer during pregnancy occurred in 0.29/1,000 deliveries (from 1996 to 2006, from 92,370 deliveries at cheil general hospital and women 's healthcare center). this high incidence could be explained by recent wide use of ultrasound examination as a routine antenatal evaluation in our hospital. several studies have reported that the histologic types of ovarian cancers during pregnancy are similar to those for nonpregnant women in the corresponding reproductive - age group (4, 9 - 11). in our series, borderline malignancy and invasive epithelial ovarian cancer were more dominant than other types of malignancies (table 4). in white women, dysgerminoma is the most common ovarian germ cell tumor coexisting with pregnancy, and constitutes 25 - 35% of all reported ovarian cancers (9, 12, 13). however, the most common subtype of germ cell tumor in our study was immature teratoma. surgical management of an adnexal mass, diagnosed during pregnancy, creates a dilemma to gynecologists. if an adnexal mass, larger than 6 cm, has a complex structure, or ascites or persists for 16 gestational weeks, surgical management is critical for obtaining a final histologic diagnosis and ruling out malignancy (14). when any surgical intervention is needed for an adnexal mass during pregnancy, the surgery should be performed during the safest period of pregnancy. elective surgery for tumors with low suspicion of malignancy should be delayed until the second trimester (16 - 18 weeks of gestation), when risk for spontaneous abortion, hormonal dependence of the corpus luteum of pregnancy is reduced, and when functional cysts are resolved in the vast majority of cases (15). in the first trimester, spontaneous abortion rate after surgery has been documented to be 10%, while 76.3% patients progress to full - term delivery (16). a mass, first noticed in the third trimester, is best managed by awaiting fetal maturity as long as the clinical suspicion of malignancy is low. premature labor is more likely to occur and pregnancy outcome is poor if surgical exploration is attempted during the third trimester (17, 18). the principles of management in ovarian cancer complicating pregnancy include surgery with adequate staging. for advanced disease, the principles of adequate staging and debulking surgery should be similar to those used for the treatment of nonpregnant women. for most early - stage ovarian cancers, the surgical principles should be unilateral oophorectomy or adnexectomy with appropriate staging. especially, in borderline ovarian malignancies and germ cell tumors, it is imperative to use conservative surgical strategies to maintain pregnancy. in our series, of 20 patients with borderline malignancies (n=15) and germ cell tumors (n=5), 9 patients received cystectomy, 8 patients received uso and 3 patients received uso with omentectomy and multiple biopsy as the initial surgical management. and all 5 patients with germ cell tumor received bep (cisplatin, bleomycin, and etoposide) chemotherapy without any feto - maternal complications. most malignant ovarian germ cell tumors could be treated by conservative surgery without compromising survival since these tumors are very sensitive to standard combinational chemotherapy (19). and borderline malignancies differ from invasive epithelial ovarian cancer in their indolent behavior and good prognosis. all six cases in gotlieb 's report (20) with immediate conservative surgery preserving pregnancy had satisfactory outcomes. since there was no established benefit from postoperative therapy for ovarian borderline malignancy, even in late - stage diseases (21), conservative surgery is safe for these tumors (20, 22). conservative surgical management for most malignant ovarian germ cell tumors and borderline malignancies diagnosed during pregnancy should be considered as the proper initial treatment. the standard management of eoc is based on the primary surgery, including hysterectomy and bilateral salpingo - oophorectomy with peritoneal sampling (peritoneal washing, omentectomy, multiple peritoneal biopsies and the removal of peritoneal implants) with lymph - node biopsy. recently, with the help of several developed diagnostic tools, and an increase in self - health monitoring among women, early detection of ovarian cancer has increased and with the widespread use of routine prenatal ultrasound, the finding of an adnexal mass during pregnancy is an increasingly common occurrence. in contrast to past strategies for managing eoc, most young patients with malignancies desire to be managed focused on the quality of life after treatment, and to maintain the pregnancy if possible. however, frequent relapse of eoc makes conservative treatment strictly limited to patients with early stage eoc and low risks. in our study, 6 of 7 patients with eoc received comprehensive rather than complete staging operations (1 cystectomy and 5 uso). our study demonstrates that in eoc complicating pregnancy, conservative management with adequate staging procedures should be considered for oncologic safety. generally, chemotherapy is compatible with the second or third trimester, when the risk of congenital malformation for fetuses exposed to chemotherapy is no greater than in the general population (23, 24). in our study, three patients with germ cell tumors received bep chemotherapy during pregnancy, all of which had healthy babies without congenital malformations. the literature contains numerous reports of bep regimen and adjuvant cisplatin and cyclophosphamide used in pregnancy with no untoward effects (8, 25 - 28). there were a few case reports describing the combined use of paclitaxel and carboplatin in human pregnancy, and there seems to be no significant fetal toxicity when these are administered during the second or third trimester (29, 30). recently, no studies have evaluated the long - term consequences for children exposed to intrauterine chemotherapy. to establish standard guidelines for treatment and improve the quality of life in patients with pregnancy complicating ovarian cancer with an oncologic safety,	the aim of this study is to evaluate the clinical feature and pregnancy outcome in patients with ovarian cancer diagnosed during pregnancy. we retrospectively analyzed the medical records of 27 patients diagnosed with ovarian cancer during pregnancy at cheil general hospital & women 's healthcare center from january 1996 to december 2006. mean age of the patients was 29.1 yr (range 23 - 40), and a mean follow - up period was 57 months (range 7 - 112 months). of 27 patients, 15 (55.5%) had borderline malignancies, 7 (25.9%) had epithelial malignancies and 5 (18.6%) had germ cell tumors. a total of 26 patients received a conservative surgery preserving pregnancy. the mean time for surgical intervention during pregnancy was 20 weeks of gestational age. of the 27 patients, 26 had full term delivery of a healthy baby without any congenital malformation. only one patient with epithelial ovarian cancer had a relapse at 19 months after the first conservative operation with adjuvant chemotherapy. there were few data for managing patients with ovarian cancer diagnosed during pregnancy. this study results could help establish a guideline for management of ovarian malignancy complicating pregnancy.
sincipital encephaloceles, also called frontal or anterior encephaloceles, are rare neural tube defects and are more common in south and southeast asian populations. in children with sincipital encephaloceles there are often significant associated intracranial anomalies, including intracranial cysts, complete / partial corpus callosal agenesis, interhemispheric lipomas, facial clefts, and migrational anomalies. occipital encephaloceles may be associated with chiari or dandy - walker malformations and callosal or migrational anomalies. it is reported that younger siblings of patients affected with encephaloceles have up to 6% risk of a congenital central nervous system abnormality. after approval from the institutional ethics review committee, a hospital - based cross - sectional study was conducted over 8 years from june 2007 to may 2015 on 28 patients in the departments of radiodiagnosis and pediatric surgery in a tertiary care hospital. the study comprised of outpatients and in patients, of both sexes presenting with sincipital encephaloceles. patients were only allowed plain water preceding the imaging so as to keep the patient ready for sedation / anesthesia if needed. the patients were evaluated by either computed tomography (ct) or magnetic resonance imaging (mri) whichever was feasible. the ct scanner used in this study was siemens somatom spirit dual slice ct scanner (siemens healthcare, erlangen, germany). unenhanced ct scans of head and faciomaxillary region were obtained in all patients. scanning parameters used were spiral mode with slice thickness of 6 mm and collimation 6 mm 2.5 mm, pitch : 1.4 ; kvp : 130 ; mas : 80. mri imaging was performed with a 1.5-t superconducting magnet system 1.5 tesla siemens magnetom avanto b15 system (siemens healthcare, erlangen, germany) using a transmit / receive body coil with a 15-mt / m gradient. the imaging parameters were t1, t2, and fluid - attenuated inversion recovery (flair) sequences obtained in all three planes : axial, coronal and sagittal. parameters for t1-wi (t1-weighted image) : 400600/15 (repetition time ms / echo time ms), 220240-mm field of view, 256 192 matrix, and slice thickness of 5 mm with a gap of 1 mm. parameters for t2-wi : 3200/428 (repetition time ms / echo time ms), 256-mm field of view, 256 256 matrix, and slice thickness of 5 mm with a gap of 1 mm. parameters for flair : 10,000/70130 (repetition time ms / echo time ms), 240-mm field of view, 256 192 matrix, and slice thickness of 5 mm with a gap of 1 mm. the areas of interest in the study were type of encephalocele, its contents, extracranial extension and effect on adjacent structures and to look for associated brain malformations. after approval from the institutional ethics review committee, a hospital - based cross - sectional study was conducted over 8 years from june 2007 to may 2015 on 28 patients in the departments of radiodiagnosis and pediatric surgery in a tertiary care hospital. the study comprised of outpatients and in patients, of both sexes presenting with sincipital encephaloceles. patients were only allowed plain water preceding the imaging so as to keep the patient ready for sedation / anesthesia if needed. the patients were evaluated by either computed tomography (ct) or magnetic resonance imaging (mri) whichever was feasible. the ct scanner used in this study was siemens somatom spirit dual slice ct scanner (siemens healthcare, erlangen, germany). scanning parameters used were spiral mode with slice thickness of 6 mm and collimation 6 mm 2.5 mm, pitch : 1.4 ; kvp : 130 ; mas : 80. mri imaging was performed with a 1.5-t superconducting magnet system 1.5 tesla siemens magnetom avanto b15 system (siemens healthcare, erlangen, germany) using a transmit / receive body coil with a 15-mt / m gradient. the imaging parameters were t1, t2, and fluid - attenuated inversion recovery (flair) sequences obtained in all three planes : axial, coronal and sagittal. parameters for t1-wi (t1-weighted image) : 400600/15 (repetition time ms / echo time ms), 220240-mm field of view, 256 192 matrix, and slice thickness of 5 mm with a gap of 1 mm. parameters for t2-wi : 3200/428 (repetition time ms / echo time ms), 256-mm field of view, 256 256 matrix, and slice thickness of 5 mm with a gap of 1 mm. parameters for flair : 10,000/70130 (repetition time ms / echo time ms), 240-mm field of view, 256 192 matrix, and slice thickness of 5 mm with a gap of 1 mm. the areas of interest in the study were type of encephalocele, its contents, extracranial extension and effect on adjacent structures and to look for associated brain malformations. fifty percent of the patients presented before the age of 3 years, with the youngest presenting on day 7. rest of the patients (42%) presented between 3 and 10 years of age. our study did not show any gender predominance, with both males and females being affected equally. nasofrontal encephalocele was the most common type, seen in 13 (46.4%) patients. the naso - orbital and the combined type were the least common with 4 (14.2%) patients each. two patients had evidence of herniation of lateral ventricle along with brain tissue through the cranium into the encephalocele sac [table 1 ]. type of sincipital encephaloceles corpus callosal agenesis with colpocephaly was the most common associated malformation in our study [table 2 ]. 6/10 archnoid cysts were hemispheric, 4/10 were inter hemispheric and 2/10 were located in posterior fossa. associated brain malformations seven patients had evidence of hydrocephalus, of which five cases were due to the obstruction by large arachnoid cysts and two cases were due to aqueduct stenosis. subdural collection was noted in 6 patients. in 4 patients it was subdural csf collection due to hyperfunctioning of shunts and in 2 patients it was blood due to rupture of bridging cortical veins. of these 6 patients, ventriculoperitoneal (vp) shunts were performed in 2 patients and cystoperitoneal shunt in 2 patients with large interhemispheric cysts causing mass effects. corpus callosal agenesis with colpocephaly was the most common associated malformation in our study [table 2 ]. 6/10 archnoid cysts were hemispheric, 4/10 were inter hemispheric and 2/10 were located in posterior fossa. associated brain malformations seven patients had evidence of hydrocephalus, of which five cases were due to the obstruction by large arachnoid cysts and two cases were due to aqueduct stenosis. subdural collection was noted in 6 patients. in 4 patients it was subdural csf collection due to hyperfunctioning of shunts and in 2 patients it was blood due to rupture of bridging cortical veins. of these 6 patients, ventriculoperitoneal (vp) shunts were performed in 2 patients and cystoperitoneal shunt in 2 patients with large interhemispheric cysts causing mass effects. encephaloceles occur due to the failure of surface ectoderm to separate from the neuroectoderm, resulting in a bony defect in the cranial and/or facial bones, which allows herniation of meninges or brain tissue. encephaloceles occur in one of every 4000 live births and have no sex predilection. according to location, these are occipital (75% of cases), sincipital (15%), or basal (10%). sincipital encephaloceles involve the mid - face and occur about the dorsum of the nose, orbits, and forehead. these sincipital encephaloceles are more common in south and southeast asian populations whereas the occipital encephaloceles are more common in the western population. the sincipital encephaloceles are further subdivided into nasofrontal, nasoethmoidal, and naso - orbital types. the nomenclature used for encephaloceles is based on the origin of their roof and floor ; thus, for example, the roof and floor of frontonasal encephaloceles are the frontal and nasal bones, respectively. nasofrontal encephaloceles result from herniation through both the foramen cecum and the fonticulus frontalis and projecting along the nasal bridge between the nasofrontal sutures into the glabella (nasofrontal region). nasoethmoidal encephaloceles occur when there is persistent herniation through the foramen cecum into the prenasal space and nasal cavity under the nasal bones and above the nasal septum (nasoethmoidal region). moreover, naso - orbital types occur along the medial orbit at the level of the frontal process of the maxilla and the ethmoid - lacrimal bone junction (naso - orbital region). affected children may present with nasal obstruction, mass in the frontal region, proptosis, or cerebrospinal fluid (csf) leaks from ruptured encephalocele sac with recurrent meningitis. the prognosis for the sincipital encephalocele patients is generally good and is usually associated with normal intelligence and motor development. surgery is the best treatment option for encephaloceles, and mri is the choice of imaging modality for defining the contents of an encephalocele prior to surgery. high - resolution ct may also be used to display the bone anatomy, but the intracranial extension is best defined with mri. the extent of cerebral tissue in an encephalocele is also better defined with mri, which aids in prognosis and surgical planning. the aim of the radiologist is to precisely define the type of the encephalocele ; delineate the size, contents, extension ; and identify associated anomalies. there are often significant associated intracranial anomalies, including intracranial cysts, complete / partial corpus callosal agenesis, interhemispheric lipomas, facial clefts, and migration anomalies. earlier studies postulated theories such as adhesive theory, which suggest failure of mesodermal migration into midline leading to herniation of the brain matter and resulting failure of closure of anterior neuropore. as corpus callosum forms near the site of final closure of anterior neuropore, many of these patients have associated corpus callosal agenesis which was also observed in our series. rapport., postulated that this condition may have several etiological factors because mesodermal abnormalities alone can not account for the observed abnormalities. lemire., thought that anterior encephaloceles result from abnormal dedifferentiation, whereas some other studies suggest neuroectodermal abnormality. from the array of associated malformations that we have observed in our series the most common associated malformation with sincipital encephaloceles is the agenesis or hypogenesis of the corpus callosum. corpus callosum is the largest cerebral commissure connecting neocortical areas and develops between 12 and 20 weeks of gestation. development occurs from front to back with the exception of the rostrum which develops after the splenium. imaging by ct and mr in complete callosal agenesis reveals widely separated and nonconverging lateral ventricles, disproportionately enlarged occipital horns (colpocephaly), and an elevated third ventricle which is continuous superiorly with the interhemispheric fissure [figure 1 ]. in sagittal mr studies, corpus callosum, cingulate gyrus, and sulcus are not seen. gyri on the medial hemispheric surface radiate outward from the high - riding third ventricle. in partial agenesis of corpus callosum, the splenium and rostrum are absent. 3-year - old male child with a swelling at nasal bridge, (a) axial computed tomography scan shows a nasofrontal encephalocele (), (b and c) axial computed tomography sections show corpus callosal agenesis as evidenced by parallel running lateral ventricles with colpocephaly (arrow in c). they are congenital lesions that occur as a result of the splitting of the arachnoid membrane. the cysts are extra - axial fluid - filled sacs, which are sharply marginated somewhat scalloped appearing lesions that parallel the csf intensity on all sequences of mri. some suprasellar cysts become very large [figures 2 and 3 ] and cause obstructive hydrocephalus. the arachnoid cysts, particularly the interhemispheric cysts, are usually associated with corpus callosal agenesis. 2-year - old male child with a forehead swelling, (a) axial computed tomography bone window shows nasoethmoidal and naso - orbital type of encephalocele (). (b) axial, (c) coronal and (d) sagittal computed tomography sections show associated large hemispheric arachnoid cyst (arrow). (b) axial ct section also reveals the parallel orientation left lateral ventricle (upward arrow) suggestive of corpus callosal agenesis. 2-year - old female child with swelling at nasal bridge, (a) sagittal t1-weighted image shows a large interhemispheric arachnoid cyst (#). (b) axial t1-weighted image in another patient with nasoethmoidal encephalocele () shows an arachnoid cyst in right temporal fossa (arrow). in our series, subdural collection of the six patients four had subdural csf collection and it was thought to be due to hyperfunctioning of the shunts [figure 4 ], (two had been operated with ventriculo - peritonial shunt for obstructive hydrocephalus and in the other two, ventricular catheter of the shunt was placed in the large interhemispheric cysts i.e. cysto - peritoneal shunt which caused mass effects). other two patients had small subdural hematomas probably due to rupture of bridging cortical veins around the herniation. 2-year - old female child with forehead swelling, (a) axial computed tomography section shows left subdural collection (arrow) in a patient with ventriculoperitoneal shunt. (b) axial computed tomography section in another patient with a large arachnoid cyst showing bilateral frontal subdural collection (arrow) with calcification of the arachnoid membrane. agyria - pachygyria complex is a defect in migration of cerebral neurons, resulting in failure of cortical gyri to develop [figure 5 ]. the extent, distribution, and detailed structure of agyria - pachygyria vary widely, and the clinical features are accordingly very diverse. rather than one entity, pachygyric cortical abnormalities form a broad spectrum that raises different clinical and genetic problems. this complex can be of generalized / bilateral or localized / unilateral gyral malformations. generalized seizures with increased incidence of spastic quadriparesis and microcephaly are associated with the generalized / bilateral type, whereas partial seizures are more common in localized / unilateral gyral malformations and hemiparesis is the most frequent neurologic deficit. patients with bilateral or generalized gyral anomalies have poor prognosis for outcome of epilepsy and neurologic disability. the recognition of these lesions with high - resolution techniques of mri is important for planning proper treatment and genetic counseling. 4-year - old child presented with swelling over the nose and seizure, (a) axial computed tomography scan shows nasofrontal type of encephalocele () with mild orbital extension. (b) axial computed tomography section also shows agyria and pachygyria complex involving left parietooccipital lobe (arrow) with corpus callosal agenesis. (c) higher axial computed tomography section also shows ventriculoperitoneal shunt (#) in interhemispheric fissure with left subdural collection (arrow). we could not perform mri in many cases, which is the investigation of choice due to cost constraints. we could not perform mri in many cases, which is the investigation of choice due to cost constraints. encephaloceles are often associated with significant intracranial anomalies, with agenesis of corpus callosum being the most common. hence, a precise analysis of each case is necessary to recognize the associated malformations and establish the prognosis and risk of recurrence.	objective : the aim of this study was to evaluate the associated intracranial malformations in patients with sincipital encephaloceles.materials and methods : a hospital - based cross - sectional study was conducted over 8 years from june 2007 to may 2015 on 28 patients. the patients were evaluated by either computed tomography or magnetic resonance imaging whichever was feasible. encephaloceles were described with respect to their types, contents, and extensions. a note was made on the associated malformations with sincipital encephaloceles.results:fifty percent of the patients presented before the age of 3 years and both the sexes were affected equally. nasofrontal encephalocele was the most common type seen in 13 patients (46.4%), and corpus callosal agenesis (12 patients) was the most common associated malformation. other malformations noted were arachnoid cyst (10 patients), hydrocephalus (7 patients), and agyria - pachygyria complex (2 patients).conclusion : capital brain malformations are frequently encountered in children with sincipital encephaloceles. detail radiological evaluation is necessary to plan treatment and also to prognosticate such rare malformations.
today, most newborns in the united states are vaccinated against the hepatitis b virus, usually in the first few days of life.1 the practice of vaccinating infants against hepatitis b, a blood - borne virus that attacks the liver, is a popular target for contemporary vaccine critics, who for the last decade have argued that the virus is a sexually - transmitted infection that poses little or no risk to babies. indeed, this is how federal health officials themselves characterized the virus back in 1981 when they first approved the vaccine. however, by 2000, 47 states had mandated the vaccine for all schoolchildren with little or no resistance from parents. the widespread adoption of hepatitis b vaccine mandates in the 1990s thus raises the question : how did a vaccine with such a limited initial target population come to be accepted as compulsory for every child in the country ? the answer lies in an examination of gradual adjustments to the image of hepatitis b infection, an image created and recreated by ever - shifting verbal descriptions, media portrayals, and textual descriptions of the disease in the two decades following the vaccine s introduction. prior to the approval of the hepatitis b vaccine by the u.s. food and drug administration (fda), the virus was rarely the object of media attention and was only faintly visible to the american public. but as scientific discoveries leading to the vaccine accumulated, hepatitis b acquired an increasingly public image. initially, in the late 1970s and early 1980s, the image of hepatitis b constructed by popular portrayals was that of a deadly but foreign infection, a scientific obscurity of little direct relevance to most americans. shifting cultural, scientific, and political preoccupations, however, acted to reshape continually this (not strictly visual) image, as evidenced in media and scientific reports on hepatitis b and the policies that made it mandatory for children in the 1980s and 1990s. in particular, the outbreak of aids in the early 1980s, the advent of genetically - engineered pharmaceuticals in the late 1980s, and the political push for health reform in the early 1990s all left an indelible imprint on the picture of hepatitis b being held up before americans. through these constantly shifting portrayals, hepatitis b became, in turn, a sexually transmitted aids - like scourge ; an example of the promise of a new era of pharmaceuticals ; a threat from which all american children had a right to be protected ; and a cancer - causing infection spread by teenage lifestyles. examination of these shifting representations of hepatitis b reveals the process by which the virus was framed as a widespread infection worth preventing by the universal vaccination of children. in the case of hepatitis b, the metamorphosis of the infection s image was reflected in constantly revolving policy recommendations regarding who should receive the vaccine in the 1980s, and was ultimately key to securing broad uptake of the infection s vaccine in the 1990s. like many twentieth - century pharmaceutical discoveries, the development of the hepatitis b vaccine was, in part, a matter of happenstance. in 1963, research physician, baruch blumberg, was investigating variations in susceptibility to infections among different ethnic and racial groups when he identified a protein in the blood of australian aborigines, which he dubbed the australia antigen. blumberg and colleagues initially surmised that the protein was inherited, but they soon noted a correlation between australia antigen in the blood and hepatitis b infection, then called serum hepatitis. gradually, the researchers came to suspect that australia antigen, which floated freely in the blood of people infected with hepatitis b, was part of the virus that caused hepatitis. in 1967, they determined that australia antigen was, in fact, a surface protein on the virus that caused the disease ; the discovery quickly led to a test to screen blood and diagnose patients. in related research, blumberg and colleagues found that monkeys injected with highly purified australia antigen (later renamed hepatitis b surface antigen or hbsag) did not progress to serum hepatitis ; the discovery suggested that non - infectious material could be separated from the virus itself. in 1969, the researchers patented the process for separating and purifying the antigen. the basis for the development of a vaccine, what blumberg called the most significant outcome of their work, had thus been discovered.2 over the course of the 1970s, the diagnostic test that blumberg s research group had developed revealed the full extent of the hepatitis b epidemic, both in the u.s. and around the world. two distinct forms of hepatitis, a and b (formerly infectious and serum hepatitis, respectively) had been known since the 1950s, but countless cases of the latter had gone undetected, in large part because of the vague symptomatic presentation of hepatitis b infection. infection may or may not cause acute symptoms, including debilitating fatigue, nausea, and loss of appetite, and acute symptoms may or may not be fatal. those who do recover may or may not become chronic carriers. carriers, in turn, may or may not become victims, decades later, of hepatitis - induced cirrhosis or liver cancer.3 furthermore, hepatitis b s fairly mundane acute symptoms meant that, for decades, it was routinely confused with other conditions. in the 1970s, however, armed with the newly developed diagnostic protocols, researchers identified for the first time more than 200,000 new cases in the u.s. each year, and more than 200 million carriers worldwide. concurrent epidemiological studies also identified those at highest risk of the disease ; in addition to health care workers, the list included hemophiliacs, prisoners, gay men, injection drug users, sex workers, native alaskans, and immigrants from sub - saharan africa, china, and southeast asia.4 as development of a vaccine against the infection accelerated in the late 1970s, blumberg and other infectious disease experts predicted that effective vaccine would save hundreds of thousands, if not millions, of lives.5 but news of the potential vaccine, and of hepatitis b itself, rarely reached lay audiences over the course of the 1970s. norman pastorek, host of the television show today s health, developed acute hepatitis b when a surgical patient s blood splashed in his eye ; he chronicled in detail the disease s mean, sneaky malevolence on air and in print.6 two years later, hepatitis b made headlines again when blumberg shared the nobel prize in medicine for his work on the disease.7 the press also reported on the 1979 outbreak of hepatitis b among youth who shared needles to inject the recreational drug methylene deoxyamphetamine.8 such stories began to illustrate the picture emerging from epidemiological studies for the public : that the disease posed a risk to specific subsets of the population including surgeons and drug users. however, the image sketched by such stories was faint, as for most of the 1970s, the public heard and likely understood very little about hepatitis b. on the eve of the hepatitis b vaccine s introduction, lay americans therefore had little reason to view the disease as a direct threat to their health. the cbs evening news reported that while hepatitis b struck developing countries in asia and africa in epidemic proportions, in the u.s., it affected mainly patients on dialysis, medical personnel, and people living in institutions.9 news anchor, dan rather, echoed this when he reported on the hepatitis b vaccine s approval in 1981, stating that a vaccine had been approved for a disease affecting health workers, male homosexuals, and drug addicts.10 on nbc, the evening news anchor described hepatitis b to the public as follows : hospital workers get it, so do drug addicts, mental patients, homosexuals, and millions of people in africa and asia. the network s subsequent segment on the vaccine focused largely on the medical adventure story of the virus s discovery, which a reporter recounted over grainy images of aborigines in native attire.11 historian william muraskin has argued that representation of hepatitis b was deliberately constructed by a medical profession acting in self - interest. having been identified as a high - risk group themselves, health care workers, in muraskin s analysis, endeavored to define hepatitis b infection as an issue private to their profession and outside the public s purview. the media, reliant on the medical community for information about the virus, reported what they were told : that gays, injection drug users, and certain immigrants and refugees were at high risk and that the spread of hepatitis in iatrogenic settings was controlled through the use of gowns, masks, gloves, and other hygienic practices. the health care profession s internal policy of voluntary testing for carrier status became the implicit policy toward the population at large, as well. the resulting public ignorance, muraskin concludes, was two sided : it prevented hysteria and discrimination of carriers, but it also hampered public awareness of the extent of the epidemic and the true risk of infection.12 muraskin s assessment assumes that the risk of infection was widespread at this time ; regardless of whether this was the case, what is clear is that in the period prior to the approval of the hepatitis b vaccine, the popular impression of the virus as posing little threat to average americans had been deeply forged. the hepatitis b vaccine approved by the fda in 1981 was an unusual product in the history of viral vaccine development. instead of containing live, weakened virus or killed, denatured virus, heptavax b, as it was called, contained purified antibodies harvested from the blood of people infected with the virus. in a display of awe and enthusiasm for scientific discovery, the same news reporters who had downplayed the disease s risk for average americans played up, in the next breath, the new vaccine s novel form. first completely new viral vaccine in 10 years, and the first vaccine ever licensed in the united states that is made directly from human blood.13 when clinical trials revealed that the vaccine was over 92% protective, newsweek called its blood - derived antibodies ingenious, and magazines from time to glamour called the vaccine a medical breakthrough.14 fervent reports in popular and scientific journals proclaimed that hepatitis b still a little - known disease to the american public would soon join such well - known pathogenic villains as smallpox and polio as a problem of the past.15 such rhetoric was quickly dampened by another discovery, however. the clinical trials that had tested the vaccine in the 1970s had included only gay men who had been identified as being at high risk of the infection. when, in 1982, the federal advisory committee on immunization practices (acip) issued its customary recommendations on who should receive the vaccine, the list included those considered to be at highest risk for the disease, including gay men, injection drug users, sex workers, health care workers with blood contact, and select immigrants. but the fda s approval of the plasma - derived vaccine had closely coincided with reports of a deadly new disease among gay men. within a year, health officials had documented a high rate of hepatitis b infection not only among gay men but also among gay men who were sufferers of aids. the announcement spurred fears that the new vaccine was contaminated with aids.16 in 1982 and 1983, newspapers reported that gay men and injection drug users were frequent blood donors for the vaccine and that many health care workers had refused the vaccine for this very reason.17 restrictions on blood donation and a lack of evidence linking aids cases to the new vaccine appeared to quell such fears.18 but the emergence of aids had a profound and lasting impact on the nature of media representations of hepatitis b. no sooner did the potential link between hepatitis b vaccination and hiv infection fade from media reports than a new link between the two viruses emerged ; this new link, a recitation of the similarities between the two infections, would persist in popular and scientific discourse for well over a decade. the analogy between aids and hepatitis b infection had been drawn early on by epidemiologists working to discover the causative agent of aids. both diseases, scientists noted, appeared to be transmitted sexually, showing a pattern of infection among injection drug users and blood transfusion recipients. when the centers for disease control (cdc) identified the groups at high - risk for aids in 1983gay men with multiple sex partners, i.v. these closely mirrored the list earlier reported to be at high risk of hepatitis b.19 such parallels were repeatedly echoed by the media. as a 1985 cover story on aids in time pointed out, both diseases were scourges of drug addicts, blood recipients and gay men, and researchers were uncertain as to whether hepatitis b was a co - agent of aids or merely [a ] tagalong infection.20 media coverage also pointed out not only how hepatitis b was similar to aids but also how it was worse : 15 times more common, 200 times more infectious, far more stable in the environment, responsible for far more deaths, and, unlike hiv, spread by casual contact.21 but the public could take solace, the vice chair of the national foundation for infectious diseases pointed out in a letter to the new york times, in the fact that a vaccine existed to keep this cousin of aids at bay.22 this message, that the hepatitis b vaccine was a beacon of hope in a time of fear, was repeated often in the press. there s no cure for aids, gay community news told readers, but there is one for hepatitis b, which kills five times as many people each year.23mademoiselle issued the same message : there is no aids vaccine yet, but there are two new ones against hepatitis b.24 that second hepatitis b vaccine, widely available by the late 1980s, was a vastly different product from the first, blood - derived vaccine. recombivax hb, the genetically - engineered hepatitis b vaccine approved in 1986, contained viral proteins not harvested from diseased patients in the clinical setting but rather manufactured by recombinant yeast in the lab. before it was licensed, press reports hailed recombivax b for its potential to prove that genetic engineering would revolutionize the pharmaceutical industry. scientists and the reporters who quoted them called biotech vaccines exciting and imaginative, and referred to the hepatitis b vaccine specifically as a pioneering product.25 researchers told the new york times that biotech shots were cutting edge weapons that would eliminate not only hepatitis b but also aids and malaria.26 the business press breathlessly reported on the race between tiny california biotech firms to produce the world s first genetically - engineered vaccine, and when chiron s recombivax was approved, venture magazine crowned it one of the best entrepreneurial ideas of 1986.27 the approval of recombivax hb the first genetically - engineered vaccine and the fifth genetically - engineered pharmaceutical to make it to market was heralded on the front pages of the new york times, the los angeles times, the wall street journal and elsewhere for ushering in what fda commissioner, frank young, called a lawsuits over polio vaccine tainted with live virus had prompted half the nation s vaccine manufacturers to pull out of the a fact the business press repeated as they reported on the new vaccine.29 the genetically - engineered hepatitis b vaccine was enthusiastically received by investors, the pharmaceutical industry, and health professionals not only because it held the promise of a new generation of vaccines (and thus new markets), but also for its potential to address safety concerns that had damaged the industry and shaken consumer confidence. in the words of the wall street journal s editorial board, chiron s discovery brought an end to the days when vaccine development was an inexact scientific art.30 we see no reason why our approach wo nt work with virtually any infectious disease, whether it is viral, bacterial or even parasitic in nature, one researcher told time.31 nodding implicitly toward past and present fears, another researcher told the magazine that genetically - engineered vaccines eliminated the risk of actually getting herpes, hepatitis b or influenza from the injection, since the viruses themselves are not present in the formula.32 because the new vaccine did not contain a whole virus, it just ca nt do any damage, period, promised fda microbiologist richard daemer.33 these sentiments were echoed by fda commissioner young in a press statement he made on the occasion of recombivax s approval. these techniques should be extended to any virus or parasite, he stated. he went on to say that while the plasma - derived vaccine had never posed a risk of aids, the new lab - made vaccine should further reassure people. he also strongly urged those at high risk of hepatitis b to take advantage of this new life - saving protection.34 young s plea came as public health officials were bemoaning stubbornly low hepatitis b vaccination rates. in the 5 years since the acip had recommended that gay men, injection drug users, health care workers, and select immigrants be vaccinated against the infection, hepatitis b prevalence had not decreased but rather increased, with rates particularly high among young adults.35 incrementally, federal recommendations evolved in response. in 1988, the acip recommended that all pregnant women be tested for hepatitis b, and, if positive, their infants vaccinated within 12 h of birth to prevent transmission to the next generation.36 when disease incidence persisted at high rates, the acip altered its strategy again, recommending universal infant vaccination against hepatitis b in 1991.37 health officials acknowledged that the new strategy was necessary because vaccinating persons engaged in high - risk behaviors, life - styles or occupations has not been feasible and because many infected people had no identifiable source for their infections.38 the message that just about everyone was at risk of hepatitis b came to dominate media reports on the disease by the early 1990s. outlets from the philadelphia tribune to good housekeeping reported that a third of people with the disease were not in any of the known risk groups.39redbook warned readers that hepatitis was spreading fast, and the boston globe noted that hepatitis was spread by sharing gum, food, toothbrushes, and razors and by body piercing.40new york magazine, in a feature titled, the other plague, recounted the stories of a young woman who contracted a fatal case by getting her ears pierced, a young man who was infected when mugged at knife - point, and a woman infected at a nail salon.41 frequent mention of the prevalence of asymptomatic carriers heightened the sense of an immediate health threat : in the words of the new york magazine reporter, anyone could be one of the u.s.s 1.5 million typhoid marys, unwittingly transmitting hepatitis b to people unaware of their risk.42 meanwhile, health officials at the cdc were considering not just revised recommendations to increase hepatitis b vaccination but a broader program to encourage higher vaccination rates overall. the worst measles epidemic in over a decade had swept the nation in 1989 and 1990, infecting more than 50,000 people and causing hundreds of deaths.43 rates of rubella and pertussis were also on the rise. when a federal vaccine advisory panel (a panel distinct from the acip) studied the measles outbreaks, their resulting white paper concluded that the epidemic was driven by low immunization rates among pre - school - aged children.44 (immunization of schoolchildren, by contrast, had reached a peak in the early 1980s following a national immunization campaign sponsored by jimmy carter s administration.) low immunization rates among the very young, the panel argued, were the result of costly vaccines, inadequate insurance policies, and other barriers to health care. the nation s failure to immunize all susceptible children was, in their assessment, the result of a broken health care system, an issue that had become a top concern for most americans on the eve of the 1992 presidential election. in that race, presidential candidate bill clinton had campaigned and was elected on a platform that emphasized the need to fix the nation s ailing health care system.45 his first month in office, he proposed a measure that amounted to health reform in miniature, a billion dollar universal purchase program, in which the federal government would become the sole buyer of all vaccines and distribute them free of cost to all children, regardless of family income.46 as congress debated the proposal, clinton spent the spring of 1993 championing the cause of childhood immunization. his administration spread the message that vaccines were a child s basic right, one being violated by greedy drug companies. our message to the drug companies today is youre not going to profit at the expense of our children, clinton said at one press conference.47 the head of the national vaccine program office told the new york times that immunization had become a privilege, with the implication that it was the right of all children to be vaccinated.48 secretary of health and human services, donna shalala, expounded on this line of thinking, equating a child s right to vaccination with the commonly accepted rights of children to clean air, clean water, and public education.49 but vaccination was not just about rights for the administration, which also saw it as a means of reining in runaway health care costs. in addition to a broken health care system, clinton had inherited a ruined economy, and he saw the two as tightly linked. in the spring of 1993, his administration promoted vaccines for children as an important way to save health care dollars. each day we delay really making a commitment to our children carries a dear cost, clinton said before a joint session of congress.50 we now know that we will save $ 10 later for every $ 1 we spend by eliminating childhood diseases. that s a good investment no matter how you measure it.51 pharmaceutical companies stringently opposed the clinton plan, arguing that it would make vaccines unprofitable and bring the development of new vaccines to a halt.52 to an extent, many in congress agreed. the vaccine proposal that ultimately passed as part of the 1993 budget act replaced universal purchase with a $ 300 million plan to provide immunizations to native american children, those on medicaid, and those who lacked insurance coverage or coverage for vaccines. the new vaccines for children program provided states with added funds for vaccination and guaranteed their ability to purchase vaccines at federal contract prices, making low - cost vaccines available to children in both public clinics and private practices.53 that same spring, clinton signed a proclamation supporting national preschool immunization week, an annual week of coordinated efforts to fully vaccinate preschoolers with all federally recommended vaccines including the vaccine against hepatitis b.54 the enactment of the vaccines for children program coincided with a broadened set of hepatitis b vaccine recommendations by the acip, which now urged that all 11- and 12-year - olds be vaccinated against the virus. the committee stressed that universal infant vaccination should continue, but that adolescent vaccination was needed to cause disease incidence to decline more quickly. vaccinating high - risk adults had proved enormously difficult, an acip statement noted, but routine childhood vaccination would obviate vaccination of adults.55 we do not feel that targeting adults for vaccination has worked, this will be the first time, she went on, that a vaccine is recommended for children to prevent a disease that primarily occurs in adults.56 as state health boards and legislatures began taking steps to mandate the hepatitis b vaccine for infants, kindergartners, and 7th graders, many directly attributed these steps to the new federal program. minnesota s vaccine task force credited the clinton changes for the extra funds and discount pricing that made it feasible to require the hepatitis b vaccine for 7th graders.57 state health officials in colorado, louisiana, pennsylvania, and elsewhere also credited the administration for making it possible to require the new vaccine for students, hold school - based drives to encourage vaccination, and enforce new mandates since all students were now guaranteed vaccine, regardless of their ability to pay.58 at the same time, the rhetoric that once tightly linked hepatitis b to aids was beginning to diminish. ruth, who continued to inform audiences that hepatitis b was 100 times more infectious than hiv.59 but when states like idaho, california, and pennsylvania mandated the vaccine in the following years, health officials and legislators cited not the opportunity to prevent an hiv - like scourge but, instead, the growing popularity of tattoos and body piercing, and the increased presence of diabetic syringes in schools, as justification.60 and although characterizations of hepatitis b as a sexually - transmitted disease persisted, they increasingly gave way to characterizations of the virus as a preventable infection linked to cancer. this is a very safe and effective way to avoid what is a terrible disease that causes cancer and other chronic problems, said the head of colorado s health board on that state s decision to mandate the vaccine for school.61 the majority of state laws mandating hepatitis b vaccination for children thus went into effect between 1994 and 1998, taking advantage of federal enthusiasm for universal vaccination and funding support for recommended vaccines. when these laws were first introduced, members of the public with a lay understanding of hepatitis b most likely viewed it as an illness comparable to aids, if not in some ways worse and yet preventable with a safe, new, cutting edge vaccine. but as hepatitis b vaccine laws became the norm, a debate over the merits of the shot began to simmer. the debate that began to emerge in the late 1990s over the safety and appropriateness of the hepatitis b vaccine for children fit into a larger series of debates over vaccine safety that spanned the entire twentieth century and accelerated toward its end. in 1982, when a media expos publicized a scientific dispute over the safety of pertussis vaccine,62 it galvanized a previously diffuse set of vaccine - anxious parents to lobby for new vaccine policies. the resulting 1986 national vaccine injury compensation act provided compensation for the families of vaccine - injured children and established the vaccine adverse events reporting system (vaers), which began tracking reports of vaccine - related harms in 1990.63 by 1999, 42 states had mandated the hepatitis b vaccine for students, and over 25,000 reports of adverse events linked to the vaccine had been reported to vaers. scientists asserted that the mere number of events was far from proof of causation of harm, but parents insisted the figures revealed a different truth. in the subsequent wave of reporting on hepatitis b, media reports focused on the perspective of patients, not scientists. in 1998, the media reported on france s decision to halt hepatitis b vaccination because of fears the shot caused neurological damage.64 early in 1999, the television news program 20/20 aired a report on adults with impairments thought to be linked to the vaccine and an infant who died shortly after receiving the vaccine.65 in the spring of 1999, a house committee held hearings on hepatitis b vaccine safety concerns. the hearings attempted to weigh the potential risks of the vaccine against the risks of what appeared, by this time, to be two very different diseases. to officials from the cdc and members of the american liver foundation and hepatitis foundation, hepatitis b was, primarily, a lethal disease that infected one in 20 americans and caused 5,000 deaths each year, many of these from liver cancer. to members of massachusetts citizens for vaccination choice and parents requesting open vaccination education, and to the doctors and parents who had witnessed blindness, deafness, seizures, and other effects in children following vaccination, hepatitis b was instead a rare, sexually - transmitted infection that threatened drug addicts and foreigners and that posed no risk to american infants from healthy families.66 when new jersey attempted to mandate the hepatitis b vaccine for its schoolchildren later that same year, some parents and a lone legislator resisted, citing reports of hazards and the inappropriateness of the vaccine for youth.67 new jersey s mandate easily passed, but in the years that followed, anti - vaccine and vaccine safety groups grew increasingly active. they were especially galvanized by news of potentially harmful levels of mercury in vaccines, the withdrawal of a new vaccine against rotavirus from the market because of safety concerns, and a now notorious study that purported to link the mmr vaccine to autism. as anti - vaccination sentiment swelled in the 2000s, the hepatitis b vaccine was often held by critics as proof that policies were not always devised in the best interest of children. critics looked to early scientific representations of hepatitis b as a sexually - transmitted disease affecting specific strata of the adult population as evidence in support of their position. you pop out of the womb and the first thing you get is a shot for a sexually - transmitted disease, one frustrated father told usa today in 1999.68 the argument provided traction for vaccine critics over the course of the ensuing decade, a decade in which popular vaccination resistance grew. do we really need hepatitis b on the second day of life ? implored actor and vaccine critic jim carey on cnn s larry king live in 2009. it s a good thing that s gone too far.69 recent opposition to the hepatitis b vaccine is, of course, just one episode in the long history of alternate acceptance and rejection of vaccination. in the early days of aids, hepatitis b was portrayed as a related infection urgently worth preventing ; this association helped place the virus on par with such dreaded but also vaccine - preventable diseases as polio and smallpox, guaranteeing its position in the arsenal of childhood vaccines. but as aids became a pharmaceutically - manageable infection, and as parental concerns turned increasingly toward the rising prevalence of autism and other neurological conditions, the perceived risks of hepatitis b infection became overshadowed, for many, by the perceived risks of vaccination. the pattern is a familiar one in the history of vaccination resistance ; historians james colgrove and dorothy porter have both described a similar trend in public attitudes toward the polio vaccine as prevalence of that disease diminished.70 progressive era anti - vaccinationists, described by historian robert johnston, likewise came to perceive the vaccine against smallpox as riskier than a chance with the disease itself.71 before hepatitis b vaccine became the target of vaccination resistors, however, it was, at least briefly, accepted widely as an addition to the spectrum of vaccines for children. the factors key to securing the vaccine s acceptance are revealed by an examination of the constant evolution of the image of hepatitis b infection before the eyes of lay americans. the hepatitis b vaccine s disease target, as this analysis has shown, was framed very differently in the shot s first decade of existence than it was in subsequent decades. textual and visual representations of the disease in the 1970s and 1980s indicate that lay observers were initially given little reason to worry about hepatitis b infection or anxiously anticipate its vaccine. over the course of the 1980s, however, the disease itself was no longer represented as the province of marginalized groups but rather as a deadly killer akin to aids albeit preventable with the fruits of the most cutting - edge scientific technology. the widespread adoption of hepatitis b vaccine laws that followed in the 1990s reflected the fears and hopes of a nation stricken by aids, briefly smitten with biotechnology, and hobbled by a broken health care system all told, a set of preoccupations that were directly reflected in ever - evolving portrayals of hepatitis b itself. the vaccination of children, specifically, presented the most expedient means of addressing these fears, as the remarks of health officials, and the compliance of legislators and communities, reveals. the nation s hopes and fears would change shape again, in the late 1990s and early 2000s, and so too would perceptions of hepatitis b and its vaccine but this would occur only after the vaccine was made mandatory for every child in the nation.	in the decade following hepatitis b vaccine s 1981 approval, u.s. health officials issued evolving guidelines on who should receive the vaccine : first, gay men, injection drug users, and healthcare workers ; later, hepatitis b - positive women s children ; and later still, all newborns. states laws that mandated the vaccine for all children were quietly accepted in the 1990s ; in the 2000s, however, popular anti - vaccine sentiment targeted the shot as an emblem of immunization policy excesses. shifting attitudes toward the vaccine in this period were informed by hepatitis b s changing popular image, legible in textual and visual representations of the infection from the 1980s through the 1990s. notably, the outbreak of aids, the advent of genetically engineered pharmaceuticals, and a democratic push for health reform shaped and reshaped hepatitis b s public image. hepatitis b thus became, in turn, an aids - like scourge ; proof of a new era of pharmaceuticals ; a threat from which all american children had a right to be protected ; and a cancer - causing infection spread by teenage lifestyles. the metamorphosis of the infection s image was reflected in evolving policy recommendations regarding who should receive the vaccine in the 1980s, and was key to securing broad uptake of the vaccine in the 1990s.
endoscopic submucosal dissection (esd) has been performed widely and safely as an established treatment for superficial esophageal neoplasms.1 to date, the indication for esd has been gradually extended to larger lesions. however, as large superficial esophageal neoplasms became candidates for esd, a substantial number of post - esd strictures have been reported.2 in particular, esophageal stricture mostly occurs after near - circumferential or whole - circumferential esd at a rate of 88% to 100%.3 post - esd stricture decreases the patients ' quality of life and usually requires multiple sessions of risky endoscopic balloon dilatation (ebd).4 recently, the efficacy of prophylactic oral prednisolone in preventing post - esd stricture was reported.5 according to the report, oral prednisolone may be effective in reducing the need for ebd. however, the cumulative dose of prednisolone was high and associated with serious adverse events. a previous study reported that intralesional injection of triamcinolone reduced the incidence of stricture and the number of required ebd sessions.6 however, there is little experience in intralesional steroid injection for near- or whole - circumferential esd ulcerations. furthermore, there is no established method of injection to date. we report a patient with superficial esophageal squamous cell carcinoma who underwent complete near - circumferential esd and then received a single session of intralesional steroid injection immediately after esd. dysphagia and the need for ebd have not developed after a follow - up period of 6 months. all the laboratory findings were within normal limits. computed tomography and positron emission tomography revealed no distant metastasis. an initial upper endoscopy showed a lugol - voiding lesion involving more than three - quarters of the circumference of the lower esophagus, extending 39 to 43 cm from the incisors (fig. histopathological findings from another hospital revealed high - grade squamous cell dysplasia, but squamous cell carcinoma could not be excluded., we needed to obtain clearer views and to control the scope in detail throughout the procedure ; therefore, this patient underwent esd with tracheal intubation and general anesthesia in an operating room. the esd procedure was performed using a single - channel upper endoscope (gif - h260z ; olympus, tokyo, japan) with a transparent cap. circumferential markings were made using a hook knife (kd-620lr ; olympus) just outside the lesion, on soft coagulation mode of a high - frequency generator for electrosurgery with an automatically controlled system for cutting and coagulation (erbe, tbingen, germany). a total amount of 80 ml normal saline solution mixed with epinephrine was injected into the submucosal layer to lift the mucosa including the tumor. after the lesion was lifted, mucosal incision and dissection was made with a hook knife and an it knife (kd-611l ; olympus) (fig. just after dissection and hemostasis, a single - session endoscopic triamcinolone injection was administered (fig. triamcinolone acetonide (triam, 40 mg / ml ; shinpoong co., seoul, korea) was diluted with saline to make a 20 mg / ml solution. a total of 80 mg (4 ml) triamcinolone was injected into the deep submucosa and the superficial proper muscle layer of the ulcer base at eight sites, with a 0.5 ml dose at each site. the depth of needling was as shallow as producing a small amount of spillage into the esophageal lumen side during injection to avoid possible abscess formation. three neoplastic lesions (3817, 117, and 73 mm) were found in the specimen (fig. the histopathologic diagnosis was well - differentiated intramucosal invasive carcinoma confined to the lamina propria without lymphovascular invasion. serial esophagography was performed 2 and 4 weeks later to assess the post - esd stricture. mild irregular segmental narrowing was seen in the lower esophagus, but no definite passage disturbance was observed (fig. follow - up upper endoscopy was performed after 4 months, which revealed only a post - esd scar (fig. the patient did not complain of dysphagia and showed a good outcome for 6 months after esd. post - esd stricture is a major complication especially in esophageal esd. in a previous report, multivariate analysis revealed that a circumferential extension of more than three - quarters and a histological depth to m2 are reliable independent predictors for post - esd stricture.3 as larger superficial esophageal neoplasms became candidates for esd, substantial post - esd strictures have been reported. indeed, the rate of esophageal strictures after esd for near - circumferential or whole - circumferential lesions has been reported to be surprisingly as high as 88% to 100%.7 however, there are no guidelines for the treatment of post - esd esophageal stricture, and most of the previous studies have been performed retrospectively. until now, multiple sessions of ebd is the preferred treatment of esophageal stricture regardless of etiology;8 however, this can increase the risk of perforation, massive bleeding, and bacteremia.4 for this reason, the development of strategies to prevent post - esd stricture is important. periodic balloon dilations and temporary stenting were used to prevent post - esd stricture in some institutes. fujishiro.9 reported that repetitive, periodic ebd was effective in controlling and preventing post - esd stricture. however, isomoto.10 reported that a scheduled preventive ebd immediately after esd was needed in a median of 30 times for circumferential cases. several studies suggested that severe inflammation, fibrosis, and scarring in the exposed submucosa after esd might play an important role in the formation of stricture. according to that theory, post - esd stricture might be prevented by controlling such inflammation.11 steroid can inhibit fibrosis by preventing the migration and activation of inflammatory cells and fibroblasts.12 a few studies reported on the effectiveness of prophylactic oral prednisolone for the prevention of post - esd stricture. as a result, the rate of stricture was decreased together with the number of needed ebd procedures.5,10 however, the cumulative dose of prednisolone administered was approximately 1,000 mg. the potential risks of such a high dose, such as immune suppression, psychiatric disturbance, peptic ulceration, and optical damages, were a cause for concern. hashimoto.6 reported that endoscopic intralesional injection of triamcinolone was safe and effective for the prevention of esophageal stricture after esd for superficial esophageal carcinoma. triamcinolone was injected into post - esd ulceration 3, 7, and 10 days after esd. however, it needed three additional endoscopic procedures, causing inconvenience and additional costs to the patients. moreover, the enrolled patients had a semicircumferential mucosal defect with a relatively low risk of post - esd stricture. in a controlled prospective study, hanaoka.13 showed promising results with a single session of intralesional steroid injection immediately after esd for preventing post - esd stricture. compared with the historical control group, the study group had a significantly lower stricture rate (10% vs. 66%). we injected triamcinolone immediately after esd in the exposed submucosa of the post - esd ulceration in the same manner. however, the other authors diluted triamcinolone solution up to 5 mg / ml. because the total amount of the injected triamcinolone was roughly similar to that in our case (100 mg vs. 80 mg), the injected volume and the number of needlings were larger than those in our case (10 ml vs. 4 ml, 20 to 40 punctures vs. 8 punctures). therefore, our method of steroid injection was much simpler than that in any previous study. furthermore, our patient had a near - circumferential mucosal defect, whereas other studies excluded patients with near - circumferential defects because of the extremely high risk of esophageal stricture. because injected triamcinolone usually remains locally active for 3 to 4 weeks, we did not repeat the injection.14 in this case, no adverse events related to triamcinolone injection occurred. however, a previous animal study reported that direct injection of triamcinolone into the exposed esophageal submucosal tissue of four pigs resulted in the complication of esophageal abscess.15 in addition, potential risks of perforation, mediastinitis, and pleural effusion may be associated with local injection therapy. to prevent excessively deep mural injection, we performed the triamcinolone injection very carefully while checking for minor spillage from the injection site. there are no standard guidelines to prevent stricture in a patient with near - circumferential or circumferential mucosal defect after esd. many methods to prevent post - esd stricture, such as repetitive ebd, temporary stent insertion, and oral steroid and intralesional steroid injection, have been used by different institutes individually. because there are no randomized controlled studies comparing those methods, any method can not be widely accepted. in this case, we showed that a single session of intralesional steroid injection into a post - esd ulcer was an effective and convenient method to prevent post - esd stricture. this method of steroid injection was also simpler and safer than those reported in other previous studies. however, the true benefit and the risks of injection have not been validated yet.	stricture frequently occurs after endoscopic submucosal dissection (esd) for superficial esophageal carcinoma with near- or whole - circumferential mucosal defects, and post - esd stricture is difficult to treat and usually requires multiple sessions of endoscopic balloon dilatation. intralesional steroid injection has previously been used to prevent stricture ; however, there have been few experiences with this method after near- or whole - circumferential esd. we present a case of a single session of intralesional steroid injection performed immediately after near - circumferential esd to prevent post - esd stricture. after a follow - up period of 6 months, the patient showed good outcome without dysphagia.
the abuse of illicit drugs such as opiates is a worldwide problem for both the users and society. drug abuse is linked with a number of diseases, including hiv and hbv infections and other communicable diseases, and many drug addicts die of either the drug itself or complications from use everyday. in china, the number of drug addicts is estimated to be more than 1,160,000 in 2005, and 78.3% of them are heroin users. there is an urgent need for a better health care aiming at prevention and treatment of drug addiction. methadone maintenance treatment for the management of opioid dependence is a substitution therapy that requires daily dosing with methadone solution under close monitoring by clinic staff. methadone treatment allows for the patients to decrease or completely stop their use of illegal drugs and drug - related high - risk behaviors. by the end of 2008, there were over 500 methadone maintenance treatment clinics, and 95,000 users receiving treatment in mainland china. in sichuan province alone, the number of illegal drug users who have received this treatment is over 5000. published studies have shown that the oral health of drug addicts is poorer than that of the general population [46 ]. for former heroin users who have received or are receiving methadone treatment, the prevalence rates of caries and periodontal diseases are higher than that in the general population. there are a number of reasons for this, including a lack of concern about oral health by the patients due to their altered mental state, damage due to the illicit drugs or methadone, or differing oral health care needs for other reasons (eg, poverty or dietary problems). there is an urgent need for effective approaches to monitoring, preventing and treating oral health problems among these individuals. although the harmful effects of drug abuse on general and mental health have been well - documented, the information about the oral health status of former heroin users receiving methadone treatment in china is generally lacking. this study was designed to investigate and analyze the oral health status of former heroin users treated with methadone in chengdu, the capital of sichuan province in southwestern china. we speculate that the information obtained can then be used to better prevent and treat various oral health issues among this population. we recruited 445 former heroin users, including 317 males and 128 females, who were receiving methadone treatment in chengdu. the mean age of these subjects was 35.21 years, ranging from 20 to 59 years old. oral health examinations using a portable dental chair were carried out using the optical fiber projection plane dental reflector and community periodontal index (cpi) probe according to the who basic oral health survey methods (version 4, 1997). the decayed / filled teeth index (dft) was used to record decayed teeth (dt) and filled teeth (ft), and the presence of decayed root (d - root), residual roots, missing teeth (mt), and dental prosthetic restoration were also recorded. because of the poor periodontal health of the former heroin users, it was difficult to determine whether the missing teeth were a result of caries or periodontal disease. for this reason, the status of missing teeth was listed separately from the caries status, which was reflected by the prevalence rate of decayed and filled teeth and the mean decayed and filled teeth score. while all 32 teeth in the oral cavity were examined in this investigation for patients who had them in order to accurately reflect the teeth and dentition status, the missing teeth rate was determined excluding the third molars because of variations in tooth eruption. the consistency of df and periodontal examinations were evaluated, and the kappa values were 0.87 and 0.85, respectively. a database of the results was set up, and the data were analyzed by test, independent t - test, or single factor analysis of variance, as appropriate, using spss 12.0 software. we recruited 445 former heroin users, including 317 males and 128 females, who were receiving methadone treatment in chengdu. the mean age of these subjects was 35.21 years, ranging from 20 to 59 years old. oral health examinations using a portable dental chair were carried out using the optical fiber projection plane dental reflector and community periodontal index (cpi) probe according to the who basic oral health survey methods (version 4, 1997). the decayed / filled teeth index (dft) was used to record decayed teeth (dt) and filled teeth (ft), and the presence of decayed root (d - root), residual roots, missing teeth (mt), and dental prosthetic restoration were also recorded. because of the poor periodontal health of the former heroin users, it was difficult to determine whether the missing teeth were a result of caries or periodontal disease. for this reason, the status of missing teeth was listed separately from the caries status, which was reflected by the prevalence rate of decayed and filled teeth and the mean decayed and filled teeth score. while all 32 teeth in the oral cavity were examined in this investigation for patients who had them in order to accurately reflect the teeth and dentition status, the missing teeth rate was determined excluding the third molars because of variations in tooth eruption. the consistency of df and periodontal examinations were evaluated, and the kappa values were 0.87 and 0.85, respectively. a database of the results was set up, and the data were analyzed by test, independent t - test, or single factor analysis of variance, as appropriate, using spss 12.0 software. the mean age of these subjects was 35.21, ranging from 20 to 59 years old. eleven, 290, 136 and 8 people had middle school, high school, college, and postgraduate education, respectively. the duration of illicit drug use ranged from 1 year to 32 years, and the duration of methadone treatment ranged from 1 to 15 months. the daily dosage of methadone was 8150 ml, which was adjusted daily, and the mean maintenance dosage was 40 ml. there were 329 smokers in this study (272 males and 57 females), and 334 subjects (227 males and 107 females) brushed their teeth (once or twice) everyday. only 10.11% of subjects (30 males and 15 females) had received dental treatment in the past year, 11 subjects had a tooth or teeth extracted, 27 subjects had a tooth filled and 7 subjects had dental prosthetic restoration performed. the detailed prevalence and scores of dft, grouped by sex, are shown in table 1. as a general trend, females had higher (worse) prevalence / scores than the males, which is similar to the results of the chinese general population. the mean scores reflecting the presence of decayed teeth and decayed / filled teeth of female subjects was higher than male subjects (table 1, p 45), were 3.49, 4.37, and 4.44, respectively. the mean age of these subjects was 35.21, ranging from 20 to 59 years old. eleven, 290, 136 and 8 people had middle school, high school, college, and postgraduate education, respectively. the duration of illicit drug use ranged from 1 year to 32 years, and the duration of methadone treatment ranged from 1 to 15 months. the daily dosage of methadone was 8150 ml, which was adjusted daily, and the mean maintenance dosage was 40 ml. there were 329 smokers in this study (272 males and 57 females), and 334 subjects (227 males and 107 females) brushed their teeth (once or twice) everyday. only 10.11% of subjects (30 males and 15 females) had received dental treatment in the past year, 11 subjects had a tooth or teeth extracted, 27 subjects had a tooth filled and 7 subjects had dental prosthetic restoration performed. the detailed prevalence and scores of dft, grouped by sex, are shown in table 1. as a general trend, females had higher (worse) prevalence / scores than the males, which is similar to the results of the chinese general population. the mean scores reflecting the presence of decayed teeth and decayed / filled teeth of female subjects was higher than male subjects (table 1, p 45), were 3.49, 4.37, and 4.44, respectively. there are a number of possible reasons for the poor oral health of the former heroin users. firstly, overall awareness of oral health care in this population is low, and few of the subjects regularly brushed their teeth the recommended two times per day. it has also been reported that the intake of sugar by drug addicts is higher than by the general population. additionally, the use of many medications is associated with xerostomia, and it has been shown that the amount of saliva produced by drug abusers is decreased, creating a favorable environment for the formation of plaque and eventual dental caries. thus, the illicit drug abuse and methadone treatment both could lead to xerostomia, which is associated with dental caries. although the effect of methadone itself on the formation of caries has not been demonstrated, it has been reported that the methadone was not sugar - free and the ph of a 1% water solution of methadone is 4.55.5, suggesting that taking methadone could increase oral acidity, thus increasing the rate of caries. it has also been shown that the immune function of methadone patients decreases, with lower levels of igg, igm, and especially iga, the decrease of which may result in an increased rate of caries compared to the general population. it is also possible that the concentration of microelements in the oral cavity may have changed, influencing the capacity of teeth to resist acid. in this study, the reason may be that the duration of using methadone was short, the longest only15 months, but the oral status did relate to the duration of drug abuse. caries prevalence and mean caries score increase with increasing age ; however, we did not observe any significant differences between groups based on age. by linear regression, the caries scores only related to sex and the duration of drug abuse. nevertheless, there was a tendency toward an increase in the prevalence and score of missing teeth as subject age increased. the detection rates of gingival bleeding, calculus, and shallow pockets and deep periodontal pockets in the former heroin users treated with methadone in chengdu were higher than in the general adult population in the same area. the cpi index was used in this periodontal examination. because only the worst condition was recorded in every zone, some periodontal conditions may have been overestimated, especially the probing bleeding, which may have been overestimated by as much as 50%. however, other reports have indicated that gingival bleeding was observed in most illegal drug users. thus, while there may have been some overestimation in this investigation, the periodontal condition of the subjects was poorer than the general population. earlier studies have shown that periodontal health continues declining with the duration of illegal drug use. in this study, in addition to local factors (the presence of bacterial plaques and dental calculi), systemic factors also influence periodontal health. because heroin contains a number of noxious substances, it is likely that the drug can injure oral tissues following systemic administration. heroin has been demonstrated to induce disorders of the endocrine system, which could change the reaction of the periodontium to the drug stimulus, possibly increasing its destruction. drug abuse can also damage the humoral and cellular immunity, further exacerbating the periodontal disease. the dental calculus average section count is often used to evaluate the overall status of oral hygiene. in this investigation, the dental calculus average section count of men was higher than that of women, which explains the poorer periodontal status of the men. in this investigation, we discovered that the oral status of former heroin users in chengdu was poorer than the general population in the same area. there was a significant increase in various markers of dental and periodontal diseases in these subjects. the majority of carious teeth were untreated and the majority of missing teeth were not repaired. the frequency of missing teeth likely accounted for the lower values compared to other reports. a large number of carious retained roots were observed, demonstrating a high level of unmet dental treatment need. the west china dental hospital, located in chengdu, is the most famous dental hospital in china and has many dental clinics that offer dental treatment to all, including methadone users. this is likely because the former heroin users have a little expendable income and can not pay the fee for dental treatment. furthermore, their chaotic lifestyle makes them unlikely to practice good oral hygiene, and the illicit drug use may have masked the pain caused by the caries or residual root. since almost all of the subjects were from disadvantaged groups, it is hoped that this investigation will provide the necessary data to serve as an impetus for increasing programs that can help them improve their oral health. dentists should understand the dental emergency of methadone users. to provide better dental services for the methadone users, dentists teach them the importance of oral health, teach them brushing and oral self - care, check their oral status regularly and without charge and, and encourage them to take care of their teeth and see a dentist when needed. additionally, we suggest the government should establish a dental clinic in the methadone treatment clinic to provide basic dental care (eg, filling teeth, root canal treatment, cleaning teeth, extracting teeth) without charge or for a very low fee. we conclude that the oral health status among the studied former heroin users in chengdu is poorer compared to the general population. dentists should provide this population with basic dental care for free or charge only a small fee, as well as teaching them self - care.	summarybackgroundthere have been few studies conducted on the oral health status of illegal drug users in china, affecting the development of preventive and therapeutic approaches. the aim of the present study was to investigate and analyze the oral health status of former heroin users treated with methadone in chengdu, the capital of sichuan province in southwestern china.material/methodsthe presence of caries (decayed tooth and root), missing teeth, residual roots, dental prosthetic restoration and periodontal health were investigated in 445 former heroin users treated with methadone (317 males and 128 females). their ages ranged from 20 to 59 years old.resultsamong the study subjects, the prevalence of decayed / filled teeth was 64.72%, and the mean of decayed / filled teeth score was 2.92. the prevalence of decayed / filled roots was 21.80%, and the mean of decayed / filled roots score was 0.62. the prevalence of missing teeth was 31.46%, and the mean missing teeth score was 0.62. the prevalence of residual roots was 42.02%, with a mean score of 1.06. the rates of gingival bleeding, calculus, shallow pockets periodontal pocket, and deep periodontal pocket were 99.55%, 96.63%, 30.34%, and 2.70%, respectively.conclusionsthe oral health status among the studied former heroin users in chengdu was poorer than the general population. better dental care for the former heroin users is needed to promote their oral health.
muscle specimens were also examined by electron microscopy, which was performed as described previously. b - cell clonality was assessed in both patients ' specimens by pcr amplification using a set of biomed-2 assays (invivoscribe technologies, san diego, ca). loci for igh [igha : fr1 (variable region framework 1)-j ; ighb : fr2-j ; ighc : fr3-j ] and for igl (v - j) were targeted. muscle specimens were also examined by electron microscopy, which was performed as described previously. b - cell clonality was assessed in both patients ' specimens by pcr amplification using a set of biomed-2 assays (invivoscribe technologies, san diego, ca). loci for igh [igha : fr1 (variable region framework 1)-j ; ighb : fr2-j ; ighc : fr3-j ] and for igl (v - j) were targeted. patient 1, a 27-year - old woman, had a 7-year history of unilateral swelling of her left biceps muscle and hand as well as wrist pain and morning stiffness of her finger joints. a contrast - enhanced mri of her left biceps showed diffuse gadolinium uptake, thickening of the muscle fascia, and muscle edema (figure 1). on neurologic examination she showed normal symmetrical muscle strength and normal fine motor skills. laboratory examination of her blood serum showed elevated immunglobulin e, c - reactive protein, and antinuclear and anti - cyclic citrullinated peptide antibodies. based on the international consensus criteria, biopsy of the left biceps gave a diagnosis of dm with accompanying b - cell - rich follicle - like structures. an initial high - dose (1 mg / kg / d) prednisolone treatment over 4 weeks followed by a low - dose treatment (5 mg / d) led to rapid improvement and persistent resolution of her symptoms over a 1-year period. contrast - enhanced mri (t1) revealed areas of hyperintensity with diffuse gadolinium uptake in the affected muscle. patient 1 : left biceps (c) ; patient 2 : right deltoid and left triceps brachii muscles (a, b). patient 2, a 17-year - old girl, was referred because of a 5-month history of painful reddish swellings of the left upper arm followed by similar symptoms in her right upper arm. three weeks prior to referral she had arthralgia of the hands, knees, and feet, and morning stiffness. on neurologic examination she had a normal serum ck and erythrocyte sedimentation rate and a positive antinuclear antibody titer. mri of the upper arms revealed hyperintensity in the right deltoid and left triceps muscles (figure 1, a and b). a biopsy of the left triceps showed a picture consistent with dm with accompanying b - cell - rich follicle - like structures. she was treated with methylprednisolone pulse therapy (1,000 mg for 3 consecutive days) and monthly iv immunoglobulin (ivig) and subsequently received 60 mg prednisone daily and methotrexate (17.5 mg) weekly. both ivig and prednisone were successfully tapered, and currently she receives subcutaneous methotrexate and hydroxychloroquine, which has led to sustained improvement of her symptoms. except for some minor alterations of the skin there remained no signs of active disease. muscle biopsies of both patients revealed extensive infiltration by inflammatory cells mainly in the perimysium and endomysium (figure 2, a mac (c5b-9) deposition was mainly found on small capillaries and on the sarcolemma of single muscle fibers (figure 2 g, arrowhead). mhc class ii was expressed by the lymphoid cells and was found on numerous muscle fibers, predominantly in the perifascicular area (figure 2i). sarcolemmal mhc class i expression was detected on all muscle fibers with perifascicular enhancement (figure 2j). combined cytochrome c oxidase (cox)/succinate dehydrogenase histochemistry revealed scattered cox - negative fibers as a sign of accompanying mitochondrial dysfunction (figure 2k). cd123 dendritic cells (figure 2l) and cd138 plasma cells (figure 2 m) were found in close proximity to t- and b - cell areas. the lymphoid follicle - like structures (lfls) were mainly composed of cd45 leukocytes (figure 2e) with cd8 (figure 2n) and cd4 (figure 2o) t cells distributed around cd79 b cells (figure 2p). expression patterns of peripheral bcl-2 (figure 2q), central bcl-6 (figure 2r), and bob.1 (figure 2s) paralleled the specific lymphoid follicle - like b - cell pattern. the ki67/mib-1 proliferation index was increased within the center of the follicle - like structures (figure 2 t). serial sections of muscle biopsy stained with hematoxylin & eosin revealed ectopic lymphoid follicle - like structures (patient 1 : a and b ; patient 2 : c and d). sections were stained with an antibody against cd45 leukocytes (e) to highlight the follicle - like inflammatory infiltrate. c5b-9 was mainly found in walls of small capillaries and on the sarcolemma of single muscle fibers (g, arrowhead). electron microscopy revealed ultrastructural evidence of undulating tubules (patient 1 : h, arrowheads, 50,000 ; patient 2 : not shown). major histocompatibility complex (mhc) class ii was expressed by the lymphoid cells and was found on numerous muscle fibers, predominantly in the perifascicular area (i). combined cytochrome c oxidase (cox)/succinate dehydrogenase staining revealed blue - stained fibers indicating reduced cox activity (k). the lymphoid follicle - like structures consisted of cd123 dendritic cells (l), cd138 plasma cells (m), and cd8 (n) and cd4 (o) t cells that were distributed around cd79a (p) b cells. expression patterns of bcl-2 (q), bcl-6 (r), and bob.1 (s) illustrate lymphoid follicle - like structures with increased proliferative activity in the b - cell areas as indicated by ki67/mib-1 immunostaining (t). scale bars : a, d, e, f, k : 200 m ; b, c, g, s : 50 m ; i, j, l electron microscopy revealed undulating tubules in endothelial cells of both patients, which supported the light microscopic diagnosis of dm (figure 2h, arrowheads). to exclude the presence of a neoplastic b - cell population as described for b - cell lymphomas within skeletal muscle, we performed a b - cell clonality analysis. muscle biopsies of both patients revealed extensive infiltration by inflammatory cells mainly in the perimysium and endomysium (figure 2, a mac (c5b-9) deposition was mainly found on small capillaries and on the sarcolemma of single muscle fibers (figure 2 g, arrowhead). mhc class ii was expressed by the lymphoid cells and was found on numerous muscle fibers, predominantly in the perifascicular area (figure 2i). sarcolemmal mhc class i expression was detected on all muscle fibers with perifascicular enhancement (figure 2j). combined cytochrome c oxidase (cox)/succinate dehydrogenase histochemistry revealed scattered cox - negative fibers as a sign of accompanying mitochondrial dysfunction (figure 2k). cd123 dendritic cells (figure 2l) and cd138 plasma cells (figure 2 m) were found in close proximity to t- and b - cell areas. the lymphoid follicle - like structures (lfls) were mainly composed of cd45 leukocytes (figure 2e) with cd8 (figure 2n) and cd4 (figure 2o) t cells distributed around cd79 b cells (figure 2p). expression patterns of peripheral bcl-2 (figure 2q), central bcl-6 (figure 2r), and bob.1 (figure 2s) paralleled the specific lymphoid follicle - like b - cell pattern. the ki67/mib-1 proliferation index was increased within the center of the follicle - like structures (figure 2 t). serial sections of muscle biopsy stained with hematoxylin & eosin revealed ectopic lymphoid follicle - like structures (patient 1 : a and b ; patient 2 : c and d). sections were stained with an antibody against cd45 leukocytes (e) to highlight the follicle - like inflammatory infiltrate. c5b-9 was mainly found in walls of small capillaries and on the sarcolemma of single muscle fibers (g, arrowhead). electron microscopy revealed ultrastructural evidence of undulating tubules (patient 1 : h, arrowheads, 50,000 ; patient 2 : not shown). major histocompatibility complex (mhc) class ii was expressed by the lymphoid cells and was found on numerous muscle fibers, predominantly in the perifascicular area (i). combined cytochrome c oxidase (cox)/succinate dehydrogenase staining revealed blue - stained fibers indicating reduced cox activity (k). the lymphoid follicle - like structures consisted of cd123 dendritic cells (l), cd138 plasma cells (m), and cd8 (n) and cd4 (o) t cells that were distributed around cd79a (p) b cells. expression patterns of bcl-2 (q), bcl-6 (r), and bob.1 (s) illustrate lymphoid follicle - like structures with increased proliferative activity in the b - cell areas as indicated by ki67/mib-1 immunostaining (t). scale bars : a, d, e, f, k : 200 m ; b, c, g, s : 50 m ; i, j, l r, t : 100 m. electron microscopy revealed undulating tubules in endothelial cells of both patients, which supported the light microscopic diagnosis of dm (figure 2h, arrowheads). to exclude the presence of a neoplastic b - cell population as described for b - cell lymphomas within skeletal muscle, we performed a b - cell clonality analysis. since both patients initially presented with unilateral muscle swellings, no detectable muscle weakness, and lack of ck elevation, the diagnostic criteria of dm, according to the european neuromuscular centre, and jdm, according to the international consensus criteria, were not present. however, the morphologic criteria to diagnose dm were perfectly fulfilled, including the presence of undulating tubules known to be detectable in early - stage dm, emphasizing the need for muscle biopsy in these patients. muscle biopsy of both patients also showed characteristic lfls with defined t- and b - cell compartmentalization, which is a very rare finding in a very small subset of patients with jdm and overlap syndrome. ectopic lymphoid follicles are a well - known histopathologic feature in other autoimmune diseases, including multiple sclerosis with meningeal inflammation, sjgren syndrome affecting salivary glands, and focal myositis. nevertheless, it remains unclear which factors lead to formation of lfls in these diseases. the lymphoid aggregates present in our muscle biopsies show a remarkable similarity to secondary lymphoid organs (slos) with distinct t- and b - cell compartmentalization. expression of specific germinal center (gc) b - cell markers (bob.1/bcl-6) in the central b - cell - rich areas suggests a gc - like reaction in dm, similarly seen in true slos. it was previously reported that jdm patients with lymphoid follicle - like organization of the inflammatory infiltrate in muscle have a more severe and difficult - to - treat disease. however, our patients, who slightly exceeded the age at onset of jdm, exhibited a rather mild clinical presentation and responded favorably to therapy. brachio - cervical inflammatory myopathy, focal myositis, and overlap syndrome were discussed as potential differential diagnoses and ruled out based on histologic, clinical, and ultrastructural (presence of undulating tubules) features. the outcome in patients with idiopathic inflammatory myopathy (iim) has been studied recently. most patients (68%) with iim (including dm) developed a polyphasic or chronic disease course. there were no significant differences between the different myositis subtypes with regard to disease course, mortality, and quality of life. we show that the presence of lfls is not necessarily evidence of an unfavorable prognosis in adult dm as opposed to reports in jdm. in conclusion goebel designed the study concept, did the analysis of data, participated in data acquisition, and drafted and revised the manuscript. schonenberg - meinema participated in the acquisition of data and drafted and revised the manuscript. heppner is on the scientific advisory board for baxter ; holds a patent application for modulators of il-12 and/or il-23 for the prevention or treatment of alzheimer disease ; is the founding scientist of myosotis therapeutics ag ; and is a consultant for sanofi and novartis. m. de visser is on the editorial board for neuromuscular disorders and clinical neurology and neurosurgery. goebel is on the editorial advisory boards for neuromuscular disorders, muscle & nerve, neuropathology and applied neurobiology, brain pathology, and clinical neuropathology. w. stenzel is on the editorial board for neuromuscular disorders and neuropathology and applied neurobiology.	objective : to investigate the clinical and morphologic spectrum of early adult onset dermatomyositis (dm), an inflammatory disease that affects small vessels of the muscle and the skin.methods:histologic evaluation of frozen muscle samples was employed to visualize the cellular organization of ectopic lymphoid structures in muscle biopsies obtained from 2 patients diagnosed with dm. clinical presentation and morphologic features, as well as treatment and follow - up, were assessed and documented. electron microscopy was used to confirm the light microscopic diagnosis of dm. clonality analysis of b - cell populations using pcr was performed.results:muscle biopsy of both patients fulfilled the morphologic european neuromuscular centre criteria of dm. analyses of muscle biopsy samples revealed ectopic lymphoid follicle - like structures that showed a remarkable similarity to secondary lymphoid organs (slos) with distinct t- and b - cell compartmentalization. our 2 patients exhibited an atypical and mild clinical presentation and responded favorably to therapy.conclusions:the clinical and histopathologic features of dm can be atypical, and the presence of slos is not inevitably linked to an unfavorable prognosis.
subclavian artery stenosis or occlusion has a low prevalence (1.9%) and is asymptomatic in most cases. not all symptomatic patients require treatment. similar to the treatment of peripheral arterial disease in the lower limb (tasc ii), we would expect endovascular revascularisation to be the preferable treatment option in subclavian artery lesions. however, even for large centers, it is difficult to compare results of patients with subclavian artery stenosis treated in a uniform way. we present a case report of a patient with upper left limb ischemia caused by subclavian artery stenosis after repetitive left clavicula trauma, treated in different ways. in the discussion we give a brief systematic review of the open and endovascular treatment options with short- and long - term patency rates and possible complications. in this way in may 2009, a 43-year old male patient was referred to our department with ischemic symptoms (pulseless, palor and rest pain) of the left hand during rehabilitation. four months prior to referral to our department, he had presented to the emergency department with a midclavicular fracture on the left side after a fall. digital subtraction angiography (dsa) confirmed total occlusion of the left subclavian artery, starting after the origin of the left vertebral artery, with good collateral circulation but with slow arterial flow to the left arm (fig. a left carotid - subclavian bypass was performed using a six - millimeter ptfe vascular graft prosthesis (maquet cardiovascular, rastatt, germany). the postoperative course was uncomplicated and the patient was prescribed dipyridamole 150 mg twice daily and acetylsalicylic acid 500 mg once daily. two and a half years later, in january 2012, the patient presented with recurrence of the ischemic symptoms and with occlusion of the carotid subclavian bypass on dsa. subsequently, denudation of the brachial artery was performed with successful recanalisation of the left subclavian artery using an opta pro 6 mm 40 mm balloon (cordis, johnson&johnson, bridgewater, new jersey, usa) and a viabahn 7 mm 100 mm endoprosthesis (w.l. dipyridamole and acetylsalicylic acid were stopped and clopidogrel 75 mg once daily was started. nine months later, in september 2012, the patient again presented with persistent ischemic complaints in the left arm. dsa showed occlusion of the stent in the subclavian artery and the already known thrombosis of the carotid - subclavian bypass (fig. a new percutaneous transluminal angioplasty was attempted, though the procedure was neither antegradely nor retrogradely successful. three months later, a subclavian - axillary crossover bypass was performed under general anesthesia using a seven millimeter fluoropassiv armed graft (vascutec, terumo nv, tokyo, japan). the patient was discharged on the second postoperative day. at this time, only acetylsalicylic acid 80 mg once daily was given as anticoagulation therapy. subclavian stenosis with or without subclavian steal syndrome is relatively rare. only symptomatic patients present for treatment, which makes it difficult to estimate its incidence or prevalence rate. if symptomatic, symptoms may vary from upper limp ischemia (1092%) to signs of vertebrobasilar insufficiency (5184%), coronary - subclavian steal syndrome or future coronary bypass surgery (4.721%) and less common, signs of hemispheric involvement (10%). only symptomatic patients should be treated. our patients suffered ischemic complaints of the left upper limb either due to intima hyperplasia or extrinsic compression caused by repetitive trauma in this region. generally, surgical decompression (i.e. clavicula, cervical and/or first rib resection) is advised in vascular thoracic outlet syndrome. we did not perform first rib resection because of the high risk surgery in this region after repetitive fixation and infection. possible constructions of extra - anatomic bypass grafting are carotid - subclavian bypass (csb), carotid - subclavian transposition (cst) and cross over bypass (cob) (axillo - axillary or subclavian subclavian bypass). there are no large studies about these procedures (n = 3961) and these studies show good long - term primary and secondary rates ranging from 82% to 100%. studies comparing csb, sct and cob show a significant shorter operating time for sct (p < 0.001) and a significant better long - term patency for sct (100% at 10 years versus 52.2% for csb and 63.7% for cob, p < 0.005). in these procedures a trend for better patency was seen in prosthetic graft versus autologous vein graft (p < 0.2). in our patient we preferred a cross - over bypass, although a valuable alternative option was a redo autologous csb. another study comparing 20 csb with 20 cst procedures showed a patency rate of 100% for sct and 66% for csb at 6 years. in the largest study of percutaneous transluminal angioplasty (pta) procedures, published by henry. (n = 237), primary and secondary patency at ten years were 78% and 85%. the study showed remarkable results for a comparison between the primary and secondary patency rate without using a stent (67% and 76%) versus the patency rates with a stent (90% and 97%) (p < 0.01). patel. routinely performed pta procedures with stenting in patients with subclavian and innominate artery obstruction (n = 177). similar results were found in this study with a primary patency rate at five years of 84% and a secondary patency rate at four years of 98%. it is difficult to compare pta with open extra - anatomic procedures because subclavian artery stenosis requiring treatment rarely occurs. moreover, comparisons are further complicated by the heterogeneity of treatment options and material used. they compared findings of 51 patients who underwent csb for subclavian disease with those of 121 patients who underwent pta with or without stenting. a significant better primary patency rate was seen in the csb group versus the pta / stent group at 1, 3 and 5 years, 100%, 98% and 96% versus 93%, 78% and 70% respectively (p < 0.0001). although pta / stent is a minimally invasive technique, a comparable complication rate is seen in the pta / stent group (2.914.9%) versus the bypass group (5.914.2%). our patient underwent an uncomplicated postoperative course and is still doing well with a patent bypass eleven months after the last surgical treatment. subclavian artery stenosis is a rare condition causing symptoms in only a minority of patients. studies comparing extra - anatomic bypass with pta / stenting show different kinds of complications but a comparable complication rate. short - term results for these groups are also comparable. however, long - term results show a better patency rate in favor of the extra - anatomic bypass. our case is a good example of difficulties involved in choosing the best treatment option for subclavian artery stenosis. this research received no specific grant from any funding agency in the public, commercial, or not - for - profit sectors. written informed consent was obtained from the patient for publication of this case report and any accompanying figures. a copy of the written consent is available for review by the editor - in - chief of this journal on request. stijn schepers wrote the paper, revised the critical important intellectual content, collected the data, and surgeon for this work.key learning pointour case is a good example of difficulties involved in choosing the best treatment option for subclavian artery stenosis. our case is a good example of difficulties involved in choosing the best treatment option for subclavian artery stenosis. our case is a good example of difficulties involved in choosing the best treatment option for subclavian artery stenosis.	introductionstenosis of the subclavian artery is uncommon and it rarely causes symptoms. only symptomatic patients should be treated.presentation of casewe report a case of chronic left upper limb ischemia caused by subclavian artery stenosis after repetitive clavicular fixation. the stenosis was first treated with carotid - subclavian bypass and soon followed by angioplasty and stenting of the subclavian artery because of occlusion of the bypass. finally, failure of these procedures necessitated a subclavian - axillary crossover bypass.discussionboth extra - anatomic bypass and percutaneous transluminal angioplasty are safe and effective. if feasible, many authors use endovascular treatment. according to literature, extra - anatomic bypass still remains the first choice of treatment for symptomatic patients. however, the introduction of routine stent implantation is equalling these results. because of its lower long - term patency rate, endovascular treatment is favorable for patients at high risk.conclusionour case is a good example of difficulties involved in choosing the best treatment option for subclavian artery stenosis.
immunotherapies have shown promising results in cancer types previously hard to cure, such as melanoma,1, 2, 3 non - small cell lung cancer, and renal cell carcinoma. in addition to checkpoint - inhibiting antibodies, patient - derived t cells are a potent approach because they can be re - targeted against tumors ex vivo, or they can be infused to the patient without modifications, when extracted from the tumor biopsy. one advantage of using biopsy - derived polyclonal tumor - infiltrating lymphocytes (tils) is the presence of neoantigen - specific clones, til infusion requires high - dose preconditioning to eradicate suppressive immune cell subsets from the tumor microenvironment and postconditioning with high - dose systemic interleukin (il)-2, both often causing severe toxicities.6, 8 instead of systemic administration of cytokines like il-2, it could be more attractive to deliver them locally with gene therapy vectors, such as viruses.9, 10 in particular, tumor - targeted replication - competent viruses (i.e., oncolytic viruses) enable a thousand - fold amplification of transgene expression, restricted to tumor tissue. with regard to immunotherapy, oncolytic adenovirus constitutes a personalized cancer vaccine generated for each patient in situ, due to release of tumor - associated antigens. of note, virus - mediated danger signaling helps the immune system to recognize tumor cells, and immunostimulatory cytokines further boost this effect.3, 13, 14 we have shown that the most promising t cell - stimulating factors, in the context of adoptive cell therapy, are il-2 and tumor necrosis factor alpha (tnf-).15, 16, 17 regarding prior knowledge about the recombinant proteins, il-2 has been widely used in treating malignant melanoma and renal cell carcinoma and it stimulates t cell proliferation and differentiation.9, 19, 20 like il-2, tnf- can activate immune cells, but it also induces antitumor inflammation and the production of other cytokines and chemokines.22, 23 moreover, it directly causes cancer cell necrosis and apoptosis. in this study, we constructed and characterized new oncolytic adenoviruses built on a backbone of ad5/3-e2f - d24 (oad) carrying human il-2 (hil2), tnf-, or both. two modifications render virus replication tumor specific : an e2f promoter and a 24-base pair (bp) deletion in the constant region 2 of e1a, make the viruses selective for cells defective in the retinoblastoma / p16 pathway including most tumor cells.24, 25 in addition, the ad5/3 chimeric capsid featuring the ad3 knob but ad5 shaft and tail has demonstrated improved cancer cell transduction as well as antitumor efficacy. importantly, safety of this configuration in humans has also been established.13, 27, 28 based on the results in an immunocompetent syrian hamster model of pancreatic cancer, these viruses emerged as strong candidates for stimulating the immune system in tumors locally, with the specific application of enabling effective and safe til therapy. ad5/3-e2f - d24-htnfa - ires - hil2 (tilt-123) rose as the leading candidate for human translation. oncolytic adenoviruses were constructed to feature a backbone carrying serotype 5 (ad5) nucleic acid with an ad3 fiber knob. in addition, a 24-bp deletion (d24) in the rb - binding region of adenoviral e1a together with the e2f promoter was established to direct the replication to rb - deficient cancer cells. the transgenes were inserted into the e3 region, to replace some superfluous adenoviral open reading frames, which links expression to virus replication (figure 1a). all cytokine - armed adenovirus constructs were able to kill a panel of human cancer cell lines with similar efficacy as the virus without transgenes (figures 1b and s1). importantly, when virus was combined with hapt1-targeting tils, the cell - killing effect was significantly increased (figure 1c). both human and hamster cells were able to produce biologically active human il-2 as well as tnf- in vitro when infected with armed viruses (figures 2a, 2b, and s2). importantly, local production of cytokines was observed with all three armed viruses in vivo while systemic levels remained undetectable (figure 2c), highlighting the feasibility of the technology from a safety perspective. to establish an optimal virus dose, immunocompromised mice bearing orthotopic human ovarian tumors (skov3-luc) received three different doses of ad5/3-e2f - d24-htnfa - ires - hil2. the best efficacy was achieved with the highest dose of 1 10 viral particles (vps), which was significantly different compared with the untreated control group (p = 0.0085) as well as with the lowest dose of 1 10 vps (p = 0.0287) on day 18 (figure 3a). when skov3-luc tumors were treated with ad5/3-e2f - d24-htnfa - ires - hil2 and control viruses, all viruses had similar antitumor efficacy (figures 3b and 3c), suggesting that adenovirus replication rates in vivo were comparable despite the inclusion of transgenes. encouraged by the ex vivo results (figure 1c), the ability of cytokine - armed viruses to enhance til therapy was investigated in immunocompetent syrian hamsters (figure 4). the unarmed virus and tils had only moderate antitumor effects when administered alone, but a significant improvement in efficacy was observed when they were combined (p = 0.002) (figure 4a). the armed viruses had tremendous efficacy even as single agents (figures 4b4d), but the percentage of cured animals was higher in groups receiving tils and virus compared with the virus - only groups (p = 0.034 ; figure 4e). in fact, cured hamsters treated with the combination of ad5/3-e2f - d24-htnfa - ires - hil2 and til therapy comprised 100% (figure 4e). the experiment was repeated with a reduced virus dose with similar results in efficacy (figure 4f). the cured animals from the second experiment stayed tumor free for the follow - up period of 3 months. to investigate immunological effects of the armed viruses, cells from tumors, spleens, and tumor - draining lymph nodes natural killer (nk) cell marker gm1 and t cell markers cd8 and cd4 were more frequent in tumors treated with il-2, whereas the cytokine combination was the only treatment capable of increasing the level of major histocompatibility complex class ii (mhc ii) and decreasing the mac-2 expression in tumors (figures 5a5e). differences in the cell composition in spleens and lymph nodes were minor (figure s3). interestingly, splenocytes exhibited greater cell proliferation ex vivo if the animals had been treated with armed viruses (figure 5f). to estimate whether the viral treatment established tumor - specific immunity, cured hamsters were re - challenged with the same cancer cells as previously (hapt1). as a control, different types of cancer cells (ddt1-mf2) the animals that had previously been cured with cytokine - coding viruses rejected hapt1, whereas the animal treated with unarmed virus had a stable condition. the number of animals in these groups differs, because the curative potential of the unarmed virus was more limited. ddt1-mf2 tumor growth in cured hamsters was comparable to growth in naive animals, indicating the induction of tumor - specific antitumor immunity. treatment - related changes in tissue structures of the heart, lung, liver, and kidney were undetectable (supplemental materials and methods). meanwhile, spleens collected from all treatment groups showed mild and minimal lymphocyte hyperplasia, slightly expanded white pulp, and a mildly increased number of heterophils in the marginal zone or red pulp. there were no differences in the severity of the changes between any of the treatment groups including mock and other controls, suggesting a lack of systemic effects linked to the transgenes as predicted by low serum concentrations (figure 2c). immuno - oncology has made some clinical breakthroughs over the years, but, currently, a minority of patients respond and single - agent treatment modalities seldom lead to lasting remissions.1, 2, 3, 4 thus, the utility of immunotherapy has been established on a proof - of - concept level, but much work remains to help the majority of patients with currently incurable cancer. checkpoint - inhibiting antibodies have received much attention due to their ability to downregulate immunosuppression, but they can not generate new immunity. by contrast, new immune reactions can be achieved with adoptive cell therapies and oncolytic immunotherapy, thus being complementary to the former. however, adoptive cell therapy of non - melanoma solid tumors has proved clinically unimpressive results, because the tumor microenvironment is able to anergize the cell graft.16, 30 this effect can be countered with the biological phenomena resulting from adenoviral oncolysis, and it can be optimized with tnf- and il-2.16, 31 we previously studied the effects of these cytokines with recombinant cytokines and with replication - defective vectors coding for murine cytokines.15, 16, 17 here, we constructed clinically applicable oncolytic adenoviruses coding for human il-2 and tnf- and used them to boost adoptive cell transfer. the viruses were capable of infecting and lysing a variety of human and syrian hamster cancer cell lines. after intratumoral virus administration, high cytokine concentrations were achieved in target tissue, while blood levels remained undetectable. in addition, signs of toxicity were absent in histopathological evaluation of all major organs. taking into consideration the potential toxicity of high systemic cytokine levels,8, 32 the results suggest a safer approach for cytokine delivery. moreover, local delivery of il-2 could replace the need for high - dose il-2 administration often included in clinical t cell therapy protocols, although detailed studies are needed to validate the concept. to investigate the efficacy of the viruses on human cancer cells in vivo, we established an orthotopic ovarian carcinoma model in immunocompromised scid mice. despite the replication competence of the virus, the problem of virus infiltration into the tumor is well known ; thus, intratumoral administration represents a more efficient way of delivering the virus. in addition, this model develops resistance to oncolytic adenovirus, seen as a reduction in antitumor efficacy at later time points. the mechanism of resistance was previously shown to be related to the induction of interferon pathways. significant differences in the efficacy between the viruses were absent, which was expected because the cytokines have few effects in an immunocompromised model lacking a complete immune system. cytokine - armed oncolytic viruses are potential enhancers of t cells and t cell - based therapies.14, 35, 36 to investigate the synergy between our viruses and tils, we chose syrian hamsters as an immunocompetent animal model. syrian hamsters provide an interesting model for studying oncolytic adenoviruses, since human adenovirus is capable of replicating in hamsters, unlike in mice. conveniently, human il-2 and human tnf- are also bioactive in hamsters, which is evident from the hamster experiment where the unarmed virus had only moderate antitumor effects as compared with the human cytokine - coding viruses that share the same backbone construct. furthermore, we have developed a method to extract and expand tils from established hamster tumors, despite the limited availability of hamster - specific reagents. unfortunately, specific expansion of, for example, cd8 t cells is unfeasible and the til pool depends on the population present in the tumor at the time of collection. even in vitro, the synergistic effect of combining the viruses and tils was evident. in vivo, the combination of unarmed virus and tils led to improvement in efficacy. with regard to the cytokine - armed viruses, good efficacy was seen with single - agent treatment in two individual experiments. however, the combination of armed virus and til therapy resulted in the highest frequency of complete responses, as confirmed by histopathological analysis of the tumors. of note, all tumors in hamsters treated with ad5/3-e2f - d24-htnfa - ires - hil2 and tils were cured, suggesting that inclusion of adenovirally delivered immunostimulatory cytokines contributes to the curative efficacy of til therapy. immunocompetent mouse models have revealed that combining adenovirus - delivered tnf- and il-2 with adoptive t cell transfer decreases immunosuppressive characteristics of the tumor microenvironment and increases the number of active cytotoxic t cells in melanoma tumors.15, 17 moreover, mouse studies have revealed that the danger signals and immunostimulation caused by adenovirus and cytokines can result in repertoire expansion by polyclonal amplification of many classes of antitumor t cells while t cell exhaustion seems to be thwarted.15, 16, 17 mouse tumor models are useful for immunological studies because many reagents are available. however, human adenovirus does not replicate productively in mouse tissues ; therefore, the hamster model has some advantages in this regard. there are only a few hamster - specific or cross - reactive antibodies, and only limited characterization of the immune cell subsets in the tumor is possible. nevertheless, we saw decreased presence of macrophage marker mac-2 in the hamster tumors, whereas the frequency of gm1 (mostly nk) cells as well as cd4 and cd8 t cells was increased following intratumoral treatment with the cytokine - coding viruses. observed from immunocompetent mice that both cd8 and cd4 cells are essential for antitumor efficacy resulting from the combination of tnf- and il-2. il-2 induces both regulatory and helper t cells ; however, in this study, the nature of cd4 cells could not be specified. interestingly, the observed upregulation of mhc ii (a marker for antigen - presenting cells) might indicate that the combination of both cytokines enables efficient tumor recognition by cytotoxic cd4 t cells,39, 41 thus contributing to the overall efficacy. in addition, the presence of tnf- stimulates the expression of il-6, a cytokine needed for helper t cell differentiation.40, 42 taken together, although systemic il-2 may cause stimulation of regulatory t cells, which may not necessarily be true for local il-2, the possibility of the cd4 population also containing cytotoxic and helper t cells can not be excluded. in addition to immunological changes seen in the tumor microenvironment, further benefit from the cytokines was seen on the systemic level. because the spleen serves as an indicator for the common status of the immune system, we investigated the proliferative capability of the splenocytes derived from the treated animals ex vivo. interestingly, the splenocytes from the cytokine - treated animals showed increased proliferative capability compared with the controls, indicating increased adaptive cellular response. the same effect has been seen in a study with oncolytic vaccinia virus coding for granulocyte - macrophage colony - stimulating factor. moreover, adenovirally encoded cytokines evidently induced the formation of immunological memory, typically mediated by t cells. currently, because there are no anti - hamster antibodies available, the presence of memory - type t cells could not be verified. the animals treated with armed viruses resisted tumor recurrence unlike the animal treated with the virus without the cytokines, as also seen with other oncolytic viruses.10, 43, 44 in conclusion, we provide evidence that oncolytic adenoviruses coding for human il-2 and tnf- appear safe in immunocompetent hamsters. in addition to direct oncolytic effects and attractive immunological effects, these viruses seem useful for enabling successful til therapy of human solid tumors. whereas the virus itself shows potent antitumor efficacy, the cytokines are useful for induction of t cells in the tumor and for immunological memory responses. the preclinical data reported here allowed tilt biotherapeutics to initiate a human trial studying the utility of ad5/3-e2f - d24-tnfa - ires - hil2 in patients with advanced cancer receiving til therapy. all cell lines were purchased from american type culture collection (atcc) unless otherwise stated. human lung adenocarcinoma a549, human pancreatic cancer panc1, human melanoma sk - mel-28, human ovarian carcinoma ovcar-3, mouse fibroblast l929, and mouse t lymphocyte cell line ctll-2 were utilized in in vitro assays. negrin, stanford medical school), hamster leiomyosarcoma ddt1-mf2 (a kind gift from dr. william wold), and hamster pancreatic cancer hapt1 (dsmz) were used both in vitro and in vivo. all cell lines were maintained in rpmi 1640 or dmem supplemented with 10% fetal bovine serum (fbs), 2 mm l - glutamine, 100 u / ml penicillin, and 100 g / ml streptomycin (all from sigma - aldrich) and cultured at + 37c and 5% co2. the transgenes in ad5/3-e2f - d24-hil2 (oad.il2), ad5/3-e2f - d24-htnfa (oad.tnfa), and ad5/3-e2f - d24-htnfa - ires - hil2 (oad.tnfa-il2, also known as tilt-123) were placed into the e3 region and they were generated with the bacterial artificial chromosome (bac)-recombineering strategy based on the selection marker galk adapted from warming., ruzsics., and mck - husl. plasmids were propagated in electromax dh5-e competent cells (thermo fisher scientific / invitrogen) and the virus genomes were released from bacs with paci restriction enzyme (thermo scientific). the genomes were transfected into a549 cells with lipofectamine 2000 reagent (invitrogen) according to the manufacturer s instructions. vp concentration was determined with optical density 260 (od260) reading and infectious units by the tissue culture infectious dose (tcid50) assay. the functionality of the viruses was confirmed by infecting human and hamster cancer cell lines and measuring cell viability with the mts assay by adding 10% celltiter 96 aqueous one solution (promega) for the cells and reading the absorbance at 490 nm after 2 hr. a549, skov3-luc, hapt1, and ddt1-mf2 cells were infected for 72 hr. human il-2 and tnf- were detected from cell supernatants using the bd cytometric bead array human soluble protein master buffer kit together with human il-2 and tnf- flex sets (bd) according to the manufacturer s instructions. the beads were detected with a bd accuri flow cytometer and the results were analyzed with fcap array software (version 3.0.1 ; bd biosciences). biological activity of the cytokines was confirmed with il-2-dependent ctll-2 cells or tnf--sensitive l929 cells. ctll-2 cells were cultured with filtered supernatants or recombinant human il-2 (r&d systems) 10 ng / ml at + 37c and 5% co2 for 72 hr. for tnf- activity experiments, l929 cells were incubated with cell growth media supernatants or 0.5 ng / ml recombinant human tnf- (r&d systems) together with 2 g / ml actinomycin d for 24 hr. in both assays, was examined by injecting established hapt1 tumors with 1 10 vps 48 hr before collection. human il-2 and tnf- levels in serum and in homogenized tumors were quantified with a cytometric bead array and normalized to total protein content. subcutaneous hapt1 tumors established on syrian hamsters (mesocricetus auratus, hsdhan : aura ; envigo) were allowed to develop for 10 days. tumor fragments (13 mm) were cultured in g - rex10 (wilson wolf) in the presence of 3,000 iu / ml human il-2 (peprotech). half of the medium was replaced with fresh medium containing 1 g / ml concanavalin a (con a) (sigma - aldrich) after 5 days and every other day thereafter until day 10. fresh tils were employed in animal experiments or in an ex vivo killing assay, where hapt1 cells were infected with 5,000 vps per cell for 72 hr before adding 2.5 10 tils extracted from hapt1 tumors. the experimental animal committee of the university of helsinki and the provincial government of southern finland approved the animal experiments performed in this study. immunocompromised female cb-17 scid mice (janvier labs), aged 46 weeks, received 5 10 skov3-luc cells intraperitoneally. to investigate the effect of different dosing of the virus, the animals were divided to four groups (n = 3) and treated with ad5/3-e2f - d24-htnfa - ires - hil2 in concentrations of 1 10, 1 10, or 1 10 vps in 300 l pbs intraperitoneally. animals were imaged once a week with an ivis 100 imaging system (xenogen). three milligrams of d - luciferin (synchem) in 100 l pbs was administered intraperitoneally 8 minutes before bioluminescent imaging as previously described. to explore the differences in efficacy of the viruses in vivo, the mice were randomized into groups of five to seven mice and treated with 1 10 vps in 300 l pbs intraperitoneally or pbs alone once a week. subcutaneous hapt1 tumors (2 10 cells per tumor) were established in 5- to 6-week - old immunocompetent male syrian hamsters. when the average tumor diameter reached 0.5 cm, the animals were randomized into groups of six to seven. intratumoral injection of 1 10 vps in 50 l pbs or pbs alone was performed. viral treatments were repeated on days 4, 8, 13, and 19. on day 2, hamsters received intratumoral administration of either 4 10 hapt1-derived tils in 50 l plain rpmi or media only. twenty - five days after the treatments began, the animals were euthanized and tumors and selected organs were collected to evaluate the histopathological characteristics and immune cell subsets present. the experiment was repeated with reduced virus dosing (1 10 vps on days 1 and 8). cured animals were re - challenged with the same tumors (hapt1) or immunologically distinct tumors (ddt1-mf2) (4 10 cells / tumor) after a 2-week rest period. tumor growth was followed for 18 days until ddt1-mf2 tumors reached the maximum tolerated diameter (2 cm). cd8b, cd4, and mhc class ii cells were detected from hamster tumors, spleens, and lymph nodes as described by siurala. polyclonal anti - asialo - gm1 af488 antibody (ebioscience) was used for detecting natural killer cells and a subset of monocytes / macrophages (0.5 g per reaction) and anti - human / mouse galectin-3 (mac-2) pe (ebioscience) for detecting macrophages and dendritic cells (0.2 g per reaction). in addition, hamster splenocytes from each treatment group were pooled and assessed for proliferation after 72 hr of cultivation. tumor samples along with tissue samples from the hamster heart, lung, liver, spleen, and kidney were collected for pathological evaluation. samples were fixed in 10% formalin for 48 hr and stored in 70% ethanol until histological processing. sections at 4-m thickness were cut from paraffin blocks and the slides were stained with hematoxylin and eosin. differences between groups were estimated with the two - tailed student s t test, non - parametric mann - whitney test, or anova with graphpad prism software (version 6.05). ibm ssps statistics software (version 22.0.0.1) was utilized when analyzing log - transformed tumor volume data from hamster experiment using a linear mixed - effects model and for analyzing the curative effect of tils with the wilcoxon signed - rank test., a.e., and a.k. provided materials for the experiments that were performed by r.h. r.h. has been supported by the university of helsinki doctoral programme in clinical research. received grants from helsinki university central hospital (huch) research funds, the sigrid juselius foundation, biocentrum helsinki, biocenter finland, and finnish cancer organizations, and he is a jane and aatos erkko professor of oncology at the university of helsinki. in addition, a.h. is a shareholder in targovax asa and an employee and a shareholder in tilt biotherapeutics, ltd.	adoptive cell therapy holds much promise in the treatment of cancer but results in solid tumors have been modest. the notable exception is tumor - infiltrating lymphocyte (til) therapy of melanoma, but this approach only works with high - dose preconditioning chemotherapy and systemic interleukin (il)-2 postconditioning, both of which are associated with toxicities. to improve and broaden the applicability of adoptive cell transfer, we constructed oncolytic adenoviruses coding for human il-2 (hil2), tumor necrosis factor alpha (tnf-), or both. the viruses showed potent antitumor efficacy against human tumors in immunocompromised severe combined immunodeficiency (scid) mice. in immunocompetent syrian hamsters, we combined the viruses with til transfer and were able to cure 100% of the animals. cured animals were protected against tumor re - challenge, indicating a memory response. arming with il-2 and tnf- increased the frequency of both cd4 + and cd8 + tils in vivo and augmented splenocyte proliferation ex vivo, suggesting that the cytokines were important for t cell persistence and proliferation. cytokine expression was limited to tumors and treatment - related signs of systemic toxicity were absent, suggesting safety. to conclude, cytokine - armed oncolytic adenoviruses enhanced adoptive cell therapy by favorable alteration of the tumor microenvironment. a clinical trial is in progress to study the utility of ad5/3-e2f - d24-htnfa - ires - hil2 (tilt-123) in human patients with cancer.
the purpose of experiment 1 was to clarify whether a smaller balloon catheter can reduce bladder damage. the purpose of experiments 2 and 3 was to determine, if the catheter based on the results of experiment 1 can be used in a nutritional trial requiring daily urine collection. for this purpose, we investigated the effect of catheterization on cows veterinary profiles in experiment 2 and on feeding profiles in experiment 3. animals : holstein cows farmed at the tokyo metropolitan agriculture and forestry research center (ome, japan) were used for all experiments. niigata agricultural research institute livestock research center (sanjo, japan) and aichi agricultural research center (nagakute, japan) were used for experiment 3. all cows were individually housed in stalls and had free access to feed and clean, fresh water. experiment 1 : we investigated two parameters : (1) the occurrence of catheter - associated hematuria ; and (2) the macroscopic and microscopic observations of the bladder into which 3 sizes of catheters were inserted. we inserted a standard balloon catheter (nb catheter : all silicone foley catheter ; 70 ml, 24 french, straight round tip ; create medic co., ltd., yokohama, japan), a smaller balloon catheter (xsb catheter : all silicone balloon catheter ; 30 ml, 24 french ; nipro, osaka, japan) or a small balloon catheter (sb catheter : norta foley catheter ; 45 ml, 24 french ; bsn medical, hamburg, germany) into the bladders of 7 cows for each type of catheter. the catheters remained in place, and the urine samples were collected for 3 consecutive days. catheterization was based on multiple techniques [5, 10, 13, 39 ]. other specific procedures were as follows : all cows were given a caudal epidural anesthetic of 35 ml of 2% lidocaine hcl (xylocaine injection ; astrazeneca international k.k., after the catheter was inserted, the end of the catheter was connected to the drainage tube. the end of the drainage tube was connected to a urine collection bag (a 45 l vinyl bag) containing 700 ml of 20% (v / v) h2so4. the drainage tube was closely held with a band and adhesion bond at the inguinal region to prevent the weight of the drainage tube from pulling on the catheter. the urine collection bag was changed every day. in this study, if a cow showed discomfort, we removed the catheter and stopped any further the use of that particular type of catheter. catheter - associated hematuria was checked on the first day (d 1) with the day of catheterization as the urine sampling start day, and four days later (d 4), when the catheter was removed. urine samples were collected from the end of the drainage tube from the catheter that had been inserted into the bladder on the morning of d 1 and d 4. the red blood cell count per high - power field at a 400 magnification (rbc / hpf) study, catheter - associated hematuria was defined as greater than 50 rbc / hpf, because 90% of catheterized humans fall under that criterion. for the second parameter, examination of the bladder, bladders were harvested after aortic exsanguination under anesthesia induced intravenously with pentobarbital sodium (somnopentyl injection ; kyoritsu seiyaku, tokyo, japan). each bladder was incised at the median line on the anterior side between the apex of the bladder and the external opening of the urethra. the inside wall of the bladder was macroscopically examined, and sections were taken from the bladder wall for light microscopic examination as well as an examination of a cross section from the bladder wall. we examined : (1) the general condition of each cow in the first trial ; and (2) incidence of uti in the second trial. each trial preceded the nutritional trial, conducted between november 2004 and july 2007, which required the collection of urine. in each trial, catheter insertion and the urine collection period were the same as in experiment 1, and the catheter used was based on the results of experiment 1. specific catheterization procedures were as follows : before cows were milked, the end of the drainage tube was removed from the urine collection bag, which was clamped shut at approximately 20 cm from the end ; then, the end of the drainage tube was sprayed and wrapped in paper towels soaked in 70% ethanol to prevent infection, before the cows were led to the milking parlor. after milking, the interior of the urine collection bag was sprayed with 70% ethanol ; then, the drainage tube was reconnected to the bag. urine ph, body temperature, blood values and body weight were measured at the pre - catheter period, one week before catheter insertion (1 w) and during the period that the catheter was in place, between d 1 and d 4. urine samples for ph were collected from spontaneous urination by manual stimulation in the groin once at 1 w and from the drainage tube once in the morning of d 4. urinary ph was measured immediately after sampling using a ph meter (model f-22 ; horiba, kyoto, japan). body temperature was recorded with a rectal mercury thermometer twice a day (8:30 and 13:30) for 3 successive days at 1 w and one day after d 1 (d 2) to d 4 of the catheterized period. the blood samples were collected from the caudal vein once at 1 w and on d 4. the samples were taken using coded heparinized vacuum tubes (venojectii vp - h100, terumo co., ltd., the samples were analyzed immediately after sampling for red blood cell count, hematocrit, hemoglobin content and white blood cell count on a well - calibrated cell counter (model pc-608 ; erma, tokyo, japan). the total urine samples (100 ml) for urinary creatinine were collected every day for three days from the urine collection bag. urinary creatinine was determined using a commercial kit analyzer (creatinine - testwako ; wako pure chemical industries ltd., for the second trial, 13 lactating cows were catheterized a total of 22 times (6 cows were catheterized once, 5 cows were catheterized 2 times and 2 cows were catheterized 3 times). when cows were catheterized more than once, a minimum interval of five weeks was used between catheterizations. incidence of uti was checked on d 1, d 4, four days (+ d 4) and 11 days (+ d 11) after d 4 (catheter removal day). cows with a positive urinary bacterial culture result before the start of the trial were excluded. urine samples on d 1 and d 4 were collected once each day from the drainage tube. the samples on + d 4 and + d 11 were also collected using a sterile nelaton catheter (urethral catheter ; fujihira industry, tokyo, japan) once each day. if a cow developed a uti on d 4 or + d 4, the cow was treated with antibiotics every five days, and a urine sample was not collected on + d 11. one month after final antibiotic administration, urine samples were collected from treated cows and cultured to investigate the progress of the uti. these samples were collected using the same procedure as on + d 4 and + d 11. a uti was defined, if the bacterial colony count for urine samples was greater than 10 cfu / ml. experiment 3 : we explored the effect of different urine collection methods vulva urine cup (normal method) or catheter on feeding profiles. this experiment preceded the nutritional trial between february 2005 and may 2006, which required the collection of urine. this feed was adjusted so as to meet the total digestible nutrient requirements for maintenance and lactation in accordance with the japanese feeding standards for dairy cows. milking, catheter insertion, catheter type and period of urine collection with catheter or vulva urine cup were the same as in experiment 2. eighteen lactating cows were fitted with a vulva urine cup (sanshin industry, yokohama, japan), and catheters were inserted into 14 lactating cows. the following feeding profiles were measured : body weight, dry matter intake (dmi), milk yield, output of urine and feces, digestibility, urinary total nitrogen and allantoin, nitrogen balance and nitrogen rate. for urine collection with the vulva urine cup, the same volume and concentration of h2so4 was used as for the urine collection bag in experiment 2. the measuring of dmi and milk yield was the same as in the study by koga.. after feces weight was recorded daily, samples were frozen at 20c until analyzed. samples of feed, urine and feces were analyzed for total nitrogen in accordance with standard methods. wet samples were dried for 48 hr at 55c, ground and passed through a 1 mm screen. statistical significance was defined as p100cow 4nb100cow 5nb100cow 6nb100cow 7nb500cow 8sb0.05). the daily concentration of urinary creatinine was 1.15 0.16 mg / ml and was 21.4 0.7 mg / kg bw per unit of body weight (coefficient value : 3.3%). the daily excretion of urinary creatinine per unit of body weight was 21.4 0.7 mg / kg bw (coefficient value : 3.3%). incidence of uti : the incidence of uti did not differ significantly between the catheterized times (p=1.000). the incidence of uti in experiment 2 is presented in table 3table 3.incidence of uti (%) itemperiodd 1d 4+d 4+d 11(n=22)(n=22)(n=22)(n=20)uti0.09.19.10.0a : number of cows on + d 11 did not include 2 cows that developed uti on d 4 and + d 4. those cows were not examined for uti on + d 11 because they had started antibiotic care after + d 4.. two cows (9.1%) had a uti on d 4. these two cows with uti were also uti positive on + d 4. other cows without uti on d 4 did not develop uti until + d 4 and + d 11. a : number of cows on + d 11 did not include 2 cows that developed uti on d 4 and + d 4. those cows were not examined for uti on + d 11 because they had started antibiotic care after + d 4. the bacterial population density in samples from the two cows with uti was greater than 10 cfu / ml. we administered an antibiotic to these two uti - positive cows (mycillin sol meiji for veterinary use ; meiji seika kaisha ltd., the antibiotic was administered as an intramuscular injection of 20 ml per cow every day for 5 days. effect on feeding : an sb catheter was also used in experiment 3 (see discussion). the effect of different urine sampling methods vulva urine cup or catheter on feeding profiles is presented in table 4table 4.effect of urine sampling method on feeding profilesitemssampling methodp - valuevulva urine cupcatheterbody weight, kg672 16645 320.547dry matter intake, kg / d23.5 0.923.4 1.70.972dry matter intake per body weight, % 3.5 0.13.6 0.30.767milk yield, kg / d40.6 1.734.2 3.40.174urine output, kg / d12.0 0.715.0 1.40.139feces output, kg / d55.0 1.951.2 3.80.465digestible ratio of dry matter, % 66.2 1.067.9 2.00.533digestible ratio of nitrogen, % 63.1 1.364.9 2.60.627urinary nitrogen, mg / ml9.5 0.611.9 1.20.145urinary allantoin, mg / ml4.7 0.45.7 0.50.250input and output of nitrogen, g / dintake554.9 22.9540.2 46.30.817milk197.5 7.1170.8 14.40.187feces203.4 8.1188.3 16.50.505urine114.2 10.1173.2 20.40.044retention39.8 15.48.0 31.20.458input and output of nitrogen, % of nitrogen intakemilk36.3 1.431.8 2.90.255feces36.9 1.335.1 2.60.628urine20.5 1.533.0 3.10.006retention6.3 2.60.1 5.20.385. only the output of nitrogen in urine and urinary nitrogen (% of nitrogen intake) differed between sampling methods (p0.1). we believed this non - catheterization period cleared up the affects of the catheter insertions upon the cows that had been catheterized more than once. therefore, the incidence of uti was computed as if 22 cows had been catheterized. second, uti was defined strictly in terms of the urinary bacterial count in this experiment. evidence of a relationship between bacteriuria and hematuria was not obtained in this experiment, because hematuria in cows with uti (bacteriuria positive) was not monitored. we assume that a few cows without uti would have a positive hematuria test after catheterization, because of the maximum of 36 rbcs / hpf found in urine samples collected using the sb catheter on d 4 in experiment 1 (table 1). third, the incidence of uti lasted only for a period of three days in this experiment. the incidence of uti under catheterization for periods of more or less than three days is undefined. three days catheterization is an appropriate period, because almost all cow catheterization periods reported in the literature were 3 days [9, 21, 36, 38 ] ; some other cases comprised 4 days and 6 days. moreover, significant risk factors for developing uti were increased during catheterization in humans and in dogs. the infecting organisms form a biofilm on the catheter surface, and these encrustations provide a refuge for bacteria that are very resistant to antibiotics. fourth, the incidence of uti with the nb catheter was not determined. from the result of experiment 1 given the risk of uti with the nb catheter, its use should not be encouraged. it should be noted that this study is the first to show the incidence of uti in cows. for that reason, we sought to compare our data against other data, such as those for humans and dogs. the incidence in this experiment (9.1%) was subequal with studies in humans, 8.7% and 9.1% for catheterization over a period of three days. furthermore, the incidence of uti was 3.0% per catheterized day in this study, which was consistent with the rate in humans, 310%, and in dogs, 4.7% per catheterized day. because the incidence of uti in sb - catheterized cows was not higher than other data, it is apparent that handlers should not be concerned about the urinary tract infection risk in sb - catheterized cows. before concluding this section, we note that many studies on the prevention of uti have presented data for humans [8, 32, 35, 37 ] we strongly recommend maintaining the hygienic conditions set forth in the cdc guidelines and surveying animals to detect uti before and after catheterization. furthermore, the length of time for catheterizing cows should be as brief as possible in trials to avoid increasing bacterial mobility. effect on feeding : in experiment 3, the results of feeding profiles show that feeding data, without the nitrogen output in urine and the urinary nitrogen (% of nitrogen intake), were not influenced by the two types of collection methods vulva urine cup and catheter. we can offer little explanation why the nitrogen output in urine and the urinary nitrogen (% of nitrogen intake) differed between sampling methods. urinary creatinine concentration was almost the same as it is when collected using a vulva urine cup (see general conditions in the discussion). there was no difference in urine output or urinary nitrogen concentration when using either of these sampling methods. however, the nitrogen output in urine and the urinary nitrogen (% of nitrogen intake) did yield higher values when using the catheter as opposed to the vulva urine cup. these differences according to sampling method can be explained by assuming that the degree of urine collection was higher for catheters and thus the loss of urinary nitrogen was lower for catheters. we suggest that there is a significant difference when using an sb catheter in the detection of urinary data in a nutritional trial. in addition, since the daily output of urine did not differ between sampling methods, total urine collection during a 3-day catheterized period was successful without urine loss and was useful for total urine collection. conclusion : results show that a 30 ml balloon catheter is not appropriate for urine collection in cows. the procedure is more useful than a previous procedure using a 75 ml balloon catheter. the procedure has three key elements : reduced bladder damage, 9.1% infection (uti) risk per three continuously catheterized days and problem - free total urine collection for nutritional trials. in the future, accurate total urinary data related to nutrition and feeding can be obtained and considered using this modified procedure which presents a starting point for a new line of research.	abstractthis study proposed a modified procedure, using a small balloon catheter (sb catheter, 45 ml), for reducing bladder damage in cows. holstein cows and the following catheters were prepared : smaller balloon catheter (xsb catheter ; 30 ml), sb catheter and standard balloon catheter (nb catheter ; 70 ml, as the commonly used, standard size). in experiment 1, each cow was catheterized. the occurrence of catheter - associated hematuria (greater than 50 rbc / hpf) was lower in the sb catheter group (0.0%, n=7) than in the nb catheter group (71.4%, n=7 ; p<0.05). in experiment 2, general veterinary parameters, urine ph, body temperature and blood values in cows were not affected before or after insertion of sb catheters (n=6). the incidence of urinary tract infection (uti) was 3.0% per catheterized day (n=22). in experiment 3, feeding profiles, daily excretion of urinary nitrogen (p<0.05) and rate from nitrogen intake in urine (p<0.01), were higher with use of the sb catheter (n=13) than with the use of the vulva urine cup (n=18), indicating that using the sb catheter can provide accurate nutritional data. from this study, we concluded that when using an sb catheter, the following results occur ; reduction in bladder damage without any veterinary risks and accuracy in regard to feeding parameters, suggesting this modified procedure using an sb catheter is a useful means of daily urine collection.
diabetes mellitus is a heterogeneous group of metabolic diseases that is characterised by hyperglycaemia. it can result in blindness, kidney and heart disease, stroke, loss of limbs, and reduced life expectancy if left untreated and is recognized as a primary threat to human health in the 21st century. type 2 diabetes mellitus is the most common form of diabetes and accounts for approximately 90% of cases ; its development is controlled by interactions between multiple genetic and environmental factors [24 ]. the genetics and pathophysiology of type 2 diabetes remain poorly understood because detailed investigations, such as genetic dissection, have been restricted in humans for practical and ethical reasons. animal models of type 2 diabetes mellitus have provided important information, and many rat strains of spontaneous diabetes have been reported [511 ]. among these strains, goto - kakizaki (gk), otsuka long - evans tokushima fatty (oletf), and spontaneously diabetic torii (sdt) rats have been widely used for elucidating the genes responsible for the development of diabetes, the physiological course of the disease, and the complications related to diabetes [1214 ]. the different diabetes model strains exhibit different aspects of the disease, and thus additional animal models are needed to elucidate the complete pathogenesis of diabetes. we have developed a novel, nonobese rat strain with spontaneous diabetes, long - evans agouti (lea) rat, which was established from a long - evans closed colony together with the long - evans cinnamon (lec) rat. they do not exhibit any signs of obesity throughout their lives but experience late onset of the disease and exhibit histological changes (macrophage infiltration and fibrosis) of the pancreatic islets. thus, lea rats may serve as a new model of nonobese type 2 diabetes mellitus caused by impairing insulin secretion. in the present study, we examined the pathophysiological characteristics of the strain and demonstrated that the lea rat is a useful model of human nonobese diabetes. lea rats, also known as lea / sendai or sendai rats, were maintained at the institute for animal experimentation, tohoku university graduate school of medicine, japan. wistar rats were purchased from japan slc (hamamatsu, japan) and were used as controls. all rats were housed in air - conditioned animal rooms at an ambient temperature of 23 3c and relative humidity of 50 10%, under specific pathogen - free conditions with a 12 h light / dark cycle. food, consisting of a labo mr standard diet (nosan, yokohama, japan), and water were available ad libitum. all animal care procedures were approved by the animal care and use committee of tohoku university graduate school of medicine, and complied with the procedures of the guide for the care and use of laboratory animals of tohoku university. urinary glucose and protein were monitored at 1-month intervals using uro - paper ag2 (eiken, tokyo, japan). the detection limits of the uro - paper were 1020 mg / dl for urine protein and 4060 mg / dl for urine glucose. the body weight (bw) and body length (bl), that is, the distance from the nose to the anus, of five male rats (6 months old) were measured. the body mass index (bmi) after 16 h fast, a dose of 2 g / kg bw of glucose was given orally, and blood samples were collected from the tail vein at 0, 30, 60, 90, and 120 min after loading. the blood glucose levels were measured with a glutest eii blood glucose monitoring meter with a monitoring range of 40500 mg / dl (sanwa kagaku, nagoya, japan). the rats were classified according to the three - grade system of diabetes mellitus (dm), impaired glucose tolerance (igt), and normal. dm was defined as 120 min blood glucose levels of 200 mg / dl. igt was defined as 120 min blood glucose levels between 140 and 199 mg / dl. the plasma insulin concentration was determined as immunoreactive insulin (iri) by elisa with a rat insulin assay kit (morinaga milk industry, yokohama, japan) using separated plasma from the blood collected from the tail vein at 0, 30, 60, 90, and 120 min after glucose loading. to study early - phase insulin secretion, an ogtt was performed on male rats (2 and 12 months of age) as described previously, and blood glucose levels (bg) and insulin concentrations (iri) at 0 and 30 min after glucose loading were measured. the insulinogenic index (i.i.) = iri/bg, where iri and bg are the differences between their respective values at 0 and 30 min. the animals were intraperitoneally challenged with a dose of 0.75 u / kg bw of human insulin (novolin r ; novo nordisk, denmark). blood samples were drawn from the tail vein at different time points, and glucose levels were determined as described previously. the tissues from rats were fixed overnight at 4c in phosphate - buffered saline (pbs) that contained 4% paraformaldehyde. they were rinsed with pbs, dehydrated, embedded in paraffin, cut into 5-m - thick sections, and stained with haematoxylin and eosin (h&e). for insulin detection, the pancreatic sections were processed for immunostaining by an indirect method using guinea pig anti - insulin polyclonal antibody (1 : 100, daco, carpinteria, ca) as the primary antibody and peroxidase - labelled goat anti - guinea pig igg antibody (1 : 200, chemicon international, temecula, ca) as the secondary antibody. the specific reactions were visualised with a dab substrate kit (vector, burlingame, ca). to distinguish the inflammatory cells infiltrated in the islets, additional deparaffinised sections of pancreas were processed for immunostaining using mouse monoclonal antibodies against rat cd4 clone w3/25 (mca 55r ; abd serotec, oxford, uk), cd8 clone ox-8 (mca48r ; abd serotec), cd45ra clone ox-33 (mca340 g, abd serotec), and macrophage antigen clone ed1 (mca341, abd serotec). specific reactions were visualised with a sab - po kit (nichirei, tokyo, japan). the volume of -cells relative to the pancreas volume was calculated as the proportion of the total area of -cells to the total area of pancreatic tissue, according to the method of bouwens. three serial paraffin sections (5-m - thick) of pancreatic tissue from three animals of each strain were obtained at intervals of 100 m. the sections were immunostained with guinea pig anti - insulin antibodies (1 : 100) and analysed under an olympus bx51 microscope (olympus, tokyo, japan) connected to a computer running the winroof software (mitani corp., tokyo, japan). the image analysis quantified the total pancreatic tissue area and the insulin - positive area, permitting the calculation of the ratio of islet -cell area to total pancreatic area. to ensure that any change in the relative -cell volume was not attributable to a change in the size of individual -cells, the density of nuclei per insulin - positive area was also measured in 20 islets of the five rats of each strain. the incidence of diabetes in lea rats as determined by ogtt is shown in figure 1(a). diabetes mellitus was observed only in male rats, and its incidence increased with age : 10%, 61%, and 86% at 6, 12, and 14 months of age, respectively. the onset of diabetes was not observed in females, although 33% of the females showed only igt at 12 months of age. as the onset of diabetes differed according to sex glucosuria appeared at 5 months of age, before the onset of diabetes in male rats, and was present in 100% of the males at 8 months of age. in female rats, glucosuria appeared at 7 months of age and was present in 100% of the females at 9 months (table 1). proteinuria appeared at 6 months of age, concomitant with the onset of diabetes, in male rats and was present in 57% of the males at 9 months of age, whereas proteinuria appeared in 20% of female rats at 9 months of age and did not exceed 30% of the females thereafter. the average bws of male and female lea rat increased gradually throughout the experimental period and were 506 37.8 g (n = 5) and 312 27.7 g (n = 5) at the 12 months of age, respectively (figure 1(b)). a significant decrease in bw the bmi of the lea rats at 6 months of age (0.57 0.02 g / cm, n = 5) was not significantly different from that of the control wistar rats (0.59 0.02 g / cm, n = 5), confirming that the lea rats were nonobese. the survival rate of lea rats was examined(supplementary figure 1 available online at http://dx.doi.org/10.1155/2013/986462). we found that 95% of the male rats survived to 12 months of age, and 50% survived to 22 months of age. the survival rate of male lea rats was not significantly different from that of normal control wistar rats, which indicates that diabetes does not influence the survival of lea rats. the results of the ogtt in male rats at different ages are shown in figure 2. two - month - old male lea rats showed impaired glucose tolerance compared with age - matched male wistar rats (figure 2(a)). at 12 and 14 months of age, the lea rats presented with typical diabetic glucose levels of 200 mg / dl at 120 min after glucose loading (figures 2(c) and 2(d)). the wistar rats did not show any change in blood glucose level in relation to age. the plasma insulin concentrations in male rats at 2 months of age showed that the pre - ogtt values did not differ among the rats, whereas the values at 30 min after glucose loading were significantly lower in lea rats (figure 2(e)). the plasma insulin level was significantly lower in lea rats at 12 months of age after glucose loading (figure 2(f)). these results indicate that lea rats have impairment of insulin secretion in response to glucose stimulation. the low insulin levels measured at 30 min after glucose loading suggest that lea rats have a decreased ability to secrete insulin at an early phase of the disease (figures 2(e) and 2(f)). the insulinogenic index (i.i.) of male lea rats was significantly lower at both 2 and 12 months of age compared with that of wistar rats (table 2), which indicates that early - phase insulin secretion is significantly impaired at an early age. we performed an insulin tolerance test on nonfasting, 12-month - old male rats by intraperitoneal injection of insulin, to examine insulin response (figure 3). before insulin injection, the blood glucose levels were significantly higher in lea rats than in wistar rats. after insulin injection, the blood glucose levels in lea rats significantly decreased by maximum 51.1% over 120 min to the same levels observed in the wistar rats. these results indicate that lea rats have a normal glycaemic response to exogenous insulin and are not insulin - resistant. the age - dependent histological changes in the pancreas were examined (figure 4). male lea rats at 2 months of age had inflammatory reaction in fraction of the pancreatic islets (figure 4(a)), although the most of islets were intact. immunostaining for insulin revealed that insulin - positive cells were irregularly distributed within inflammatory foci (figure 4(b)). the inflamed islets were infiltrated by cells positive for anti - rat macrophage antibody (figure 4(c)) but not for antibodies against cd4 +, cd8 +, or cd45ra (data not shown). however, inflammatory reactions disappeared by 6 months of age, and the islets had been replaced by fibrotic remnants in rats over 12 months of age (figure 4(d)). the number of -cells was reduced, although -cells were present at 12 months of age (figure 4(e)). a few tiny islets without fibrosis, which appeared to be regenerative islets, were intermingled in the affected pancreas in rats from 12 months of age (figures 4(f) and 4(g)). the pancreatic islets from female lea rats had no pathological changes, including fibrosis and inflammatory reaction. the volume of -cells relative to the gross volume of the pancreas was determined using the winroof software to analyse sections that were immunostained for insulin (figure 4(h)). in male lea rats, the volume of -cells significantly decreased from 0.74 0.23% at 2 months to 0.52 0.08% at 6 months. the control wistar rat strain showed a rising, albeit not statistically significant, trend from 0.79 0.05% at 2 months to 0.96 0.24% at 6 months. these results reveal that the significant decrease in the insulin - positive area with age appears in lea rats owing to severe fibrosis of the islets. to identify whether the diminished -cell mass in lea rats was caused by a reduction in cell number or atrophy of the cells, we counted the number of nuclei in the insulin - positive areas of 20 islets in five of each of 6-month - old lea and wistar rats. the number of nuclei did not differ significantly between the two strains (117.97 4.13 m / nuclei versus 128.82 13.35 m / nuclei for lea versus wistar rats, resp.). these results indicate that a reduction in the cell number (not atrophy of -cells) is responsible for the reduction of -cell volume in lea rats. in lea rats, glucosuria and proteinuria were present in 100% at 8 months of age and in 66% of males at 12 months of age, respectively. the histopathological analysis was performed to examine the renal lesions in 12-month - old male lea rats (figure 5). the large dilatation of tubular lumen was present, mostly in superficial cortex regions (figure 5(a)). atrophy of tubular epithelium and flattend / detached renal tubules were also observed (figure 5(b)). the intracytoplasmic hyaline droplet accumulation and the disappearance of tubular epithelial cell layer associated with thickening of basement membrane were evident in proximal tubules (figure 5(c)). there were no obvious pathological changes in the glomeruli at 12 months of age (figure 5(d)). two inbred strains, long - evans agouti (lea) and long - evans cinnamon (lec), which were selected for coat color, were established from a closed colony of long - evans rats at the center for experimental plants and animals, hokkaido university (japan). the lea rat has been known as the control strain for the lec rat, an animal model of wilson disease. however, the large amount of urine and the strong odor of urine, a possible indicator of diabetes, are often observed in lea rats during long - term breeding. in 1996, we found three male rats that were positive for glucosuria and hyperglycaemia among littermates from an inbred colony of lea / hkm. the lea rats exhibit several distinctive diabetes - related characteristics : (1) onset of diabetes is observed only in male rats, not in female, at over 6 months of age ; (2) the early - phase insulin secretion is impaired at 2 months of age ; (3) the progressive fibrosis in islet in an age - dependent manner ; (4) a normal glycaemic response to exogenous insulin ; (5) nonobese. from these results, we conclude that the lea rat is a new rat model for nonobese type 2 diabetes mellitus. several rat model strains of spontaneous type 2 diabetes mellitus have been identified to date, and they are classified into two types, obesity and nonobesity models. the obesity models of type 2 diabetes, such as sand, wistar fatty, and oletf rats, are characterised by hyperglycaemia, hyperinsulinemia, and insulin resistance. in contrast, the nonobesity models, such as gk, wbn / kob, and spontaneously diabetic torii (sdt) rats [11, 19 ], are characterised by hyperglycaemia, hypoinsulinemia, and noninsulin resistance. we classify the lea rat as a nonobesity model because bmi of lea rats is not different from that of control rats. however, there are several differences between the gk and lea rats. in gk rats, there is no sex difference with respect to the occurrence of diabetes and no age - dependent deterioration of impaired glucose tolerance ; in addition, hyperglycaemia occurs 8 days after birth [8, 20 ]. the sdt rat is a new model of nonobese, severe type 2 diabetes mellitus with hyperglycaemia, hemorrhage in and around the islets, and hyposecretion of insulin (hypoinsulinemia) resulting from a significantly decreased number and size of islets. although lea rats displayed no hemorrhage in and around the islets, macrophage infiltration was present around the islets, leading to the progressive fibrosis of islet (figure 4). the glucose intolerance in correspondence with the impairment of the insulin secretion was observed in male lea (figure 2 and table 2), suggesting that the main cause of diabetes in lea rats is hypoinsulinemia attributable to a decreased number of -cells in the islets (figure 4). it is also likely that significant decreased capability of early - phase insulin secretion is caused by the hypofunction of -cells. although there was inflammatory reaction in fraction of the pancreatic islet in male lea rats at 2 months of age, the volume of -cells in lea rats was comparable to that of control rats (figure 4(h)), suggesting that lea rats have congenital defect of insulin secretion in addition to the progressive reduction of -cell mass in age - dependent manner. the ability to secrete insulin in the early phase reflects the first phase of insulin secretion from pancreatic -cells and contributes to the suppression of gluconeogenesis in the liver. therefore, we suggest that the lea rat is unable to suppress the increasing blood glucose concentration that occurs after feeding because of a decreased ability to secrete insulin in the early phase, which eventually leads to the deterioration of -cell function by glucose toxicity and causes the rats to experience chronic hyperglycaemia. the reduction in the number of -cells is thought to be the main cause of type 2 diabetes in lea rats, and this is supported by previous observations in human type 2 diabetes patients and rat models of type 2 diabetes mellitus [2226 ]. both congenital and acquired factors are involved in the mechanism of -cell reduction. in regard to congenital factors, mutations in transcription factor genes, such as insulin promoter factor-1 (ipf-1) and hepatocyte nuclear factor-1 (hnf-1), have been verified. have revealed that apoptosis reduces -cells in human diabetes and that it progresses by amyloid deposition and hyperglycaemia. zhu. have reported that the impairment of -cell proliferation causes a decrease in -cells under hyperglycaemic conditions in oletf rats, and movassat. free - fatty - acid- (ffa-) induced -cell apoptosis has been proposed by shimabukuro. as the underlying cause in zucker diabetic fatty (zdf) rats. although it is speculated that the impairment of -cell proliferation, progression of apoptosis by hyperglycaemia, and impairment by cytokines produced by macrophages cause the reduction in -cells in lea rats, further analyses are required to clarify the pathogenesis of diabetes in lea rats. the abnormality of the renal tubules at 12 months of age was observed in the lea rats with glycosuria and proteinuria (figure 5 and table 1). onset of diabetes as determined by ogtt was observed in only male lea rats (figure 1(a)), and its incidence increased with age : 10% and 61% at 6 and 12 months of age, respectively. however, glucosuria appeared at 5 months of age before the onset of diabetes in male rats, and was present in 100% of the females at 9 months (table 1), which did not develop diabetes (figure 1(a)). based on these findings, it is unlikely that the onset of diabetes and impairment of glucose intolerance are not associated with glucosuria and proteinuria. although the lea rat is used as the control strain of the lec rat since they do not harbor atp7b mutation, the several phenotypes such as hypersensitivity to x - rays and the lack of d - amino acid oxidase (dao) activity, which is involved in the degradation of d - serine, a key coagonist for n - methyl - d - aspartate (nmda) receptor, have been reported. we are now performing quantitative trait locus (qtl) analyses for impaired glucose tolerance and urinary glucose to lead us to identification of genes for glucose intolerance, renal glucose excretion, and the development of diabetes in lea rat. in conclusion, the lea rat has distinctive characteristics that are different from the previously described model rats. the lea rats develop late onset diabetes in correspondence with the impairment of the insulin secretion, which is caused by progressive fibrosis in pancreatic islets in age - dependent manner. in japan, the prevalence of type 2 diabetes mellitus is increasing rapidly, and more than 10% of individuals over 40 years of age are affected. relatively few diabetic individuals in japan are obese, and impairment of insulin secretion often develops before onset of diabetes. the unique characteristics of lea rat are a great advantage to analyze the progression of diabetes mellitus with age. the additional studies are expected to disclose the genes involved in type 2 diabetes mellitus.	animal models have provided important information for the genetics and pathophysiology of diabetes. here we have established a novel, nonobese rat strain with spontaneous diabetes, long - evans agouti (lea) rat derived from long - evans (le) strain. the incidence of diabetes in the males was 10% at 6 months of age and 86% at 14 months, while none of the females developed diabetes. the blood glucose level in lea male rats was between 200 and 300 mg / dl at 120 min according to ogtt. the glucose intolerance in correspondence with the impairment of insulin secretion was observed in male rats, which was the main cause of diabetes in lea rats. histological examination revealed that the reduction of -cell mass was caused by progressive fibrosis in pancreatic islets in age - dependent manner. the intracytoplasmic hyaline droplet accumulation and the disappearance of tubular epithelial cell layer associated with thickening of basement membrane were evident in renal proximal tubules. the body mass index and glycaemic response to exogenous insulin were comparable to those of control rats. the unique characteristics of lea rat are a great advantage not only to analyze the progression of diabetes, but also to disclose the genes involved in type 2 diabetes mellitus.
carcinoid tumors represent a group of well - differentiated tumors originating from the diffuse endocrine system outside the pancreas and thyroid. the overall prevalence of carcinoid tumors in the united states is estimated to be one to two cases per 100,000 persons. various sites of origin of this neoplasm are appendix - 30 - 45%, small bowel - 25 - 35% (duodenum 2%, jejunum 7%, ileum 91%, multiple sites 15 - 35%), rectum 10 - 15%, caecum - 5%, and stomach - 0.5%. here we report a case of primary jejunal carcinoid tumor in a 66-year - old woman metastasizing to liver with ultrasonography, computed tomography, and diffusion - weighted magnetic resonance imaging (dwi) findings. dwi can help in the differential diagnosis of hepatic hypervascular metastatic mass lesions from benign ones, as well as in the diagnosis of carcinoid tumor. carcinoid tumors represent a group of well - differentiated tumors originating from the diffuse endocrine system outside the pancreas and thyroid. the overall prevalence of carcinoid tumors in the united states is estimated to be one to two cases per 100,000 persons. carcinoids most frequently occur in the gastrointestinal tract (66.9%), followed by the tracheobronchial system (24.5%). in rare cases, they may arise in the liver, gallbladder, ovary, testis, and thymus. at the time of diagnosis, 58%-64% of patients with small intestinal carcinoids have disease that has spread beyond the intestine to regional lymph nodes or the liver. here we report a rare case of jejunal carcinoid tumor metastasizing to liver with ultrasonography (us), computed tomography (ct), and diffusion - weighted magnetic resonance imaging (dwi) findings. physical examination revealed hepatomegaly and a mass alining from right upper quadrant to epigastrium. on laboratory examinations blood analysis revealed deep anemia (hemoglobin : 3.6 ; red blood cell : 2.34), and blood transfusion was induced to the patient. on abdominal us, hepatic echogenous mass lesions the biggest being 14x10 cm in size having cystic components were demonstrated. also a 54 cm hypoechoic mass lesion neighboring to pancreatic tail protruding into the lumen from the jejunal wall was noted, which contained calcifications. abdominal ct revealed heterogeneously enhancing hepatic mass lesions having central necrotic areas and a lobulated contour (fig. 1a, b), and a heterogenously enhancing jejunal mass lesion having calcified areas (fig. pre (a) and post contrast (b) ct images reveal peripherally enhancing liver masses (stars) with central necrotic areas. abdominal contrast enhanced ct images reveal heterogeneously enhancing proximal jejunal mass (carcinoid tumor, arrows) with central calcifications. on the other hand, dwi was performed for the differential diagnosis of hepatic masses from cavernous hemangiomas, which revealed restriction in diffusion and an apparent diffusion coefficient (adc) value of 1,3310 which was compatible with the adc values of hepatic metastatic lesions (fig. us - guided tru - cut biopsy of the biggest hepatic mass lesion was evaluated as carcinoid tumor metastasis histopathologically. the metastatic liver lesions (stars) and the primary jejunal carcinoid tumor (arrow) show restriction (hyperintense signal) on diffusion - weighted magnetic resonance imaging. it accounts for more than 95% of all carcinoids and 1.5% of all gastrointestinal tumors. the tumor arises from the endochromaffin cells of kulchitsky i.e. neural crest cells situated at the base of crypts of lieberkuhn. various sites of origin of this neoplasm are appendix - 30 - 45%, small bowel - 25 - 35% (duodenum 2%, jejunum 7%, ileum 91%, multiple sites 15 - 35%), rectum 10 - 15%, caecum - 5%, and stomach - 0.5%. most carcinoids occur in patients older than 50 years, however, appendiceal carcinoids occur in young patients in their second to fourth decade. most patients are asymptomatic but symptoms can vary from pain, intestinal obstruction (19%), weight loss (16%), palpable mass (14%), intussusceptions, perforation, or gastrointestinal hemorrhage (rare). carcinoids of the jejunum and ileum occur equally in men and women at a mean age of 65,4 years[14 ]. our patient was 66 years old, compatible with the literature, and presented with symptoms of gastrointestinal bleeding that is quite rare in case of carcinoid tumor. the majority of jejunal and ileal carcinoids are argentaffin - positive, serotonin - producing, and substance p containing ec - cell tumors that produce carcinoid syndrome when liver or retroperitoneal nodal metastases are present. similar to all carcinoids, those that are primary to the jejunum and ileum vary in their biologic behavior and ability to metastasize. in general, ec - cell carcinoids of the small intestine behave in a malignant fashion, producing lymph node and liver metastases. the primary tumor is small however it frequently evolves a fibrotic reaction with foreshortening and thickening of the mesentery. the tumor also elaborates serotonin and other histamine - like substances than can cause carcinoid syndrome which is characterized by abdominal cramps, diarrhea, hepatomegaly, flushing etc. carcinoids also have the tendency to metastasize to lymph nodes, liver and rarely bone. the diagnosis is made when the tumor has spread through the bowel wall into the peritoneum and mesentery and is achieved by several complementary imaging techniques. plain abdominal scenogram is the initial screening modality and it may show curvilinear calcification. however, it is highly non - specific. barium studies (enteroclysis, follow through examination) may show intramural or intraluminal filling defects in the distended ileum. there can also be narrowing of the lumen with stricture formation, thickening of the valvulae conniventes and increase in the interbowel loop distance due to wall thickening. further, tumor spread to the root of mesentery may encase the vessels and produce infarction. sonography of the bowel can depict pseudokidney sign due to wall thickening, associated lymphadenopathy and liver metastasis. however, the findings are non - specific to offer a confident diagnosis. diagnosis can be made fairly confidently on ct, which is used for demonstrating the intestine, mesentery, lymph nodes and liver in a single examination and for staging the neoplasm as well as follow up after surgery or chemotherapy. it reveals a soft density mass with speculated borders and radiating strands with or without calcification in 80% of cases[58 ]. in our patient, us revealed wall thickening and calcification in the proximal jejunum, as well as liver masses, and ct demonstrated jejunal and hepatic masses obviously. other findings on ct can be lymphadenopathy (retroperitoneal, retrocrural) and metastasis commonly to the liver. they also depend on size of the primary tumor : tumors smaller than one cm metastasize in two percent of cases, tumors 1 - 2 cm metastasize in 50% and > 2 cm are clinically silent but they may produce carcinoid syndrome. on ct, hepatic metastases are hypervascular enhancing lesions and may assume variety of patterns such as finely nodular, coarsely nodular, mixed single large mass and rarely, pseudocystic appearance probably due to ischemic necrosis. liver metastases from jejunal and ileal carcinoids are generally hypervascular and as such are generally best seen during the arterial phase of intravenous contrast material administration on ct and mr images. centrally located tumor necrosis and degeneration may produce central nonenhancing regions within the metastases on ct and mr images, producing a rimlike pattern of enhancement. the hepatic masses in our patient were also hypervascular, with cystic changes in the tumor located on the left hepatic lobe. for the differential diagnosis of hepatic lesions, we considered hemangioma and performed dwi with adc mapping. the adc value obtained from the hepatic mass was 1.33 10 - 3, supporting metastasis. in a study, the mean adc of hemangioma (7.58 l0 - 3 mm2/sec 1.88) was the highest, followed by hcc (3.15 l0 - 3 mm2/sec 1.80) and metastasis (2.55 l0 - 3 mm2/sec 1.65). we also performed us - guided tru - cut biopsy to the mass on the right hepatic lobe, which was evaluated as carcinoid tumor metastasis histopathologically. bone metastases are rare and most osseous metastases have been reported from carcinoid of the stomach and rectum. carcinoid tumors may also be associated with a high incidence of other primary malignant neoplasms e.g. in the colon, breast, pancreas, kidney, lip, vulva, cervix, larynx, rectosigmoid and histiocytic lymphoma. the imaging appearance of jejunal and ileal carcinoids varies by tumor size, extent of mesenteric involvement, and presence or absence of lymph node or liver metastases. small, polypoid and nodular carcinoids located in the mucosa and submucosa of the intestinal wall are best evaluated with enteroclysis or small bowel series. in many cases, small nodular carcinoids when identified, they are characteristically solitary or multifocal, smooth, rounded nodules or mucosal elevations in the distal ileum. similarly, small primary tumors may be difficult to visualize with magnetic resonance (mr) imaging. they are best visualized on gadolinium enhanced t1-weighted mr images obtained with fat suppression, where they manifest as nodules or focal areas of mural thickening with moderately intense gadolinium enhancement. the primary tumor may also manifest on ct scans as asymmetric or concentric mural thickening. mural thickening is secondary to infiltrating tumor and desmoplastic submucosal fibrosis, which thickens and stiffens the intestinal wall, producing mural and irregular fold thickening. in our patient, the jejunal mass was observed as irregular wall thickening. at the time of diagnosis, 58%64% of patients with small intestinal carcinoids have disease that has spread beyond the intestine to regional lymph nodes or the liver., hepatic metastases were accompanying the primary jejunal carcinoid tumor at the time of diagnosis. the imaging differential diagnosis for small intestinal carcinoids includes metastatic disease, primary small intestinal adenocarcinomas, lymphoma, and gastrointestinal stromal tumors. the characteristic curvature of the small intestine and spiculated margins of the mesenteric nodal metastases strongly suggest carcinoid. in cases without the characteristic fibrotic changes, somatostatin receptor scintigraphy or biopsy will help in establishing the diagnosis. nonneoplastic diseases such as crohn 's disease and localized ischemic enteritis may produce inflammatory changes in the distal ileum and mesentery that simulate carcinoid[111418 ]. dwi and adc values can help in the differential diagnosis of hepatic hypervascular metastatic mass lesions from benign ones, as well as in the diagnosis of carcinoid tumor.	context : carcinoid tumors represent a group of well - differentiated tumors originating from the diffuse endocrine system outside the pancreas and thyroid. the overall prevalence of carcinoid tumors in the united states is estimated to be one to two cases per 100,000 persons. various sites of origin of this neoplasm are appendix - 30 - 45%, small bowel - 25 - 35% (duodenum 2%, jejunum 7%, ileum 91%, multiple sites 15 - 35%), rectum 10 - 15%, caecum - 5%, and stomach - 0.5%. liver metastases from jejunal and ileal carcinoids are generally hypervascular.case report : here we report a case of primary jejunal carcinoid tumor in a 66-year - old woman metastasizing to liver with ultrasonography, computed tomography, and diffusion - weighted magnetic resonance imaging (dwi) findings.conclusion:primary jejunal carcinoid tumor is a rare entity. dwi can help in the differential diagnosis of hepatic hypervascular metastatic mass lesions from benign ones, as well as in the diagnosis of carcinoid tumor.
subtilisin solutions were prepared by mixing and vortexing of 1.25, 5, 8, 12.5, 15, 17.5, 20, and 25 l of subtilisin stock solution (sigma aldrich, catalogue number p4860 ; lot 056k1213) per ml of 100 mm phosphate buffer (ph = 8, filtered with a 0.25 m anotop filter), which yielded enzyme solutions at concentrations of 0.05, 0.2, 0.3, 0.5, 0.6, 0.7, 0.8, and 1 mg ml. these concentrations correspond to 1.5, 6, 9.6, 15, 18, 24, and 30 u ml, expressed in activity units used in our previous report. saxs measurements were carried out at beamline 7t - mpw - saxs at bessy ii synchrotron source in berlin, germany, using an x - ray energy of 9650 ev. enzyme solutions at various concentrations as well as pure buffer (as background) were injected in glass capillaries and placed in a capillary holder. the capillary holder was then mounted on the sample cell chamber, evacuated, and heated to 25 or 55 c. the individual samples were then exposed to the x - ray beam to record scattering intensity patterns by using a gas detector placed in the vacuum chamber. the integration and processing of the scattering data was carried out by using the software provided at the beamline. static and dynamic light scattering measurements were carried out using the 3ddls instrument (ls instruments, fribourg, switzerland) using vertically polarized he ne laser light (25 mw with a wavelength of 632.8 nm) with an avalanche photodiode detector at angles between 15 and 135 at 55 c. the background scattering intensities (from pure buffer) were subtracted from the scattering intensities of the enzyme solutions. at each scattering angle, dynamic light scattering measurements were also performed by recording the intensity autocorrelation function g2() as a function of lag time. the autocorrelation functions were analyzed by means of the cumulant method in order to determine the initial decay rate and the corresponding average apparent diffusion coefficient dapp. the decay of the normalized autocorrelation function was modeled as g1() = (g2() 1) = exp(), where = dappq is the initial decay rate, q = (4n/) sin(/2) is the scattering vector magnitude, n is the refractive index of the solvent, and is the wavelength of the laser. einstein equation, rh = kbt/6dapp, where kb is the boltzmann constant, t is the absolute temperature, and is the solvent viscosity at the given temperature. the scattering intensity patterns from static light and x - ray scattering experiments can be described as i(q) kp(q)s(q), where k is an instrument- and sample - dependent constant, p(q) is the form factor, which depends on the size and shape of the primary particles, and s(q) is the structure factor giving information about the spatial arrangement of the primary particles at length scales larger than that of the primary particles (radius of rp). please note that q = (4/) sin(/2) for saxs measurements. in the limit of qrg < 1, the mean radius of gyration rg of randomly distributed (e.g., freely diffusing) primary particles or clusters can be determined from the measured scattered intensity i(q) by using the guinier analysis. in the limit of 1/rg q 1/rp, where rg is the mean radius of gyration of a sufficiently large cluster composed of primary particles with radius rp, the structure factor for fractal clusters with fractal dimension df scales with q through a power law relation as i(q) s(q) q.	the structural characterization of subtilisin mesoscale clusters, which were previously shown to induce supramolecular order in biocatalytic self - assembly of fmoc dipeptides, was carried out by synchrotron small - angle x - ray, dynamic, and static light scattering measurements. subtilisin molecules self - assemble to form supramolecular structures in phosphate buffer solutions. structural arrangement of subtilisin clusters at 55 c was found to vary systematically with increasing enzyme concentration. static light scattering measurements showed the cluster structure to be consistent with a fractal - like arrangement, with fractal dimension varying from 1.8 to 2.6 with increasing concentration for low to moderate enzyme concentrations. this was followed by a structural transition around the enzyme concentration of 0.5 mg ml1 to more compact structures with significantly slower relaxation dynamics, as evidenced by dynamic light scattering measurements. these concentration - dependent supramolecular enzyme clusters provide tunable templates for biocatalytic self - assembly.
porous ni materials, such as plaques and foams, are widely used in industry for the fabrication of electrodes for rechargeable batteries. nanostructured active materials are impregnated chemically or electrochemically into the porous ni structures, which are used as current collectors. ni plaques are current collectors of choice for battery applications demanding high power and reliability, along with long cycle life, such as batteries for aerospace and railway applications, power tools and some portable electronics. the smaller pore size decreases the distance for electrons to travel from the current collector into the active material during cell discharge and consequently improves the discharge rate characteristics for high power applications. significant advances in the development of ni plaques were achieved by the use of filamentary ni particles with high surface area, which improved contact with active materials. a new wave of interest in the application of porous ni materials is related to the development of electrochemical supercapacitors (es). es can complement or replace batteries in electrical energy storage applications when high - power delivery is required. the interest in the application of manganese dioxide in es is related to low cost of this material, which exhibits high sc in environmentally friendly aqueous electrolytes. the charging mechanism of manganese dioxide electrodes of es is described by the following reaction : where a = li, na, k, h. equation 1 indicates that high electronic and ionic conductivity of the electrode material are important in order to utilize the high theoretical capacitance (1,370 f g) of manganese dioxide. the properties of mno2 are influenced by crystalline structure, particle size, porosity and surface area. a complicating factor in the application of mno2 in es is low electronic conductivity of this material. this problem can be addressed by the use of advanced current collectors, such as ni plaques. in a previous investigation, manganese dioxide nanofibers were prepared by a chemical precipitation method and utilized for the fabrication of composite manganese dioxide the composite films deposited on stainless steel foils showed high sc in a voltage window of 1.0 v. however, the sc decreased significantly with increasing film thickness. the results presented below indicated that relatively high sc can be achieved at high active material loading using ni plaques as current collectors. in this approach, the high surface area and porous structure of the ni plaques provided improved electrical contact of the current collector with active material and enabled good electrolyte access to the active material. we presented experimental results on the fabrication of composite electrodes, investigation into electrode microstructure and electrochemical behavior. manganese dioxide nanofibers were prepared by the method described in the previous investigation. in this approach, precipitation was performed by the reduction of 0.2-m kmno4 (aldrich) solutions with ethanol using the following reaction : mwcnt were provided by arkema company. ni plaques with mass of 0.1 mg cm were provided by inco company and impregnated with a manganese dioxide slurry containing 15 mass% of mwcnt. xrd studies were performed with a diffractometer (nicolet i2) using monochromatic cu k radiation at a scanning speed of 0.5 deg min. tga and dta of the manganese dioxide nanofibers were carried out in air at a heating rate of 5 c / min using a thermoanalyzer (netzsch sta-409). electron microscopy investigations were performed using a jeol 2010f transmission electron microscope and a jeol jsm-7000f scanning electron microscope equipped with energy - dispersive spectroscopy. capacitive behavior of the electrodes was studied using a potentiostat (parstat 2273, princeton applied research) controlled by a computer using a powersuite electrochemical software. electrochemical studies were performed using a standard three - electrode cell containing 0.5 m - na2so4 aqueous solution degassed with purified nitrogen gas. the counter electrode was a platinum gauze, and the reference electrode was a standard calomel electrode (sce). cyclic voltammetry (cv) studies were performed within a potential range of 01.0 v versus sce at scan rates of 2100 mv s. the sc was calculated using half the integrated area of the cv curve to obtain the charge (q), and subsequently dividing the charge by the mass of the active material (m) and the width of the potential window (v) : impedance spectroscopy investigations were performed in the frequency range of 0.1 hz100 khz at amplitude voltage of 5 mv. simulations of the impedance behavior were performed on the basis of the equivalent - circuit models using zsimpwin 3.10 commercial software. in the previous investigation, manganese dioxide nanofibers were prepared by the reduction in kmno4 solutions with ethanol, followed by stirring of obtained suspensions during 20 h, and then washing and drying of the suspension. as an extension of the previous investigation, the stirring time was varied in the range of 120 h. figure 1a, 1b show typical tem images of the nanofibers obtained after 1 h of stirring. it was found that the nanofibers were not individual crystals but were composed of small nanoparticles with typical size less than 5 nm. the increase in the stirring time from 1 to 20 h resulted in partial agglomeration of the nanofibers, which formed bundles (fig. this result was in a good agreement with the results of investigation into other nanomaterials, which indicated that stirring of nanoparticles can promote agglomeration. tem images of the nanofibers a, b after stirring during 1 h and c typical bundles formed after stirring during 20 h xrd studies (fig. 2) showed changes in the diffraction patterns of the nanofibers with increasing stirring time. the xrd pattern of the nanofibers stirred for 1 h showed xrd peaks of birnessite, corresponding to the jcpds file 87 - 1497. according to the literature, birnessite has a two - dimensional layered structure that consists of edge - shared mno6 octahedra with cation and water molecules occupying the interlayer regions. the birnessite formula is generally expressed as axmno2+y(h2o)z, in which a represents an alkali metal cation. the average oxidation state of mn usually falls between 3.6 and 3.8, which represents a predominance of mn with minor amounts of mn and mn. the intensity of the peaks of the birnessite phase decreased with increasing stirring time, indicating lower crystallinity. x - ray diffraction patterns for powders, containing nanofibers stirred during a 1 and b 20 h (inverted filled triangle jcpds file 87 - 1497) it was found that the precipitated manganese dioxide nanofibers contained potassium and adsorbed water. eds studies showed the k / mn atomic ratio of 0.16 0.03 and 0.14 0.03 for nanofibers stirred during 1 and 20 h, respectively. figure 3 shows tga and dta data for the nanofibers stirred during 1 h. the tga studies of the powders showed the mass loss related to the dehydration at temperatures below 400 c. small mass gain in the range of 400420 c in tga data and corresponding dta exothermic peak can be attributed to oxidation of non - stoichiometric manganese dioxide. the mass loss in the range of 880900 c and corresponding endotherm can be attributed to reduction of manganese dioxide in agreement with the literature data. similar behavior was observed for the nanofibers stirred during 20 h. a tga and b dta data for nanofibers stirred during 1 h (exo shows exothermic effects) the slurries containing manganese dioxide nanofibers and mwcnt were used for the impregnation of ni plaques. figure 4a shows a schematic of the cross - section of a ni plaque, which consists of a perforated ni foil and sintered ni particles. the voids between the particles provide a space for the loading of the plaques with an active material. figure 4b, 4c show sem images of the surface of a ni plaque at different magnifications. the plaques exhibited porous microstructure with pore size in the range of 150 m (fig. 4c) showed small particle size of ni particles in the range of 0.53 m, which formed a conductive porous matrix. it was suggested that high surface area, small particle size, porosity and conductivity of ni plaques are beneficial for application in es. 5) containing manganese dioxide nanofibers and mwcnt. a schematic of a cross - section of a ni plaque, containing perforated ni foil (thickness 0.1 mm) and sintered ni particles (13 m) and b, c sem images of the surface of a ni plaque at different magnifications sem image of a composite manganese dioxide nanofibers mwcnt material impregnated into a ni plaque in the previous investigation, it was found that manganese dioxide nanofibers and mwcnt formed a porous fibrous network, which was beneficial for the electrolyte access to the active material. however, the sc of the films deposited on metal foil substrates decreased significantly with increasing film mass from 50 to 300 g cm. in contrast, the results presented below showed that relatively high sc can be obtained for active material loading of 715 mg cm using ni plaque current collectors. in this approach, porous ni current collectors provided improved contact with active material, whereas mwcnt was used as a conductive additive. figure 6 shows typical cvs for the composite electrode with material loading of 7 mg cm. within the potential range of 01.0 v versus sce, the composite electrode exhibited capacitive - like current - potential responses, indicated by the box shape of the cvs. that there are no redox peaks in the range between 0 and 1.0 v. figure 7 shows sc at different scan rates for the composite electrodes. the electrodes prepared using powders stirred during 1 h showed higher sc compared to the powders stirred during 20 h. this can be attributed to low agglomeration of the powders stirred during 1 h. this result indicated that further optimization of the powder processing conditions can be beneficial for the improvement in electrode performance. the highest sc of 185 f g was achieved at a scan rate of 2 mv s for material loading of 7 mg cm. the reduction in the sc is especially evident at a scan rate of 100 mv s. turning again to the results of the previous investigation, it is seen that due to the use of ni plaque current collectors, the material loading can be increased by 12 orders of magnitude, and relatively high sc can be obtained. moreover, the sc (fig. 8) at a scan rate of 100 mv s for the 7 mg cm loading of ni plaque (sc = 111 f g) was higher compared to the sc of the 0.24 mg cm film (sc = 82 f g) deposited on a foil substrate. the high sc at high scan rates is important for the fabrication of efficient es. cvs for a composite electrode containing manganese dioxide nanofibers stirred during 1 h, mixed with mwcnt and impregnated into a ni plaque with material loading of 7 mg cm at scan rates of a 10, b 20 and c 50 mv s sc versus scan rate for composite electrode containing manganese dioxide nanofibers stirred during a 1 h and b 20 h, mixed with mwcnt and impregnated into ni plaques with material loading of 10 mg cm sc versus scan rate for composite electrodes containing manganese dioxide nanofibers stirred during 1 h, mixed with mwcnt and impregnated into ni plaques with material loading of a 7 mg cm and b 14 mg cm figure 9 shows charge discharge curves obtained at different current densities were nearly linear and indicated good capacitive behavior in agreement with box shape cvs shown in fig. the equivalent circuit of es was discussed in the literature and included rc transmission line, describing the porous electrode. cn elements represent double - layer capacitance and pseudo capacitance, whereas rn elements represent electrolyte resistance in pores, faradaic resistance and equivalent series resistance of the electrodes. conway and pell described the impedance of porous electrode using 5-element (n = 5) circuit. a constant phase impedance (cpe) the equivalent circuits should allow an optimum representation of the measured spectra with a minimum set of model parameters. 10) was found for the equivalent circuit similar to that proposed for composite ruthenium oxide - graphite electrodes. in this circuit, the high frequency z value of the complex impedance z = z iz showed that r = r1 + r2 is about 0.4 ohm for the sample with an area of 1 cm. manganese dioxide nanofibers stirred during 1 h, mixed with mwcnt and impregnated into ni plaques with material loading of 10 mg cm at current densities of a 60 b 40 and c 20 ma cm impedance spectroscopy data for composite electrode containing manganese dioxide nanofibers stirred during 1 h, mixed with mwcnt and impregnated into ni plaques with material loading of 10 mg cm manganese dioxide nanofibers with length ranged from 0.1 to 1 m and a diameter of about 46 nm were prepared by a chemical precipitation method. composite electrodes for es, containing two different fibrous materials, were fabricated by impregnation of slurries of the manganese dioxide nanofibers and mwcnt, as a conductive additive, into porous ni plaque current collectors. the composite electrodes with total mass loading of 715 mg cm showed good capacitive behavior in the 0.5 m na2so4 solutions. the reduction in stirring time of the precipitated nanofibers resulted in lower agglomeration and higher sc. the highest sc of 185 f g was obtained at a scan rate of 2 mv s for materials loading of 7 mg cm. testing results indicated that ni plaques are promising current collector materials for application in es. the authors gratefully acknowledge the financial support of the natural sciences and engineering research council of canada. this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.	manganese dioxide nanofibers with length ranged from 0.1 to 1 m and a diameter of about 46 nm were prepared by a chemical precipitation method. composite electrodes for electrochemical supercapacitors were fabricated by impregnation of the manganese dioxide nanofibers and multiwalled carbon nanotubes (mwcnt) into porous ni plaque current collectors. obtained composite electrodes, containing 85% of manganese dioxide and 15 mass% of mwcnt, as a conductive additive, with total mass loading of 715 mg cm2, showed a capacitive behavior in 0.5-m na2so4 solutions. the decrease in stirring time during precipitation of the nanofibers resulted in reduced agglomeration and higher specific capacitance (sc). the highest sc of 185 f g1 was obtained at a scan rate of 2 mv s1 for mass loading of 7 mg cm2. the sc decreased with increasing scan rate and increasing electrode mass.
gmc, also named injection contracture, is a clinical syndrome characterized by gait abnormality and limb dysfunction including restriction of adduction and internal rotation of the hip joint. it was first found by valderrama at a scientific meeting of the british orthopedic association in london in 1969. its pathologic change typically presents with fibrosis and contracture of the gluteus and its fascia. the knees can not be brought together and are separated in a frog - leg position. as younger patients with gmc continue to develop and grow, pathologic changes increase in prevalence and severity including leg length discrepancy, pelvic oblique, compensatory scoliosis, and, in severe cases, bilateral dislocation of the hip joints. now, arthroscopic release, a minimally invasive surgery which was reported by zhang for the first time, has became the gold standard of treatment in gmc patients. compared with developed countries, gmc is more widely reported in china. so far, numerous studies have suggested that gmc usually was associated with repeated intramuscular injection into the gluteal region during childhood. however, it remains unclear that, the reasons why these children are weak and have no choice to accept repeated intramuscular injection. the paper reveals that chd children are prone to catching cold, repeated respiratory tract infection, or pneumonia and have to take frequently intramuscular injection which use benzyl alcohol as a dissolvent for penicillin in some regions of china in the 1970s and 1980s, leading to degeneration, necrosis and fibrosis of the gluteal muscles and fascia, as well as serious limitation of hip movements. we identified 4 gmc adolescents with chd from january 2013 to march 2014. the duration of symptoms ranged from 6 to 10 years. there were 2 males and 2 females with age ranging from 14 to 17 years (table 1). although 3 of 4 patients (patients 1, 3, and 4) had limitations with activities of daily living, they believed their activity levels were lower than those of healthy people. three of 4 patients (patients 1, 3, and 4) had an abnormal gait with out - toe walking, and could not crouch with both knees close to each other or sit down with their legs crossed. patient 1 was diagnosed as chd for the first time, while the other 3 had been cured yet. two representative patients (patients 1 and 4) were selected for further discussion of their clinical and radiographic findings (table 2). clinical characteristics of the patients radiographic findings of the patients each author certifies the study had approved, by ethics committee of peking university shenzhen hospital, for the reporting of these cases and that all investigations were conducted in conformity with ethical principles of research. patient 1 was a 16-year - old girl who had bilateral diseases with gluteal muscle atrophies, abnormal gait with out - toe walking, dimpling of skin around the gluteal regions, snapping hips, adduction and internal rotation dysfunction of hips, ober 's signs. the most thing affected her was adduction and internal rotation dysfunction of hips, which made her not to do competitive sports (actually, it was mainly influenced by chd which was diagnosed later.) like other healthy girls. 3d reconstruction ct showed that her gluteus maximus and gluteus medius were obviously dysplastic and part of them turned to contractile bands. we heard a systolic ejection murmur at the second left intercostal space in preoperative examinations. furthermore, chest x - ray revealed pulmonary vascularity, cardiac enlargement and aortic knob shrinking. finally, cardiac ultrasound proved the diagnosis of asd. because of this, we sent her to cardiosurgery department for atrial septal defect closure and partial anomalous pulmonary venous drainage correction on beating heart. the arthroscopic release and rehabilitation program for gmc were operated 110 days later, when she was completely rehabilitated from cardiosurgery. three months later, the patient gained a satisfactory result without complications, snappings, and dysfunctions of hip (figure 1). (a) a 3d reconstruction ct shows the gluteal muscles of patient 1, a 16-year - old woman who had gmc with severe gluteus maximus and gluteus medius atrophy. (b) and (c) show the contracture bands (white arrow) in left and right side, respectively. (e) chest x - ray reveals pulmonary ascularity, cardiac enlargement and aortic knob shrinking. patient 4 was a 17-year - old boy who had bilateral diseases with gluteal muscle atrophies, abnormal gait with out - toe walking, dimpling of skin around the gluteal regions, snapping hips, adduction and internal rotation dysfunction of hips, ober 's signs. the most things affected him was adduction and internal rotation dysfunction of hips, as well as snapping hips. due to a failed open surgery before when he was very young, he was taken ligation of patent ductus arteriosis (the detailed data of that surgery were unavailable). 3d reconstruction ct showed that his gluteus maximus and gluteus medius were obviously dysplastic and part of them turned to contractile bands. three months later, the patient gained a satisfactory result without complications, snappings, and dysfunctions of hip (figure 2). (a) a 3d reconstruction ct shows the gluteal muscles of patient 4, a 17-year - old man who had gmc with mild gluteus maximus and gluteus medius atrophy. (b) and (c) show the contracture bands (white arrow) in left and right side, respectively. patient 1 was a 16-year - old girl who had bilateral diseases with gluteal muscle atrophies, abnormal gait with out - toe walking, dimpling of skin around the gluteal regions, snapping hips, adduction and internal rotation dysfunction of hips, ober 's signs. the most thing affected her was adduction and internal rotation dysfunction of hips, which made her not to do competitive sports (actually, it was mainly influenced by chd which was diagnosed later.) like other healthy girls. 3d reconstruction ct showed that her gluteus maximus and gluteus medius were obviously dysplastic and part of them turned to contractile bands. we heard a systolic ejection murmur at the second left intercostal space in preoperative examinations. furthermore, chest x - ray revealed pulmonary vascularity, cardiac enlargement and aortic knob shrinking. finally, cardiac ultrasound proved the diagnosis of asd. because of this, we sent her to cardiosurgery department for atrial septal defect closure and partial anomalous pulmonary venous drainage correction on beating heart. the arthroscopic release and rehabilitation program for gmc were operated 110 days later, when she was completely rehabilitated from cardiosurgery. three months later, the patient gained a satisfactory result without complications, snappings, and dysfunctions of hip (figure 1). (a) a 3d reconstruction ct shows the gluteal muscles of patient 1, a 16-year - old woman who had gmc with severe gluteus maximus and gluteus medius atrophy. (b) and (c) show the contracture bands (white arrow) in left and right side, respectively. (e) chest x - ray reveals pulmonary ascularity, cardiac enlargement and aortic knob shrinking. patient 4 was a 17-year - old boy who had bilateral diseases with gluteal muscle atrophies, abnormal gait with out - toe walking, dimpling of skin around the gluteal regions, snapping hips, adduction and internal rotation dysfunction of hips, ober 's signs. the most things affected him was adduction and internal rotation dysfunction of hips, as well as snapping hips. due to a failed open surgery before when he was very young, he was taken ligation of patent ductus arteriosis (the detailed data of that surgery were unavailable). 3d reconstruction ct showed that his gluteus maximus and gluteus medius were obviously dysplastic and part of them turned to contractile bands. three months later, the patient gained a satisfactory result without complications, snappings, and dysfunctions of hip (figure 2). (a) a 3d reconstruction ct shows the gluteal muscles of patient 4, a 17-year - old man who had gmc with mild gluteus maximus and gluteus medius atrophy. (b) and (c) show the contracture bands (white arrow) in left and right side, respectively. we present a series of 4 gmc patients with chd, including asd, vsd, tof, and pda respectively. accordingly, we suggest that some gmcs are related to chds, or rather are derived from chds. to be specific, children suffered from chds are easy to acquire respiratory infections (nearly one - quarter to one - third), which may be result in frequent use of benzyl alcohol as a dissolvent for penicillin for intramuscular injections in some regions of china in 1970s1980s, as several researchers have suggested that benzyl alcohol is the main cause of gmc. this series is limited by its small size relating to the decreasing of gmc for abandoning benzyl alcohol as a dissolvent. patient 1 was admitted to hospital for releasing gmc, however, asd was found in preoperative examinations. this warns that surgeons should attach more importance to the cardiac examination, especially identifying the real cause of can not do competitive sports. when chd is repaired and physical condition is fully recovered, it is time to release gmc. at last patient 4 was hospitalized in order to cure gmc through a minimally invasive surgery for a noneffective open surgery 4 years ago. with the normal results of physical examinations and chest x - ray, we did arthroscopic release directly, like other common gmc patients without chd. result relationship between the 2 diseases, we propose 3 recommendations : (1) as to chd children, in addition to treating the primary disease as early as possible, it is essential to decreasing the use of intramuscular injection, and (2) paying more attention to cardiac examination especially cardiac ultrasound in perioperative period, and (3) taking 3d - ct to reconstruct gluteal muscles for observing contracture bands clearly in preoperation.	abstractgluteal muscle contracture (gmc), presented with hip abduction and external rotation when crouching, is common in several ethnicities, particularly in chinese. it remains unclear that the reasons why these children are weak and have no choice to accept repeated intramuscular injection. here, we found some unique cases which may be useful to explain this question.we describe a series of special gmc patients, who are accompanied with congenital heart disease (chd). these cases were first observed in preoperative examinations of a patient with atrial septal defect (asd), which was proved by chest x - ray and cardiac ultrasound. from then on, we gradually identified additional 3 gmc patients with chd. the original patient with asd was sent to cardiosurgery department to repair atrial septal first and received arthroscopic surgery later. while the other 3 were cured postoperative of ventricular septal defect (vsd), tetralogy of fallot (tof), patent ductus arteriosus (pda), respectively, and had surgery directly.the study gives us 3 proposals : (1) as to chd children, it is essential to decrease the use of intramuscular injection, (2) paying more attention to cardiac examination especially cardiac ultrasound in perioperative period, and (3) taking 3d - ct to reconstruct gluteal muscles for observing contracture bands clearly in preoperation. however, more larger series of patients are called for to confirm these findings.
non - traumatic convexal subarachnoid hemorrhage (csah) observed at the convexity of the brain is a relatively uncommon entity with various vascular and nonvascular causes. : cerebral venous thrombosis (cvt),1) reversible cerebral vasoconstriction syndrome (rcvs),4) vascular malformations, vasculitides,12) infectious aneurysms,10) moyamoya disease or syndrome,22) severe carotid atherosclerosis,6) posterior reversible encephalopathy syndrome (pres),24) cerebral amyloid angiopathy (caa),5) and nonvascular disorders, such as primary and secondary brain neoplasms7) or abscess.18) cerebral infarcts in the territory of the anterior cerebral artery (aca) are reported to comprise 0.5 - 3% of all ischemic strokes9)11) and few studies have specifically assessed the clinical characteristics of stroke patients with aca infarction.2) we report a case of aca cerebral infarction with spontaneous csah lead to stenosis of the aca. it is a infrequent case, but is worth consideration that the management and evaluation of these patients. a 59-year - old female patient visited emergency room with right side subjective weakness spontaneously. however, the national institute of health stroke scale (nihss) was 0. initial computed tomography angiography at one and half hours after symptom onset demonstrated subarachnoid hemorrhage (sah) localized in the left frontal convexity and mild focal stenosis at both a2 segments. also the magnetic resonance diffusion - weighted images (dwi) revealed an acute infarction of anterior cerebral arterial territory (fig. 1). we could not find any vascular lesion on computed tomographic angiography (cta) and magnetic resonance angiography (mra) and treated with dual antiplatelet drugs (cilostazole [otsuka pharmaceutical co., ltd. tokyo, japan ] 50 mg bid / day plus aspirin [bayer pharma ag., she has done well for a follow - up period and her modified rankin scale was 0 at day 5. the follow - up evaluation was performed at out - patient clinic after 5 months. cta shows that the sah was washed out and encephalomalatic change in left aca territory. mri also reveals that no evidences of acute infarction in dwi and hemorrhagic transformation in susceptibility weighted imaging (swi) (fig. although the prevalence of non - traumatic convexal sah is reported to be 7.5% of all spontaneous sah patients,13) little has been conscious of concerning the incidence of csah accompanying with acute infarction among many different etiologies of csah. demostrated the image protocol to cover the wide spectum of entities potentially responsible for csah that brain cta with a paired channel at both arterial and venous phases and mr imaging including gre t2 sequences, flair, dwi, mra 3d tof, contrast - enhanced venogram, and pre- and postgadoliniumt1-weighted imaging.3) however, we evaluated the cta and mr imaging cluding t2, dwi, swi and 3d tof mra. cta and mra are useful to determine the vascular causes of csah such as vascular malformations, rcvs, vasulitides, high - grade stenosis, moyamoya disease, and septic aneurysms. in addition, mr imging is ascertainable diagnositic tools between dural and cortical cvt, and non - vascular causes such as, caa, pres, neoplasm, abscess and cavernoma. in this case, aca territory infarction seemed to occur hemodynamically because of stenotic lesion on a2 segment and hypoperfusion on brain spect with tc-99 m hmpao. nakajima. assumed that the accompanying an infarction and non traumatic csah might demonstrate that hemodynamic insufficiency because of arterial stenosis or occlusion get to the critical point. for instance, after an acute infarction due to occlusive disease by the embolic or hemodynamic mechanism, ensuing dynamic changes of intracranial perfusion pressure might bring about the csah.14) also cuvinciuc. explained that the main mechanism of csah because of chronic arterial occlusive diseases is considered to be the rupture of dilated vulnerable compensatory pial vessels.3) a large randominzed controlled trial studied clopidogrel and aspirin in high - risk patients with acute nondisabling cerebrovascular events (chance) was finished in china. the result revealed that antiplatelet double therapy with clopidogrel plus aspirin was better than aspirin alone for reducing the risk of recurrent stroke and not increase the risk of bleeding among patients with high risk transient ischemic attack (tia) or minor infarction23) due to our patient 's acute hemorrhage on the frontal convexity, we were concerned the hemorrhagic transformation and rebleeding because dual antiplatelet therapy (clopidogrel plus aspirin) was associated with a significant trend to increase moderate bleeding.17) so we treated with cilostazol plus aspirin successfully. the patient 's symptom was improved and there was no evidence of rebleeding on following studies. tan. reported that cilostazol, alone or with aspirin, decrease recurrence of ischemic stroke significantly, poststroke intracranial hemorrhage, and extracranial bleeding in patients with a prior ischemic stroke as compared with other antiplatelet treatments.21) cilostazol, a selective inhibitor of cyclic nucleotidephosphodiesterase 3, increases activated intracellular cyclic adenosine monophosphate (camp) concentrations and thus inhibits platelet aggregation.19) cilostazol is a known direct arterial vasodilator,15) and has antiatherosclerotic effect,8) which can strengthen the endothelial barrier20) and additionally may play a role in neuroprotection.16) acute infarction with spontaneous convexal sah is rare but it is worth to work up to identify the etiology of csah using the cta or mra and mr image including t2, dwi, swi. and we suggest that dual antiplatelet dugs (cilostazole and aspirin) are taken into account for treatments that improved the neurologic symptoms and reduced the concerns about stroke recurrence and rebleeding.	non - traumatic convexal subarachnoid hemorrhage (csah) is a comparatively infrequent with various vascular and nonvascular causes, it rarely occurs concomitant to acute ischemic stroke. we report a case of a 59-year - old woman, visited emergency room with right side subjective weakness spontaneously. magnetic resonance diffusion - weighted images revealed an acute infarction of anterior cerebral arterial territory. computed tomographic angiography showed a left frontal csah without any vascular lesions. and other laboratory studies were non - specific. we treated with dual antiplatelet drugs (cilostazole [otsuka pharmaceutical co., ltd. tokyo, japan ] and aspirin [bayer pharma ag., leverkusen, germany ]). she has done well for a follow - up period. (5 months) this case demonstrates the csah with acute infarction is rare but need to work up to identify the etiology and antiplatelet dugs are taken into account for treatments.
adult obesity has become a disease of epidemic proportions, and it is well known as a root cause of multiple medical comorbidities. conservative treatment options for the obese, such as low - calorie diets, behavior modifications, and exercise, are still the mainstay of therapy. unfortunately, the results of these measures remain disappointing, with most patients either losing an inadequate amount of weight or, for those able to lose a significant amount of weight, experiencing total weight regain within a few years. currently, surgical intervention offers the best means to achieve significant and durable weight loss in the morbidly obese. the widely accepted indications for bariatric surgery today are severe obesity with a body mass index (bmi) greater than 40 or greater than 35 in the presence of weight - related comorbid disease. many bariatric surgical procedures have been tried with a wide spectrum of success and complications. among them, laparoscopic roux - en - y gastric bypass has become the most often performed bariatric operation in the united states. easier recovery, lower morbidity, shorter hospital stay, less pain, less disability, and superior cosmetic results have made minimally invasive surgery the preferred surgical approach for the treatment of morbid obesity. however, the procedure is not risk - free, especially during the learning curve. major complications include bowel obstructions, anastomotic leaks and strictures, gastrogastric and gastro - cutaneous fistulas. gastrogastric fistula is a communication between the proximal gastric pouch and the distal gastric remnant. fistula formation, although a commonly described phenomenon in various gastric operations and disease processus, rarely has been described in the realm of bariatric procedures. we have performed an extensive literature review using the pubmed database, and we did not find any article reporting the surgical technique for the repair of this condition. the aim of this article is to review the current literature about gastrogastric fistula and to describe the surgical steps of its laparoscopic management. in 1995, the greenville group documented an 11.8% rate of staple - line dehiscence after staple partition of the stomach in roux - en - y gastric bypass procedures. these staple - line breakdowns resulted in failure of the procedure because the restrictive quality of the proximal pouch was no longer present. because the patient could eat again without discomfort, and regained weight, revision of the failed operation remained the only option. to avoid this high incidence of staple - line dehiscence, they postulated that resultant separation of gastric pouches would free them of the dependence on staple lines and avoid this cause of failure. nevertheless, the new operation also has introduced a new complication, the gastrogastric fistula, which was documented in 6% of the 100 patients evaluated in this series. in 2000, schauer observed 2 cases of gastrogastric fistula in their series of 275 laparoscopic roux - en - y gastric bypass. two years later, blachar analyzed 463 patients who underwent the same procedure, and they found one case of gastrogastric fistula. recently, gould studied their first 100 cases in a new minimally invasive bariatric surgery program, observing a gastrogastric fistula in 2 patients, and dresel reported one patient with a fistula from her gastric pouch to the gastric remnant among 120 patients who underwent laparoscopic roux - en - y gastric bypass. even after transection of the stomach during gastric bypass, late staple - line failure can occur developing fistulous communication between the proximal pouch and the distal stomach if the pouch remains close to the bypassed stomach. because the 2 portions of the stomach are completely separate, these are probably caused by a leak with abscess formation adjacent to the distal staple line, which then drained into the distal stomach, forming the gastrogastric fistula. a gastric leak usually is a catastrophic complication, leading to sepsis and necessitating reoperation for drainage. by developing a fistula via the divided edge of the distal stomach, these patients probably internally drain their leaks or abscess, which explains their delayed presentation and absence of systemic sepsis. analyzing 6 patients with gastrogastric fistulas, cucchi identified a leak or an abscess before the discovery of the gastrogastric fistula in all patients but one. on the other hand, according to gould 's observations, gastrogastric fistula is a purely technical complication that occurred on 2 occasions early in their experience. these fistulas resulted from the incomplete division of the stomach during the creation of the pouch. the angle of his is sometimes very difficult to clearly visualize during laparoscopic roux - en - y gastric bypass. clear and definitive visualization of the completely divided stomach is essential to avoid this complication. cucchi observed 6 patients with gastrogastric fistulas, and they had similar postoperative symptoms. failure in weigth loss is a late clinical sign observed in patients with gastrogastric fistula because they can eat again without discomfort. we highlight this situation, which must be considered in those patients regaining weight after a roux - en - y gastric bypass. following open roux - en - y gastric bypass at most bariatric centers, postoperative upper gastrointestinal contrast studies (ugi) are routinely performed to detect a leak or early obstruction, because the subtle early clinical signs of these complications after open procedures are obscured by incisional pain and narcotic administration. laparoscopic surgeons have adopted the practice of routine postoperative radiological contrast studies, perhaps because of this custom following open bariatric surgery. gould perform a limited upper gastrointestinal dilute - barium radiographic examination routinely on the morning of postoperative day 1 after laparoscopic rouxen - y gastric bypass. the diet is initiated after a normal gastrointestinal examination in patients without clinical indicators of potential leak (ie, tachycardia, low urine output, fever). in their series, a gastrogastric fistula was identified on the postoperative barium leak study in 2 patients. in both cases, lyass analyzed the efficacy of selective use of radiological studies after laparoscopic roux - en - y gastric bypass instead of routine postoperative upper gastrointestinal contrast studies, and they concluded that the selective approach is safe (figure 1). they studied 368 patients, and 41 patients (11%) developed symptoms and signs in the early postoperative period suggestive of complications. most of the symptomatic patients had abdominal pain, tachycardia, nausea or vomiting, and elevated white cell count. of 4 patients with 4 clinical signs, 2 had complications. of 10 patients with 3 clinical signs, 4 (40%) had complications. with 1 or 2 signs, radiological contrast studies in the early postoperative period were performed in 39 of the 41 symptomatic patients. computed tomography was performed in 34 (87%), upper gastrointestinal contrast studies in addition to computed tomography in 6 (15%), and upper gastrointestinal contrast study alone in 5 (13%). gastrogastric fistula was diagnosed in one patient by computed tomography and confirmed by upper gastrointestinal contrast study. huang studied all adult patients referred for endoscopic evaluation of upper gastrointestinal symptoms at any time after roux - en - y gastric bypass. barium contrast upper gastrointestinal radiography was useful in the evaluation of patients with symptoms after surgery when performed in conjunction with upper endoscopy (figure 2). they state that it provides important anatomic information, which may be particularly helpful to endoscopists with limited experience with this patient population. barium contrast radiography is particularly useful and is the preferred initial study for the detection of staple - line dehiscence, which may be small and overlooked during endoscopy. all 8 cases of staple - line dehiscence were correctly diagnosed by upper gastrointestinal radiography, and one case of gastrogastric fistula was identified. gastrogastric fistula demonstrated by upper endoscopy. in 1995, cucchi studied 6 patients with gastrogastric fistula. in all cases, patients had either fever or tachycardia in the early postoperative course, although routine postoperative day 3 meglumine diatrizoate swallows were negative in each case. for them, this fact emphasizes the insensitivity of contrast studies in detecting leaks, and additional evaluation or treatment of suspected leaks in these patients often must be guided by their clinical presentations. once identified, gastrogastric fistulas may be treated surgically, by laparoscopy or open surgery, when diagnosed in the early postoperative setting and when patients fail to loose weight or experience late regain of weight in their postoperative follow - up. in the era of minimally invasive surgery as demonstrated by other laparoscopic operations, minimally invasive approaches offer significant advantages compared with open surgery, such as reduced postoperative pain and complications, decreased length of hospital stay, and earlier return to normal activity. even revisional bariatric surgeries can be approached laparoscopically with acceptable morbidity compared with patients whose revision is approached in an open manner. when diagnosed in the acute postoperative setting, as demonstrated by gould, they elected to laparoscopically divide the fistula on the first postoperative day in their 2 cases. when the fistula is identified late in the postoperative course, as a cause of poor weight loss, surgical approach is more difficult. we did not find any report in the literature describing the laparoscopic approach for these cases and herein we describe it, step - by - step. the patient is placed in a supine position with the surgeon on the right and the assistant on the left, and 2 monitors above the patient 's shoulders. after creation of carbon dioxide pneumoperitoneum (14 mm hg) using the veress needle technique, trocars are placed : 10-mm supraumbilical (for the 30-degree laparoscope), 5-mm subxiphoid, 10-mm in the left subcostal region, 5-mm in the right subcostal region, and 12-mm in the right flank. adhesions from the previous surgery are released using blunt dissection, electrocautery and ultracision harmonic scalpel (or ligasure vessel sealing system). dissection is performed to identify the gastric pouch, the gastrojejunal anastomosis, and the gastric remnant. after exposure of these structures, a fibrous area can be visualized between the gastric reservoir and the gastric remnant. it is best demonstrated by turning off the laparoscope light and keeping the endoscope light on (figure 3a). the endoscope light is guided to the gastrogastric fistula and when the laparoscope light is turned off, the fistula can be observed (a). after complete identification, the gastrogastric fistula is transected with an endo gia stapler (figure 4a). the closure of the fistula can be observed under direct view by flexible endoscopy (figure 4b). the staple line is subsequently reinforced with continuous 3 0 polypropylene sutures, and the integrity of the gastric pouch staple line is determined intraoperatively by 2 methods. first, the pouch is endoscopically inspected and tested for leakage after insufflation and submerging it in irrigation fluid. second, the methylene blue is introduced through the nasogastric tube, and the anastomosis is examined once again for leaks of the dye. a tubulo - laminar drain is placed next to the gastric pouch and brought out through the left subcostal port site. the following recommendations are established to minimize this complication : oversewing of the long divided staple lines of the proximal pouch and distal stomach with closely spaced sutures at 2 mm between stitches ; construction of an 8-mm to 10-mm anastomosis, taking care to include strong bites of both gastric and jejunal mucosa with every suture ; avoidance of possible obstruction of the efferent jejunal limb by ensuring that the limb lies without tension and without kinks during its course for the transverse mesocolon to the gastric pouch ; intraoperative tests of pouch and anastomosis integrity with air and methylene blue ; from our results with these 6 patients, this obviously will not prevent all leaks, but hopefully will reduce the incidence ; interposition of omentum or a limb of jejunum between the gastric segments. in 1995, the greenville group documented an 11.8% rate of staple - line dehiscence after staple partition of the stomach in roux - en - y gastric bypass procedures. these staple - line breakdowns resulted in failure of the procedure because the restrictive quality of the proximal pouch was no longer present. because the patient could eat again without discomfort, and regained weight, revision of the failed operation remained the only option. to avoid this high incidence of staple - line dehiscence, they postulated that resultant separation of gastric pouches would free them of the dependence on staple lines and avoid this cause of failure. nevertheless, the new operation also has introduced a new complication, the gastrogastric fistula, which was documented in 6% of the 100 patients evaluated in this series. in 2000, schauer observed 2 cases of gastrogastric fistula in their series of 275 laparoscopic roux - en - y gastric bypass. two years later, blachar analyzed 463 patients who underwent the same procedure, and they found one case of gastrogastric fistula. recently, gould studied their first 100 cases in a new minimally invasive bariatric surgery program, observing a gastrogastric fistula in 2 patients, and dresel reported one patient with a fistula from her gastric pouch to the gastric remnant among 120 patients who underwent laparoscopic roux - en - y gastric bypass. even after transection of the stomach during gastric bypass, late staple - line failure can occur developing fistulous communication between the proximal pouch and the distal stomach if the pouch remains close to the bypassed stomach. because the 2 portions of the stomach are completely separate, these are probably caused by a leak with abscess formation adjacent to the distal staple line, which then drained into the distal stomach, forming the gastrogastric fistula. a gastric leak usually is a catastrophic complication, leading to sepsis and necessitating reoperation for drainage. by developing a fistula via the divided edge of the distal stomach, these patients probably internally drain their leaks or abscess, which explains their delayed presentation and absence of systemic sepsis. analyzing 6 patients with gastrogastric fistulas, cucchi identified a leak or an abscess before the discovery of the gastrogastric fistula in all patients but one. on the other hand, according to gould 's observations, gastrogastric fistula is a purely technical complication that occurred on 2 occasions early in their experience. these fistulas resulted from the incomplete division of the stomach during the creation of the pouch. the angle of his is sometimes very difficult to clearly visualize during laparoscopic roux - en - y gastric bypass. clear and definitive visualization of the completely divided stomach is essential to avoid this complication. cucchi observed 6 patients with gastrogastric fistulas, and they had similar postoperative symptoms. shoulder pain, tachypnea, and anorexia were each noted in one patient. failure in weigth loss is a late clinical sign observed in patients with gastrogastric fistula because they can eat again without discomfort. we highlight this situation, which must be considered in those patients regaining weight after a roux - en - y gastric bypass. following open roux - en - y gastric bypass at most bariatric centers, postoperative upper gastrointestinal contrast studies (ugi) are routinely performed to detect a leak or early obstruction, because the subtle early clinical signs of these complications after open procedures are obscured by incisional pain and narcotic administration. laparoscopic surgeons have adopted the practice of routine postoperative radiological contrast studies, perhaps because of this custom following open bariatric surgery. gould perform a limited upper gastrointestinal dilute - barium radiographic examination routinely on the morning of postoperative day 1 after laparoscopic rouxen - y gastric bypass. the diet is initiated after a normal gastrointestinal examination in patients without clinical indicators of potential leak (ie, tachycardia, low urine output, fever). in their series, a gastrogastric fistula was identified on the postoperative barium leak study in 2 patients. in both cases, lyass analyzed the efficacy of selective use of radiological studies after laparoscopic roux - en - y gastric bypass instead of routine postoperative upper gastrointestinal contrast studies, and they concluded that the selective approach is safe (figure 1). they studied 368 patients, and 41 patients (11%) developed symptoms and signs in the early postoperative period suggestive of complications. most of the symptomatic patients had abdominal pain, tachycardia, nausea or vomiting, and elevated white cell count. of 10 patients with 3 clinical signs, 4 (40%) had complications. with 1 or 2 signs, radiological contrast studies in the early postoperative period were performed in 39 of the 41 symptomatic patients. computed tomography was performed in 34 (87%), upper gastrointestinal contrast studies in addition to computed tomography in 6 (15%), and upper gastrointestinal contrast study alone in 5 (13%). gastrogastric fistula was diagnosed in one patient by computed tomography and confirmed by upper gastrointestinal contrast study. huang studied all adult patients referred for endoscopic evaluation of upper gastrointestinal symptoms at any time after roux - en - y gastric bypass. barium contrast upper gastrointestinal radiography was useful in the evaluation of patients with symptoms after surgery when performed in conjunction with upper endoscopy (figure 2). they state that it provides important anatomic information, which may be particularly helpful to endoscopists with limited experience with this patient population. barium contrast radiography is particularly useful and is the preferred initial study for the detection of staple - line dehiscence, which may be small and overlooked during endoscopy. all 8 cases of staple - line dehiscence were correctly diagnosed by upper gastrointestinal radiography, and one case of gastrogastric fistula was identified. gastrogastric fistula demonstrated by upper endoscopy. in 1995, cucchi studied 6 patients with gastrogastric fistula. in all cases, patients had either fever or tachycardia in the early postoperative course, although routine postoperative day 3 meglumine diatrizoate swallows were negative in each case. for them, this fact emphasizes the insensitivity of contrast studies in detecting leaks, and additional evaluation or treatment of suspected leaks in these patients often must be guided by their clinical presentations. once identified, gastrogastric fistulas may be treated surgically, by laparoscopy or open surgery, when diagnosed in the early postoperative setting and when patients fail to loose weight or experience late regain of weight in their postoperative follow - up. in the era of minimally invasive surgery as demonstrated by other laparoscopic operations, minimally invasive approaches offer significant advantages compared with open surgery, such as reduced postoperative pain and complications, decreased length of hospital stay, and earlier return to normal activity. even revisional bariatric surgeries can be approached laparoscopically with acceptable morbidity compared with patients whose revision is approached in an open manner. when diagnosed in the acute postoperative setting, as demonstrated by gould, these fistulas they elected to laparoscopically divide the fistula on the first postoperative day in their 2 cases. mean operating time for these 2 reoperative cases was 30 minutes. when the fistula is identified late in the postoperative course, as a cause of poor weight loss, we did not find any report in the literature describing the laparoscopic approach for these cases and herein we describe it, step - by - step. the patient is placed in a supine position with the surgeon on the right and the assistant on the left, and 2 monitors above the patient 's shoulders. after creation of carbon dioxide pneumoperitoneum (14 mm hg) using the veress needle technique, trocars are placed : 10-mm supraumbilical (for the 30-degree laparoscope), 5-mm subxiphoid, 10-mm in the left subcostal region, 5-mm in the right subcostal region, and 12-mm in the right flank. adhesions from the previous surgery are released using blunt dissection, electrocautery and ultracision harmonic scalpel (or ligasure vessel sealing system). dissection is performed to identify the gastric pouch, the gastrojejunal anastomosis, and the gastric remnant. after exposure of these structures, a fibrous area can be visualized between the gastric reservoir and the gastric remnant. it is best demonstrated by turning off the laparoscope light and keeping the endoscope light on (figure 3a). the endoscope light is guided to the gastrogastric fistula and when the laparoscope light is turned off, the fistula can be observed (a). the gastrogastric fistula is isolated using a penrose drain (b). after complete identification, the gastrogastric fistula is transected with an endo gia stapler (figure 4a). the closure of the fistula can be observed under direct view by flexible endoscopy (figure 4b). the staple line is subsequently reinforced with continuous 3 0 polypropylene sutures, and the integrity of the gastric pouch staple line is determined intraoperatively by 2 methods. first, the pouch is endoscopically inspected and tested for leakage after insufflation and submerging it in irrigation fluid. second, the methylene blue is introduced through the nasogastric tube, and the anastomosis is examined once again for leaks of the dye. a tubulo - laminar drain is placed next to the gastric pouch and brought out through the left subcostal port site. the following recommendations are established to minimize this complication : oversewing of the long divided staple lines of the proximal pouch and distal stomach with closely spaced sutures at 2 mm between stitches ; construction of an 8-mm to 10-mm anastomosis, taking care to include strong bites of both gastric and jejunal mucosa with every suture ; avoidance of possible obstruction of the efferent jejunal limb by ensuring that the limb lies without tension and without kinks during its course for the transverse mesocolon to the gastric pouch ; intraoperative tests of pouch and anastomosis integrity with air and methylene blue ; from our results with these 6 patients, this obviously will not prevent all leaks, but hopefully will reduce the incidence ; interposition of omentum or a limb of jejunum between the gastric segments. in this article, we reviewed the scarce literature regarding gastrogastric fistula, and we described the surgical steps of its laparoscopic management. we believe that in the modern era of laparoscopy, patients need and want to be treated in the most possible minimally invasive manner. in the hands of the skilled and gifted laparoscopists, laparoscopic correction of the gastrogastric fistula can be performed with no additional harm to the patient. in this report, we focused on the surgical technique, and we do think a larger study must be carried out to confirm the feasibility and safety of the described technique.	background : gastrogastric fistula is a communication between the proximal gastric pouch and the distal gastric remnant, rarely described in the realm of bariatric procedures. the aim of this study was to review the existing literature about this topic and to demonstrate its laparoscopic treatment.methods:an extensive literature review found several articles reporting this complication. however, no citation was found describing the steps of the laparoscopic management of this situation.results:gastrogastric fistula occurs in up to 6% of rouxen - y gastric bypasses. two theories exist for fistula formation : (1) it is a technical complication derived from the incomplete division of the stomach during the creation of the pouch, and (2) it occurs after a staple - line failure, developing a leak with an abscess, which then drains into the distal stomach forming the fistula. early symptoms include fever, tachycardia, and abdominal pain. failure in weigth loss is a late clinical sign observed in these patients. diagnosis is based on radiologic study, upper endoscopy and computed tomography. when identified in the acute postoperative course, laparoscopic treatment is easy. chronic fistulas are difficult to manage, and the laparoscopic approach is an alternative to open surgery.conclusions:gastrogastric fistula is a possible complication of roux - en - y gastric bypass and its laparoscopic treatment is feasible.
hepatitis g virus (hgv) belongs to the flaviviridae, which includes three genus and more than 70 members. hepatitis g virus members are widely variable and biologically different (1, 2). despite the gene structure and duplication similarities, there is no antibody cross - reactivity among hgv members proteins (3, 4). hepatitis g virus is an envelope and spherical shaped virus of 40 - 60 nm diameters that e - protein, the most important protein of hgv, is necessary for the virus adhesion and fusion (5) ; therefore, determination is important in case of the anti - e2 antibodies presence. the hgv genome is composed of a single stranded rna with the length of 11 kb, caped on 5 ' without poly - a tail at the 3 ' end (6). hepatitis g virus can not be cultivated and a sensitive and suitable cell type of its culture is not developed yet. diagnosis of hgv is according to the revers transcription polymerase chain reaction (rt - pcr) and enzyme linked immunosorbent assay (elisa) in biological samples ; however, rt - pcr technique is valuable to detect current infections (8). two different techniques, rt - pcr and elisa, consider different targets to diagnose hgv ; rt - pcr only detects hgv rna molecules in the patient samples, but elisa measures antibodies against e2-proteins. therefore, a patient may have antibody titers for e2 proteins but its rt - pcr result may became negative because of an active immune response. prevalence of hgv varies in blood donors ranging from 0.9% to 10%. besides blood products transfusion, other routes for transfection include placental and needle sticking, especially for drug users (8 - 16). hepatitis g virus is mostly concomitant with hepatitis b and c viruses (hbv and hcv). anyway, hgv has no definitive impact on the patient status (17, 18). however, there are reports on the pathogenesis of hgv that make the prevalence studies essential, especially for healthcare providers and authors. according to the reports by the investigators, hgv could develop fulminant hepatitis, which its causes are manifested sporadically (19 - 28). since the patients with renal failure who undergo dialysis receive blood products and transfusion, the current study measured the prevalence of hgv assay in the patients undergoing hemodialysis and kidney transplantation in khuzestan province, iran. the current study aimed to investigate the prevalence of hgv using determination of e2, viral envelope antigen, antibodies and its rna by elisa and rt - pcr techniques. to evaluate the prevalence of hgv antibody and rna, 516 serum samples were collected from the patients undergoing hemodialysis and kidney transplanted and stored at -70 c until the test running day. also other data including gender, hospitalization or outdoor monitoring, and place of residence were recorded for each patient ; 86 cases were from ahvaz kidney transplantation center and the rest were from other cities of khuzestan province ; also 60 sera belonged to the patients undergoing hemodialysis from ahvaz. all patients were oriented and informed with the study purpose and signed written consent letter. furthermore, this study was reviewed, accepted and approved by the ethical committee of ahvaz jundishapur university of medical sciences. to diagnose seropositive patients, the test was performed according to the manufacturer s instruction and the steps briefly were : serums were diluted by sample buffer (1/10 ratio), the diluted samples were added to wells and stored at 37 c for 30 minutes and then washed with antihuman antibody and conjugated with horse radish peroxidase (hrp) and again storing at 37 c. after well rewashing, substrate was added and the chromogenic reaction was blocked by stopping solution. optical density of each sample was measured in 450 nm and 630 nm as reference filter. to evaluate hbv and hcv involvement in the patients, serological determination was done using hepatitis b surface antigen (hbs - ag) and hcv - ab elisa kits (diaplus, usa) according to the kit manufacturers. to measure hgv rna positive / negative in patients, sera rt - pcr was performed. to extract viral rna, tripure rna extraction kit (roche, germany) and to synthesize cdna sensiscript kit (qiagen, usa) were used. briefly, after viral rna extraction, microtubes were kept at 65 c for five minutes and transferred into the ice cold dishes to prepare cdna as template rna. then cdna synthesis was performed as follows : rt buffer 2 l, dntps (5 mm) 2 l, random primer (10 picom) 2.5 l, rnasin (10 u/l) 1 l, reverse transcriptase 5 l, distilled water 6.5 l were added, then mixed and incubated at 37 c for one hour. primers used for the first step of pcr were 58 (58f-5 ' cag ggt tgg tag gtc gta aat cc-3 ') and 75 (75r-5 ' cct att ggt caa gag aga cat-3 '). the first step was performed as follows : pcr buffer 5 l, dntps (5 mm) 1 l, forward primer (58f) 1 l, reverse primer (75r) 1 l, taq - polymerase 0.3 l, cdna 5 l, and distilled water 36.7 l were added ; then thermocycler apparatus (techne, uk) was programmed as follows : 94 c for 30 seconds, 60 c for 30 seconds, 72 c for 30 seconds in 30 cycles. in the second step, 5 l of the first step final product was used with 131 (131f-5'aag aga gac att gaa ggg cga cgt-3 ') and 134 primers (134r- 5 ' ggt cat ctt ggt agc cac tat agg-3 '). ultimately, 5 l of amplified product was added to the wells of a 2% gel agarose and the electrophoresis was derived at 100 v for one hour. the pcr final product immersed into a dish containing 20 l ethidium bromide in 200 ml distilled water. transiluminator apparatus (vilber - lurmat, french) was used to visualize the bands and imaging. statistical analysis was conducted using chi - square test, and the prevalence determination of each investigated variable ; the confidence interval of 95% (ci = 0.95) was considered to estimate significant results. to diagnose seropositive patients, elisa tests were performed using diagnostic kit (diaplus inc. the test was performed according to the manufacturer s instruction and the steps briefly were : serums were diluted by sample buffer (1/10 ratio), the diluted samples were added to wells and stored at 37 c for 30 minutes and then washed with antihuman antibody and conjugated with horse radish peroxidase (hrp) and again storing at 37 c. after well rewashing, substrate was added and the chromogenic reaction was blocked by stopping solution. optical density of each sample was measured in 450 nm and 630 nm as reference filter. to evaluate hbv and hcv involvement in the patients, serological determination was done using hepatitis b surface antigen (hbs - ag) and hcv - ab elisa kits (diaplus, usa) according to the kit manufacturers. to measure hgv rna positive / negative in patients, sera rt - pcr was performed. to extract viral rna, tripure rna extraction kit (roche, germany) and to synthesize cdna sensiscript kit (qiagen, usa) were used. briefly, after viral rna extraction, microtubes were kept at 65 c for five minutes and transferred into the ice cold dishes to prepare cdna as template rna. then cdna synthesis was performed as follows : rt buffer 2 l, dntps (5 mm) 2 l, random primer (10 picom) 2.5 l, rnasin (10 u/l) 1 l, reverse transcriptase 5 l, distilled water 6.5 l were added, then mixed and incubated at 37 c for one hour. primers used for the first step of pcr were 58 (58f-5 ' cag ggt tgg tag gtc gta aat cc-3 ') and 75 (75r-5 ' cct att ggt caa gag aga cat-3 '). the first step was performed as follows : pcr buffer 5 l, dntps (5 mm) 1 l, forward primer (58f) 1 l, reverse primer (75r) 1 l, taq - polymerase 0.3 l, cdna 5 l, and distilled water 36.7 l were added ; then thermocycler apparatus (techne, uk) was programmed as follows : 94 c for 30 seconds, 60 c for 30 seconds, 72 c for 30 seconds in 30 cycles. in the second step, 5 l of the first step final product was used with 131 (131f-5'aag aga gac att gaa ggg cga cgt-3 ') and 134 primers (134r- 5 ' ggt cat ctt ggt agc cac tat agg-3 '). ultimately, 5 l of amplified product was added to the wells of a 2% gel agarose and the electrophoresis was derived at 100 v for one hour. the pcr final product immersed into a dish containing 20 l ethidium bromide in 200 ml distilled water. transiluminator apparatus (vilber - lurmat, french) was used to visualize the bands and imaging. statistical analysis was conducted using chi - square test, and the prevalence determination of each investigated variable ; the confidence interval of 95% (ci = 0.95) was considered to estimate significant results. elisa and rt - pcr tests were performed on 516 sera gathered from different cities of khuzestan province to evaluate the prevalence of hgv. table 1 shows the obtained results of the two evaluating techniques. according to elisa test results for different cities, from 126 samples of ahvaz hospitals, 4 (1.95%) samples were positive for hgv antibody. from 26 samples of sosa and shushtar cities, each had one positive sample. from 36 samples of mahshahr, and 17 samples of andimeshk, all other samples from masjid soleiman, shadegan, dezfoul, khoramshahr, and baghmalek were negative for e2 antibody. distribution of hgv was significantly different among the different cities (ci = 0.95, p = 0.004). out of the 516 sera, 285 (55.23%) samples were from males and 231 (44.77%) samples from females. positive and negative cases of e2 antibodies between males and females are presented in table 2. there was no significant difference between gender of the patients considering hgv antibody (ci = 0.95, p = 0.313). for the association between blood receiving frequency and hgv antibodies, data showed no considerable difference (mean sd = 23.68 15.9 ; ci = 0.95, p = 0.99) ; note that the minimum data is not shown. concurrent infection of hgv with hcv or hbv was also investigated using elisa test for hcv antibody and hbs antigen. 438 sera were negative for both hgv and hcv and from the rest of 516 samples, 40 cases were hcv positive and hgv negative ; 37 sera were hcv negative and hgv positive. only one sample was positive in both hgv and hcv, which was considered as co - infection. from the 516 sera, 470 samples were both hbv and hgv negative ; eight hbv positive and hgv negative ; 36 sera were hbv negative and hgv positive ; however, two samples were positive for both hbv and hgv. patients with transplantation were more positive for hgv immunoreactive antibodies in comparison with the renal failure ones (p = 0.000 ; ci = 0.95). furthermore, likelihood ratio (lr) was 25.116, which implied the conclusive increase of disease likelihood in patients with transplantation. high value of lr also implied the importance of performing hgv immune - assay for kidney donors. however, calculated odd s ratio for the patients was 0.178 (0.95 ci = 0.089 - 0.353), which implied that the chance of hgv positivity, using immunoassay, was higher among patients with transplantation but lower in patients with renal failure. in fact, patients with transplantation were 16% positive for hgv antibody ; meanwhile other patients with renal failure were only 3.3% positive (table 3). patients with transplantation were more positive for hcv immunoreactive antibodies compared to the ones with renal failure (p = 0.001 ; ci = 0.95). furthermore, lr was 13.475, which implied the conclusive increase of disease likelihood in patients with transplantation. high value of lr also implies the importance of performing hcv immune - assay for kidney donors. calculated odd s ratio for negative / positive patients was 7.381 (0.95 ci = 1.76 - 30.952), which implied that the chance of hgv positivity, using immunoassay, was lower in patients with transplantation but higher in those with renal failure. in fact, patients with transplantation were only 1.3% positive for hgv antibody compared to other patients with renal failure who were 9.1% positive (table 4). prevalence of hbv antibody positivity was insignificantly different between patients with transplantation and those with renal failure (p = 0.763 ; ci = 0.95). likelihood ratio for hbv elisa test was 0.088, which implied conclusive decrease of disease likelihood in patients with renal failure. lower values of lr also emphasized that performing hbv elisa test was not essential for patients with renal failure ; this is because of vaccination or higher rate of blood transfusion episodes in such patients. furthermore, the obtained odd s ratio for hbv elisa test in patients with transplantation and those with renal failure was 0.811, and the chance of negative / positive result could range from 0.207 to 3.177 (table 5). rt - pcr results showed a negative reaction for all 38 serum samples which were seropositive for hgv antibody ; 478 samples were negative for hgv rna, and 16 (3.14%) were positive for rt - pcr assay. considering the low frequency of rt - pcr positive cases, the frequency was not reported for each city. since 1995 that hepatitis g virus was identified for the first time, a series of studies considered the epidemiology and diagnosis of this virus and its pathogenesis, especially in hepatitis. different social groups were tested for hgv and healthy blood donors, patients undergoing dialysis or kidney transplantation recipients, patients with acute, chronic, idiopathic hepatitis, and those with cirrhosis or cancer were included. all patients were subjected to two techniques ; elisa, a serological test to measure hgv antibody in the serum samples ; and rt - pcr that is a molecular test to search viral rna in the serum samples. all the sixteen sera that were positive using rt - pcr belonged to the patients undergoing hemodialysis (3.14% of all the patients undergoing hemodialysis). the prevalence of hgv in patients undergoing kidney transplantation was reported 24% in italy (18). the frequency of hgv is reported 50% between the patients undergoing hemodialysis in germany (19), 12.8% in brazil (20), 4.5% in japan (21) and 17% in taiwan (22). reported data showed a 24.3% frequency of hgv among the patients undergoing hemodialysis in south africa (15). the present study showed a 3.7% frequency of hgv among the patients undergoing hemodialysis in khuzestan province, which was close to the japanese reported data (21). however, overall prevalence of hgv among the patients undergoing hemodialysis could vary from 1.3% to 55%. on the other hand, according to the reported data, concomitant infection of hgv with hcv or hbv was not similar. even in some cases reports there was only one case of concomitant infection of hgv and hcv ; but hgv and hbv co - infection was found in two cases. however, a previus study showed the frequency of chronic hbv and hcv concomitant infection with hgv as 55% and 18%, respectively (25). other epidemiological studies in iran reported the prevalence of 12.6% for the patients undergoing hemodialysis in tehran (29). the other study reported 4.8% hgv rna positive cases among the blood donors, who were negative for hiv - ag / ab, hcv - ab, and hbs - ag (30). however, the current study reported the prevalence of 3.14% hgv rna among the patients undergoing hemodialysis, which was different from the results obtained in other countries, but agreed with the previous studies in iran. it is noteworthy that up to now no definitive pathogenesis has been found for hgv. however, it is necessary to detect hgv in the societies since, despite its questionable role for hepatitis and non - pathogenic feature, it may be converted to a dangerous pathogen in the future ; its role may also be revealed in its healthy carriers. the current study emphasized that transplantation may be an important way of hgv transmission in the iranian kidney transplantation recipients ; therefore, hgv should be considered routinely for kidney donors. higher rate and chance of hcv positivity in the patients with renal failure than transplantation recipients may be due to more episodes of blood transfusion and exposure to blood transmitted hcv. since the studies newly started considering hgv in iran, it is necessary to continue such studies especially hgv sequencing in the patients undergoing hemodialysis and those with hepatitis b and c infections. investigating the association of hgv and clinical manifestations of the aforementioned patient groups could be valuable for a clear concept about their disease severity and treatment. regarding the hgv transmission through blood or blood products	background : hepatitis g virus (hgv) is a member of flaviviridae. prevalence of hgv in healthy people is very low, but this virus is more prevalent in patients with hepatitis. besides, relative frequency of hgv in patients undergoing hemodialysis, and kidney recipients is very high. the role of hgv in pathogenesis is not clear. since this virus can not be cultivated, molecular techniques such as revers transcription polymerase chain reaction (rt - pcr) is applied to detect hgv.objectives:the current study aimed to investigate the prevalence of hgv using determination of e2, viral envelope antigen, antibodies and the rna by enzyme linked immunosorbent assay (elisa) and rt - pcr techniques. the rational of the study was to determine the prevalence of hgv in patients undergoing hemodialysis and kidney transplantation in khuzestan province, iran.patients and methods : five hundred and sixteen serum samples of the patients undergoing hemodialysis and kidney transplantation from various cities of khuzestan province were collected. anti - hepatitis g e2 antibodies were investigated by elisa method. rnas were extracted from serums and hepatitis g rna was detected by rt-pcr.results:of the 516 samples, 38 (7.36%) specimens were positive for anti - hgv by elisa. all of these elisa positive samples were negative for hgv genome by rt - pcr. of the remaining 478 elisa negative samples, 16 (3.14%) samples were positive by rt-pcr.conclusions:hepatitis g virus was not prevalent in the patients undergoing hemodialysis and kidney transplantation in khuzestan province. although reports indicated high frequency of co - infection of hgv with hepatitis b and c viruses, in the current research, co - infection of hgv with b and c was not considerable. since different groups and subtypes of hgv are reported, periodic epidemiologic evaluation of hgv and its co - infection with other hepatitis viruses is suggested in other populations such as the patients with thalassemia ; however, periodic epidemiologic monitoring of hgv may be helpful to control future potential variations of the virus.
while stoke incidence has declined in developed countries over the last decade, its incidence rose during the same period in low- and middle- income countries. although stroke is an ever - increasing problem in developing countries, little is known about its incidence and outcomes in sub - saharan africa (ssa). the interstroke study showed the commonality of the main risk factors for stroke worldwide, including ssa : hypertension, obesity, diabetes mellitus (dm), smoking, alcohol abuse, hypercholesterolemia, diet and physical activity. in addition, genetic factors may act either by predisposing to vascular risk factors, such as hypertension, heart disease, dm, hyperlipidemia or by a direct independent effect on stroke risk. these genes include apolipoproteine (apoe), plasminogen activator inhibitor 1 (serpine1), platelet receptor gp1ba, angiotensin - converting enzyme (ace), and methylenetetrahydrofolate reductase (mthfr). case - control studies of stroke candidate genes in different ethnic groups have produced contrary results. a meta - analysis of chinese, japanese, and korean ancestry reported significant association of polymorphisms in the apoe, mthfr and ace genes with ischemic stroke. however, in other studies, only minor or non - significant risk associations were observed, and it was concluded that it is a combination of several genotypes interacting which increases the odds ratio for risk of ischemic stroke. studies on stroke conducted within african populations mainly focus on the prevalence, risk factors, subtypes and outcome of stroke. to date, there are only a handful of studies examining genetic association of apoe, mthfr, ace or other candidate genes with cardiovascular disease in african populations. these candidate genes provide some of the most likely genetic variants which are involved in stroke risk and have been among the most studied. common apoe alleles has been shown to affect risk of cardio- and cerebrovascular disease and is considered an established genetic risk factor, however common variation in the mthfr gene (related to folate metabolism) and the ace gene although implicated in disease risk are not confirmed. the aim of the present study therefore, was to investigate association of the apoe, mthfr and ace gene polymorphisms with stroke in zambian patients. forty one consecutive patients with mean age 5416 years admitted to the university teaching hospital (uth) lusaka, zambia, between june and december 2010 with a diagnosis of stroke, were enrolled. we included only patients who had stroke confirmed by neurological examination and brain ct scan. daily physical checks / reviews were made of emergency room, admission ward and in - patient wards for stroke patients. within 24 hours of admission, we obtained medical history, conducted general and neurological examination and brain ct scan was performed. the patient s demographic details and risk factors for stroke were documented on a data collection sheet by study physician. risk factors for stroke included : hypertension, dm, elevated cholesterol, heart disease, tobacco smoking / sniffing, alcohol consumption, history of transient ischemic attack, and a family history of stroke. as a risk factor of stroke hypertension was defined as blood pressure of 140/90 mmhg and current use of antihypertensive medication. elevated cholesterol was considered if serum fasting cholesterol was > 5.2 mmol / l or prestroke treatment with a cholesterol lowering agent. the severity of stroke was evaluated on admission using the national institutes of health stroke scale (nihss). data on risk factors and stroke severity in our study population was published in our previous article. as a control population, 116 subjects (68 women, 48 men) matched for age, sex, free of cerebrovascular diseases and other risk factors including hypertension, dm and hiv, were recruited from the department of internal medicine of uth. only indigenous zambian patients and control subjects were included in the study. all participants gave written informed consent and the study was approved by the biomedical research ethics committee of the university of zambia. whole blood was collected in 4 ml edta tubes and stored at 4 degrees celsius prior to dna extraction which was performed using silica - gel - based membrane with vacuum technology qiaamp dna mini kit (250) (qiagen, london, uk). the common apoe epsilon alleles defined by the p.c112r (rs429358) and p.r158c (rs7412) substitutions were selected for analysis, as well as the common mthfr c677 t (p.a222v ; rs1801133) variant and the ace exonic synonymous variant p.t202 t (rs4343 ; a / g) ; which acts as a surrogate marker for the common intronic alui insertion / deletion (i / d ; rs4646994) polymorphism. genotyping was performed with abi predesigned taqman probes and chemistry (applied biosystems, foster city, ca) with analysis performed using sds 2.2.2 software on an abi 7900 for taqman. positive and negative controls where included on all taqman assay plates. positive or ambiguous results in the taqman assay were also resolved with direct sequencing. the fishers test was used to calculate for p - value and the two tailed probability levels for statistical significance with p 5.2 mmol / l or prestroke treatment with a cholesterol lowering agent. the severity of stroke was evaluated on admission using the national institutes of health stroke scale (nihss). data on risk factors and stroke severity in our study population was published in our previous article. as a control population, 116 subjects (68 women, 48 men) matched for age, sex, free of cerebrovascular diseases and other risk factors including hypertension, dm and hiv, were recruited from the department of internal medicine of uth. only indigenous zambian patients and control subjects were included in the study. all participants gave written informed consent and the study was approved by the biomedical research ethics committee of the university of zambia. whole blood was collected in 4 ml edta tubes and stored at 4 degrees celsius prior to dna extraction which was performed using silica - gel - based membrane with vacuum technology qiaamp dna mini kit (250) (qiagen, london, uk). the common apoe epsilon alleles defined by the p.c112r (rs429358) and p.r158c (rs7412) substitutions were selected for analysis, as well as the common mthfr c677 t (p.a222v ; rs1801133) variant and the ace exonic synonymous variant p.t202 t (rs4343 ; a / g) ; which acts as a surrogate marker for the common intronic alui insertion / deletion (i / d ; rs4646994) polymorphism. genotyping was performed with abi predesigned taqman probes and chemistry (applied biosystems, foster city, ca) with analysis performed using sds 2.2.2 software on an abi 7900 for taqman. positive and negative controls where included on all taqman assay plates. positive or ambiguous results in the taqman assay were also resolved with direct sequencing. the fishers test was used to calculate for p - value and the two tailed probability levels for statistical significance with p<0.05 being considered significant. an odds ratio (or) was calculated as a measure the strength of association the genotype and allele frequencies in the study and control groups are shown in table 1. there was no significant difference between the two groups in the frequencies of apoe, mthfr and ace genotypes and alleles. the apoe, mthfr and ace genotype frequencies for the case group were compatible with the hardy - weinberg equilibrium. the apoe 22 and the mthfr tt genotypes were absent in the zambian population. however, in the control group, the apoe, mthfr genotype frequencies were compatible with the hardy - weinberg equilibrium (hwe), while the ace genotype was not in agreement with hwe. the frequency of control subjects homozygous for apoe 4 was 7.7% compared to 2.4% for stroke patients. the mthfr t allele frequency was 8.6% in control subjects and was observed at a frequency of 11.0% in patients. the frequency for ace rs4343 g - allele corresponding to the d allele was 13.8% for subjects in the control group compared to 12.2% for patients. there was significant genotypic variations between cases and controls for apoe 24 and 23 genotypes, statistical analysis demonstrated a higher risk for hemorrhagic stroke in apoe 24 carriers [or of 4.45 (p<0.05) ] while the presence of apoe 23 genotype seemed to significantly lower the risk for hemorrhagic stroke (or 0.097 ; 0.006 - 1.67, p<0.05) (table 2). apoe 22 and mthfr 677tt polymorphisms were absent in both ischemic and hemorrhagic stroke. though not significant, the frequency of the mthfr 677 t allele was higher (double) in ischemic stroke (17.4%) compared to hemorrhagic stroke (8.3%). studies conducted over the last decade has shown that among blacks, stroke is more common, more severe, and carries higher mortality when compared with other races ; blacks also have increased frequency of risk factors such as dm, hypertension, and obesity. in this study we have analyzed for the first time common polymorphisms of the apoe, mthfr and ace genes in zambian stroke patients and in controls. this constitutes an approach to start characterization of the genetic risk architecture of zambian patients with cerebrovascular disease. the study also describes the prevalence of apoe, mthfr and ace polymorphisms corresponding to stroke in the zambian population. we observed a relatively high frequency of the apoe 4 allele and a complete absence of the apoe 22 genotype in both patients and controls. we did not find any association between the apoe 4 allele and stroke in our patients. however, there was an increased risk to hemorrhagic stroke by the apoe 24 genotype with or at 4.45 p<0.05. this may suggest that zambian carriers of apoe 2 and 4 have an increased risk of hemorrhagic stroke. of note, in our previous study, the prevalence of hemorrhagic stroke was observed to be higher than in reported western cohorts. a similar high frequency for hemorrhagic stroke has been reported for other african countries with estimates up to 52% in democratic republic of congo, up to 60% in ghana, and tanzania. according to the literature, carriers of apoe 2 and 4 have an increased risk of intracerebral hemorrhage perhaps because of the effects of these gene variants on risk of cerebral amyloid angiopathy. a recent metaanalysis in caucasian populations found apoe 2 genotypes to be associated with increased risk of hemorrhagic stroke. we found the effect of apoe 2 on carriers to be inconclusive as it increased hemorrhagic stroke risk in apoe 24 subjects while this risk seemed to be reversed in the apoe 23 subjects. these conflicting results may be due to a possible synergistic interaction effect of the 2 allele towards the 4 or simply the low sample size and low frequency of these alleles. found the frequency of 2 allele to be generally higher among sub - saharans (0.116) compared to europeans with the range of 0.044 - 0.108 (with the exception of swedish with frequency 0.119). hence, it would be plausible to hypothesize that an increase in the apoe 2 allele frequency (0.122, table 1) increases the risk for development of hemorrhagic stroke in a population with a generally high 4 allele frequency (0.256 in the zambian population). we observed low minor allele frequencies for the mthfr c677 t and ace i / d polymorphisms in the zambian population. no significant association were found between the mthfr and ace genotypes and stroke risk in the zambian population. association between these polymorphisms and stroke have, however, been demonstrated in other populations including chinese population, hungarian population, and the northern irish population. this may reflect population heterogeneity or specificity in disease risk. to clarify the role of these variants in stroke risk in sub - saharan populations it is important to note that the major limitation of this study is the sample size which does not exclusion of apoe, mthfr and ace polymorphisms as a risk factor in zambian patients with stroke, and therefore, large scale prospective studies are needed to confirm these findings. however, as genetic technologies become more available to african countries and in particular those between the sahara and south africa we will gain a better understanding of how genetics will determine health outcomes. these data will help ease health - related social and economic burdens within these developing nations.	the aim of the present study was to investigate the association of apoe, mthfr and ace polymorphisms with stroke in the zambian population. we analyzed 41 stroke patients and 116 control subjects all of zambian origin for associations between the genotype of the apoe, mthfr and ace polymorphisms and stroke. the apoe 24 genotype showed increased risk for hemorrhagic stroke (p<0.05) and also a high risk for ischemic stroke (p=0.05). there was complete absence of the apoe 22 and the mthfr tt genotypes in the zambian population. the difference between cases and controls was not significant for the other genetic variants when analyzed for relationship between stroke, stroke subtype and genotype. we show that genetic variation at the apoe locus affects susceptibility to stroke. no detectable association were observed for the mthfr and ace genotypes and stroke in the zambian population.
chronic venous insufficiency (cvi) is characterised by the retrograde flow of blood in the lower extremity. cvi and its sequel of varicose veins and venous hypertension are among the most common disorders that affect daily life1. the annual incidence of cvi is 26% and 1.9% in women and men, respectively2. the main risk factors for cvi include family history, prolonged standing, history of deep venous thrombosis, age, obesity, and pregnancy2. the venous hypertension of cvi usually begins with oedema and varicose veins and progresses to stasis cellulitis and pigmentation of skin leading into lipodermatosclerosis and ulceration3. since established venous insufficiency can not be cured by pharmacological interventions alone, it is most commonly treated by non - invasive methods4, 5. the compression therapy relieves cvi symptoms by improving venous function and hence is preferred among other physical therapies to treat venous and lymphatic insufficiency of lower limbs. the compression stockings (cs) and calf muscle pump exercises increase venous return and are mainstay of conservative treatment4, 6,7,8. ct exerts the highest compression at the ankle which gradually decreases towards knee, thereby ensuring the upward flow of blood in the direction of heart and preventing reflux towards foot or in lateral superficial veins. the compression pressure gradient of cs, by reducing diameter of major veins, increases velocity and volume of blood flow and decreases the venous hypertension and symptoms9, 10. cvi considerably affects patient s health - related quality of life (hrqol). in many patients, the pain and discomfort of cvi affects physical mobility, working capacity and social life11. the international society for vascular surgery recommended to expand outcome measures for venous disease studies to include patient - reported measures of functioning and hrqol. it noted that comprehensive evaluation of venous disease should include assessment of clinical outcomes and hrqol12. for chronic conditions like cvi, the assessment of hrqol can provide important information on disease burden that may not be adequately captured with traditional physician - based measures of morbidity or mortality13. due to chronic nature of cvi, the long term usage of the cs may be rough considering the compliance of the patient. therefore, the present study was aimed to analyse the effect of short term use of cs on symptoms of cvi and hrqol in cvi patients. the patients visiting outpatient clinic, at department of cardiovascular surgery, abant izzet baysal university hospital bolu, turkey were enrolled in this study. the study was approved by the clinical research ethics committee of abant izzet baysal university faculty of medicine (ib.30.2.ab.0.20.05.04050.01.0460). inclusion criteria were patients without prior treatment for cvi or varicose vein surgery and for those the disease was categorised as c2 and c3 according to ceap classification. exclusion criteria included presence of deep vein thrombosis, congestive heart failure, malignancy, patients having more advanced skin changes such as lipodermatosclerosis skin ulcers or other dermatological disease with pruritus, and patients who had ongoing use of cs. all patients enrolled were given detailed explanation of the study following which informed consent was obtained from them. a questionnaire including demographic information, occupational details, and relevant medical history the generic, disease specific hrqol, and depression - related questionnaire was requested from 126 patients. the turkish version of nottingham health profile (nhp) was used to measure a generic hrqol. it contains 38 questions with assigned individual score from six different categories such as energy level (el ; n=3), pain (p ; n=8), emotional reaction (er ; n=8), sleep (s ; n=5), social isolation (si ; n=5), and physical abilities (pa ; n=8). for calculation of final score in each category, variation in number of items per category was rationalised by computing percentage score (i.e., each sum was multiplied by 100 and divided by the number of items in the category). possible scores ranged from 0 (indicating all no answers in that category or absence of distress) to 100 (all yes answers indicating maximal distress). the questionnaire on 26 items from 2 categories of veines - qol / sym measured severity of symptoms of cvi and its impact on hrqol from patient s perspective. the five different frequencies (always, a few times a week, once a week, once in a few weeks, and never) for symptoms fullness of lower extremities, pain, swelling, night cramps, heat / burning sensation, restless legs, itching, tingling / stinging sensation, and throbbing under 10 different items were assessed. limitations in daily activities (9 items), greatest intensity during the day (1 item), change over the past year (1 item), and psychological impact (5 items) were covered by the hrqol scale of 2 to 7 points for response on intensity, frequency, or agreement. the highest score indicated better outcomes in veines - qol and on veines - sym scale. veines - qol / sym ; turkish validity and reliability were established by study of kutlu. the beck depression inventory (bdi) consists of 21 items ; it is a self - report rating inventory that measures characteristic of attitude and symptoms of depression. the study group included 44 patients who regularly used cs and remaining 82 patients who did not use cs were included in control group. the class 2 cs (jobst brand) of height below knee and pressure gradient of 2332 mmhg were used. the patients were asked to wear cs as soon as they wake up in the morning and take it off before going to bed. they were asked to use cs for a period of four weeks, exercise regularly and apply skin care moisturiser (ph 5.5). the exercises prescribed were ankle pumping and heel lifts (3 sets of 10 repeats) and daily walk of 3045 minutes. the patients were warned that they should not wear cs while sleeping due to high resting pressure. the patients (without use of cs) in the control group were also prescribed same exercises and skin care. all assessments were repeated after a four - week treatment. the statistical program for social sciences (spss 18.0, spss inc., quantitative data were presented as mean and standard deviation and qualitative data as percentage (%) and frequency (n). an independent t - test was used to compare the differences in venous symptoms, depression and hrqol scores between groups. of the 203 patients with cvi interviewed for this study, 77 were excluded due to habitants of town or village (n=33), absence of inclusion criteria (n=27), and rejection to participate into study (n=17). the remaining 126 patients were enrolled into the study : study group (n=44) and control group (n=82). nine patients (2 from study group and 7 from control group) did not undergo last evaluation. thus, the study was completed with 117 patients (73 females and 44 males) : study group (n=42) and control group (n=75). the average age of patients was 60.9 9.4 years in study group and 63.1 4.9 years for patients in control group. the height (cm), bodyweight (kg) and body mass index (bmi) (kg / m) for patients in both groups were comparable (table 1table 1.demographic and clinical features of the patientsstudy groupn=42x sdcontrol groupn=75x sdage (years)60.9 9.4 63.1 4.9height (m)1.63 0.51.63 0.0weight (kg)81.1 11.679.8 9.5bmi (kg / m)30.2 values are mean sd, p<0.05 there were significant differences between groups for veines / sym sub - parameters, nhp, bdi, veines - qol and veines / sym total scores before and after receiving treatment (p<0.001) (table 2table 2.comparison of differences for veines - qol, nhp and bdi measurements of the patients between groups (n=117)study groupn=42x sdcontrol groupn=75x sdveines 18.2bdi3.8 8.2 1.3 1.6values are mean sd, p<0.05). furthermore, pre- and post - treatment total score differences for each group were evaluated and maximum differences were found in study group for the scores of nhp, bdi, veines - qol, and veines / sym (table 3table 3.comparison of pre and post treatment scores for groups (n=75)pre treatmentpost treatmentx sdx sdveines / symstudy group22.4 7.528.5 9.7control group29.2 10.127.6 9.8veines - qolstudy group67.8 13.376.7 15.5control group76.8 16.773.3 16.0nhpstudy group213.8 157.1166.1 124.2control group182.0 118.6198.0 120.5bdistudy group11.0 8.57.1 7.7control group12.1 values are mean sd, p<0.05 values are mean sd, p<0.05 the present study showed that short - term use of cs in cvi patients has decreased symptoms and significantly improved quality of life in these patients. the compression treatment for venous insufficiency in lower extremity has been used for hundreds of years since hippocrates. the compression treatment that applies the reduction in venous reflux due to application of compression below knee is well known19. in 112 cvi patients, the use of cs (pressure 3040 mmhg) for one month significantly improved lower extremity pain, oedema, activity tolerance, depression, sleep disorders and qol9. in this study, cs of below knee length was preferred due to its comfort, improvement in cs calf muscle pump, and low cost. cs decreases venous symptoms such as pain, oedema, stasis, and skin changes20. the venous symptoms were improved by the use of class 2 (3040 mmhg) and class 3 (4050 mmhg) cs for six weeks21. similarly, a significant decrease in symptom severity (pain, depression, swelling, sleep) and increase in activity tolerances were observed in cvi patients after use of cs for one month9. the use of cs improved pain and symptoms in 90% patients with cvi22. in line with this, the decrease in venous symptom scores suggests importance of short - term cs usage. the difficulty in wearing cs, financial burden, discomfort of heat and tightness of stocking are responsible for low compliance of cs use in cvi patients. based on review of patients self - report, the mediterranean climate characteristics of hot weather, high cost of compression stockings, and difficulty to wear were the possible reasons. it is known that venous hypertension increases after removing stockings. in a study comparing the duration of cs usage, the use of cs for entire day is more beneficial than for only half day24. in order to decrease ambulatory venous hypertension and to obtain maximum benefits, patients were asked to wear cs all day long. the cs usage in patients was regularly checked via telephone interviews. although venous diseases are highly prevalent, its effect on daily activities and quality of life are not investigated thoroughly. both general and disease - specific qol are accepted as a standard to determine changes in qol. the qol measurement enables better understanding of impact of the disease while evaluating specific problem25. in this study, disease - specific qol was assessed along with veines - qol whereas general qol was assessed with nhp. at the end of one month, significant improvement was observed in venous symptoms, veines - qol scores and nhp parameters in group that used cs than that did not use cs. thus cs usage improved overall qol suggested by improvement in both disease - specific and general qol scores. in earlier study, the use of two different cs for a two - week duration was found to be beneficial for qol in both groups26. the use of class 1 (1015 mmhg) cs for four weeks significantly increased qol compared with and placebo pressure (36 mmhg) stockings27. in a similar study, it was observed that a four - week use of cs increases qol and improves symptoms of cvi28. the decrease of qol in cvi is based on physical function and mobility in addition to depression and social isolation. depression limits performance in physical, social, and personal activities leading to disability27. it was reported that 68% of the patients with venous ulceration suffer fear, depression, social isolation, anger, anxiety, and negative self - image29. however, there is inadequate research on evaluation of depression level in venous diseases and use of scales in directly evaluating depression. in earlier study in patients with venous ulcers and bdi, the scale is used to determine presence of various levels of depression30. in this study, depression was assessed by bdi and short - term use of cs decreased venous symptoms in cvi patients, improved daily activities, independence levels and thus significantly decreased depression levels when baseline and final bdi results were compared in the study group. therefore, regular and long - term use of cs will improve well being of cvi patients. this study was aimed to assess short - term effects of cs use in cvi patients. although these patients should have been followed up for longer term, long - term monitoring was not possible due to transportation difficulties, family reasons, and financial problems. nevertheless, we contacted the patients from control group and educated them about the short - term benefits of cs use. due to risk of development of venous ulcers in the advanced stages of cvi, the regular use of stocking starting from the early stages is recommended to prevent occurrence of cvi symptoms. further studies are necessary to evaluate the effect of cs use by cvi patients in turkish economy and financial burden on cvi individuals in turkey. based on the findings of this study, if the patients could be convinced to use cs for short - term duration, the favourable effects will promote long - term use.	[purpose ] this study was aimed to analyse the effects of short - term use of compression stockings (cs) on symptoms and qol in patients with chronic venous insufficiency (cvi). [subjects and methods ] based on the ceap classification c2 and c3, 117 patients with cvi were enrolled in this study. the participants were divided into two groups. the control group refused to use cs, however, was advised to do exercises and take skin care whereas the cs group used cs and performed exercise. the data were collected by using nottingham health profile (nhp), venous insufficiency epidemiological and economic study (veines - qol / sym) and beck depression inventory (bdi) at baseline and after four weeks of treatment and compared within and in between groups. [results ] the comparison of pre- and post - treatment differences between groups was statistically significant for all parameters. in the study group, pre- and post - treatment scores for each parameter were significantly different. however, elevated scores in the control group suggested worsening of the illness. [conclusion ] this study established that short - term use of cs in patients with cvi significantly improved disease specific and general qol by reducing venous symptoms. further studies with larger sample size are necessary to confirm these findings.
a 30-year - old caucasian woman presented to the emergency department with self - limited severe chest heaviness, which started at rest and lasted for three hours. medication usage included an oral contraceptive (ocp), alprazolam, citalopram, and lansoprazole. laboratory data were significant for a white blood cell count of 11.10 th / mm, serum potassium of 3.4 mmol / l, aspartate transaminase of 70 u / l, and human chorionic gonadotropin was negative. cardiac enzymes were significant for elevated troponin i 27.64 ng / ml (normal value [nv ] : 00.78), creatine kinase (ck) of 484 u / l (nv : 10150 u / l), and ck - mb of 56 ng / ml (nv : 05 ng / ml). initial electrocardiogram (ekg) was unremarkable. the following day the patient underwent transthoracic echocardiogram, which demonstrated mild hypokinesis of the inferior and septal walls with a normal ejection fraction. there was a 70% stenosis in the proximal rca and diffuse luminal irregularities of the vessel (fig. a 3.0 x 38 mm drug eluting stent and 4.0 x 38 mm bare metal stent were inserted in the rca. angiographic image of right coronary artery demonstrating diffuse luminal irregularities and double lumen (indicated by black arrows) diagnostic of spontaneous coronary artery dissection. a 38-year - old caucasian woman presented to an outpatient cardiology clinic with sudden, severe, midsternal chest pressure that began after a rigorous exercise session. she was admitted and coronary angiogram was performed with suspicion of acute coronary syndrome, coronary spasm, and/or scad. patient was found to have a 20% stenosis in mid - lad (fig., contrast was seen to swirl and stay longer than usual, consistent with self - limiting scad. no interventions were performed and patient was discharged on life - long aspirin and clopidogrel for 1 year. avoidance of strenuous exercise was recommended. angiographic image demonstrating left anterior descending artery with 20% stenosis in the mid - section. black arrow indicates area where the contrast was seen to swirl and stay longer than usual, consistent with self - limiting spontaneous coronary artery dissection. (left - hand panel) optical coherence tomography image of the left anterior descending artery demonstrating intimal tear (indicated by white arrow). (right - hand panel) optical coherence tomography image of the left anterior descending artery demonstrating intramural hematoma (asterisk) compromising the true lumen. a 65-year - old caucasian woman presented to the emergency department with worsening intermittent substernal chest pain radiating to her left arm for 2 days. she had significant past medical history of hypertension, unruptured brain aneurysm, and a previous cardiac arrest due to asystole. risk factors for cad included advanced age, family history of premature cardiovascular disease, and tobacco exposure. physical examination was unremarkable with regular heart sounds without murmurs, rubs, or gallops. initial laboratory data were within normal limits without evidence of hematologic, hepatic, or renal disease. patient was transferred to the catheterization lab and a coronary angiogram was obtained with the suspicion of non - st elevation myocardial infarction. the rca was noted to be dominant and the left circumflex artery did not have significant disease. 4). an oct revealed an isolated limited dissection composed of only the intimal layer (fig. patient had a favorable clinical course and was discharged on low dose aspirin, clopidogrel, atorvastatin, low dose lisinopril, and metoprolol. angiographic view of left anterior descending artery with delayed clearing of the contrast suggesting spontaneous coronary artery dissection (indicated by black arrow). optical coherence tomography image of the left anterior descending artery demonstrating intimal flap (indicated by white arrow). a 30-year - old caucasian woman presented to the emergency department with self - limited severe chest heaviness, which started at rest and lasted for three hours. medication usage included an oral contraceptive (ocp), alprazolam, citalopram, and lansoprazole. laboratory data were significant for a white blood cell count of 11.10 th / mm, serum potassium of 3.4 mmol / l, aspartate transaminase of 70 u / l, and human chorionic gonadotropin was negative. cardiac enzymes were significant for elevated troponin i 27.64 ng / ml (normal value [nv ] : 00.78), creatine kinase (ck) of 484 u / l (nv : 10150 u / l), and ck - mb of 56 ng / ml (nv : 05 ng / ml). initial electrocardiogram (ekg) was unremarkable. the following day the patient underwent transthoracic echocardiogram, which demonstrated mild hypokinesis of the inferior and septal walls with a normal ejection fraction. there was a 70% stenosis in the proximal rca and diffuse luminal irregularities of the vessel (fig. a 3.0 x 38 mm drug eluting stent and 4.0 x 38 mm bare metal stent were inserted in the rca. angiographic image of right coronary artery demonstrating diffuse luminal irregularities and double lumen (indicated by black arrows) diagnostic of spontaneous coronary artery dissection. a 38-year - old caucasian woman presented to an outpatient cardiology clinic with sudden, severe, midsternal chest pressure that began after a rigorous exercise session. she was admitted and coronary angiogram was performed with suspicion of acute coronary syndrome, coronary spasm, and/or scad. patient was found to have a 20% stenosis in mid - lad (fig., contrast was seen to swirl and stay longer than usual, consistent with self - limiting scad. no interventions were performed and patient was discharged on life - long aspirin and clopidogrel for 1 year. beta - blocker was not indicated because of sinus bradycardia. avoidance of strenuous exercise was recommended. angiographic image demonstrating left anterior descending artery with 20% stenosis in the mid - section. black arrow indicates area where the contrast was seen to swirl and stay longer than usual, consistent with self - limiting spontaneous coronary artery dissection. (left - hand panel) optical coherence tomography image of the left anterior descending artery demonstrating intimal tear (indicated by white arrow). (right - hand panel) optical coherence tomography image of the left anterior descending artery demonstrating intramural hematoma (asterisk) compromising the true lumen. a 65-year - old caucasian woman presented to the emergency department with worsening intermittent substernal chest pain radiating to her left arm for 2 days. she had significant past medical history of hypertension, unruptured brain aneurysm, and a previous cardiac arrest due to asystole. risk factors for cad included advanced age, family history of premature cardiovascular disease, and tobacco exposure. physical examination was unremarkable with regular heart sounds without murmurs, rubs, or gallops. initial laboratory data were within normal limits without evidence of hematologic, hepatic, or renal disease. patient was transferred to the catheterization lab and a coronary angiogram was obtained with the suspicion of non - st elevation myocardial infarction. the rca was noted to be dominant and the left circumflex artery did not have significant disease. 4). an oct revealed an isolated limited dissection composed of only the intimal layer (fig. patient had a favorable clinical course and was discharged on low dose aspirin, clopidogrel, atorvastatin, low dose lisinopril, and metoprolol. angiographic view of left anterior descending artery with delayed clearing of the contrast suggesting spontaneous coronary artery dissection (indicated by black arrow). optical coherence tomography image of the left anterior descending artery demonstrating intimal flap (indicated by white arrow). the presented case series described three women of differing ages, who presented with chest pain. coronary angiography, in conjunction with oct, was used for scad diagnosis in two of the cases. one of the patients had involvement of the proximal rca and underwent percutaneous coronary intervention (pci), whereas the other two patients had mid - lad disease and were treated conservatively with medical therapy. treatments were in accordance with the degree of stenosis in the involved vessel. from 1931 to 2013, more than 490 cases of scad have been reported in the medical literature (5). recently, however, with the availability of advanced intracoronary imaging, more subtle dissections are being detected and the overall incidence of scad is as high as 14% in patients with acute coronary syndrome. the average age for the incidence of scad is 42 years, though there have been cases reported from age 14 to about the seventh decade of life. around 80% of the cases are females and of those, 2025% occur in the peripartum period. scad has also been found to account for up to 40% of heart attacks in women under the age of 50. almost 50% present as st elevation myocardial infarction and about 25% present with multivessel involvement (6). patients are separated according to underlying condition : 1) non - atherosclerotic / peripartum period group ; 2) atherosclerotic group ; and 3) idiopathic or heterogeneous group in which one of the following conditions are present : connective tissue disorders (marfan syndrome, ehlers danlos syndrome, polyarteritis nodosa, systemic lupus erythematous and fibromuscular dysplasia,) ; medication (ocp, fluorouracil, fenfluramine, cyclosporine) ; cocaine use ; hypertension ; cystic medial necrosis ; variant angina ; and physical exercise (7). the most common conditions related to scad are coronary atherosclerosis and those associated with the peripartum period. in the atherosclerotic group, atherosclerotic plaque inflammation and rupture may induce disruption of the intimal - medial junction, leading to an intimal flap and subsequent intramural hematoma formation (7, 8). eosinophils infiltrate the uterus and serum collagenase levels increase during labor and the peripartum period. eosinophilic infiltration in the coronary artery adventitia breaks down the medial - adventitial layer, leading to scad (9, 10). in this case series, the first case only had the risk factor of obesity for atherosclerotic disease and was not in the peripartum period. however, ocp therapy may have played a role in the condition. in the second case, the patient had minimal atherosclerotic risk factors and was not in the peripartum period, but had strenuous exercise at the time of chest pain onset. in contrast, the last patient had significant risk for cad and fell into the atherosclerotic group. the clinical presentation of scad depends on the extent and flow limiting severity of the coronary dissection. it can range from asymptomatic, to unstable angina, acute myocardial infarction, ventricular arrhythmias, or sudden cardiac death. in the above described cases, one patient presented with the classic features of non - st elevation myocardial infarction and two patients presented with unstable angina. all three patients underwent invasive imaging studies, which contributed to the diagnosis of scad. scad is typically diagnosed with coronary angiography, however it can be underestimated using angiography alone (11). coronary angiography can indicate a radiolucent intimal flap or a delayed clearance of contrast from the false lumen. if there is not an intimal tear, the medial hematoma may appear as a narrowed vessel (3). therefore, in some instances it can be difficult to distinguish atherosclerotic stenosis or spasm from intramural hematoma or dissection. the use of oct is seen as a complementary technique to identify the exact location of the dissection as well as the extent and thickness of the hematoma. histological and clinical studies have shown that oct can identify the microstructure of coronary wall and atherosclerotic plaques (12). in addition ivus has been used to evaluate the relation between the false and the true lumen and dissection. in the first case, the diagnosis was made using coronary angiography alone given the degree of stenosis and the evidence of false lumen seen and corroborated by the inability to advance a guide wire through the rca. in the second case, scad was diagnosed by angiogram, though oct was performed to evaluate the extent of dissection. in contrast, in the third case the lesion was first suspected to be due to spasm because there was no intimal flap or false lumen visible when coronary angiography was performed. optimal treatments for scad have not been clearly determined, with no consensus as to recommendations. therapeutic strategies depend upon the clinical presentation, location, and extent of dissection as well as the ischemic myocardium area at risk (13). options include conservative medical therapy, pci, and coronary artery bypass graft (cabg) (14). conservative medical therapy can be a reasonable initial option in mid- or distal - single vessel dissection with a lumen diameter limitation < 50% (5, 9, 15). some studies have demonstrated that early intervention with either pci or cabg following the diagnosis of scad lead to better outcomes (5). the role of thrombolytics is controversial, and may be associated with the extension of dissection due to increased blood in the vessel wall or hematoma causing compression of the true lumen (9). pci can be the therapy of choice in single vessel disease, especially proximal dissection, in which there are ongoing symptoms and persistent restriction of coronary blood flow. however, pci can be associated with several complications relating to the passage of the coronary wire into the false lumen of the dissected vessel or the loss of coronary flow through the propagation of dissection and displacement of stents, resulting in the propagation of hematoma (15). adjunctive - imaging technologies such as ivus and oct should be considered to determine extent of dissection and to provide real - time guidance for intervention. finally, cabg is the treatment of choice in multiple vessel disease, especially where there is left main stem involvement with ongoing ischemia refractory to medical or interventional therapy (9). in the first case, pci was performed given 70% stenosis of the lumen involving the proximal rca. in the subsequent cases, medical management was pursued due to mild involvement of mid - lad. close and long - term follow up is indicated in scad patients given that 10-year recurrence rates up to 20% have been reported (6). our three patients are being followed up on a 6-month basis in an outpatient cardiology clinic. consideration of scad can be very important in young healthy females, especially when it is difficult to explain the pathogenesis of the stenotic lesion and no atherosclerotic risk factors. oct and ivus can be important tools for obtaining additional information on extent and location of the dissection. lad is the most common site of dissection ; however, as seen within this case series, patients can have rca involvement. the authors have not received any funding or benefits from industry or elsewhere to conduct this study.	spontaneous coronary artery dissection (scad) is a rare and often lethal cause of acute coronary syndrome, which typically affects young women and otherwise healthy individuals. scad can be diagnosed in patients undergoing coronary angiography and can be underestimated. special techniques such as optical coherence tomography (oct) and intravascular ultrasound should be used when there is suspicion of the condition. in the majority of cases, the left anterior descending (lad) artery is involved ; however, a few cases of the right coronary artery (rca) involvement have been reported. this article describes three cases of scad in women of different ages, all presenting with chest pain. coronary angiography in conjunction with oct was used for diagnosis in two of the cases. one of the patients had involvement of the proximal rca and underwent percutaneous coronary intervention, whereas the other two patients had mid - lad disease and were treated conservatively with medical therapy. presently, there are no specific guidelines for the treatment of scad, and therapy is individualized according to extent and severity of the condition.
multiple myeloma (mm) is a malignant disorder characterized by multifocal proliferation of clonal, long - lived plasma cells within the bone marrow (bm). mm presents clinically with a broad range of manifestations, including skeletal destruction, immune suppression, and end - organ sequelae. it is estimated that there will be 22 350 newly diagnosed mm cases and 10 710 mm deaths in the united states in 2013. over the past decade, the outcome of mm patients has been dramatically improved after the development of novel agents and the introduction of autologous stem cell transplantation. however, mm remains an incurable disease and significant efforts aim to unravel its biology. mm is preceded by a premalignant condition termed monoclonal gammopathy of undetermined significance (mgus). mgus is present in more than 3% of the population above the age of 50 years. its annual progression rate to mm is of 0.5% to 3%. in some patients, an intermediate but more advanced premalignant stage, to date, the biological alterations that lead from mgus to smm and finally to symptomatic disease have not been elucidated. mm evolution is characterized by an avascular stage of slow tumor progression followed by angiogenic switch when the balance between pro- and anti - angiogenic factors in the tumor microenvironment is deranged to promote neovascularization and disease progression. angiogenesis is a multistep and tightly regulated process of the formation of new blood vessels, which occurs physiologically during embryonic growth and wound healing, but also in tumor growth and metastasis. our previous study elucidated the role of angiogenic cytokines in mm pathogenesis, and our results were consistence with those of other groups [1115 ]. these cytokines mainly include the vascular endothelial growth factor (vegf), the angiogenin, the angiopoietins, and the basic fibroblast growth factor (bfgf). angiopoietin-1 (ang-1) and its natural antagonist, angiopoietin-2 (ang-2), are involved in the biology of mm. we have shown that the ang-1 to ang-2 ratio is an independent prognostic factor for survival in newly diagnosed mm patients. however, to our knowledge, no previous studies have evaluated the above angiogenic cytokines in a large population of each of the mm stages. the aims of this study were to : i) evaluate the circulating levels of important angiogenesis inducers vegf - a, angiogenin, ang-1, and ang-2 as representative angiogenesis inhibitors in patients with mgus, smm, and symptomatic mm ; and ii) to evaluate the gene expression of vegf - a and determine if any variability of this predominant angiogenic cytokine is present during the evolutionary process of precursor stages to symptomatic mm, contributing to better understanding the underlying mechanisms of this transition. we studied 109 consecutive patients, of whom 55 were newly diagnosed and symptomatic with mm (n=55), and 54 asymptomatic mm patients (n=54). we also evaluated 27 patients with mgus at diagnosis and, as an internal control group, 22 healthy controls of similar age and gender (12m/10f ; median age : 67 years, range : 3983 years). the study was conducted after approval from the institutional ethical committee and under the guidelines of the declaration of helsinki. the medical history of all subgroups was reviewed to ensure that there was no disease (e.g., cardiovascular disorder, inflammatory disease, renal impairment, or infection) or drug administration that could alter angiogenesis at the time of sampling. serum samples were collected at the time of diagnosis from all patients, before the initiation of any antimyeloma treatment, including supportive treatment (e.g., bisphosphonate administration) and stored at 80c until the day of measurement. patient and control group characteristics are depicted in table 1. peripheral blood mononuclear cells (pbmcs) and plasma cells from bone marrow aspirates used in gene expression study briefly, pbmcs were isolated by using ficoll - plaque density sedimentation from freshly drawn peripheral blood as previously described. mm plasma cells from bone marrow aspirates were purified by positive selection with anti - cd138 magnetic activated cell - sorting (macs) separation microbeads, as described by the manufacturer (macs, miltenyi biotec, auburn, ca). purity, as confirmed by flow cytometry cd38 and cd45 staining (facscalibur cytometer), was above 95% in all mgus and mm cases, and above 90% in all mm patients. the gene expression of vegf - a in pbmcs and in mm plasma cells was estimated in a total of 10 patients with mgus, 10 patients with smm, and 10 patients with symptomatic mm. the gene expression of vegf - a in pbmcs was also studied in 10 healthy volunteers. circulating levels of ang-1, ang-2, vegf, and angiogenin were evaluated in all patients, using an elisa methodology (r&d systems, minneapolis, mn,), according to the manufacturer s instructions. total rna was extracted with trizol according to the manufacturer s instructions (invitrogen) and purified from salts and residual dna using the rnease mini kit (qiagen). quantity of rna in each sample was measured by spectroscopy and integrity was determined by gel electrophoresis. only rnas with clear 18s and 28s peaks were used. vegf - a expression was studied using semi - quantitative rt - pcr as described previously. briefly, rt - pcr was performed as a multiplex using the titan one tube rt - pcr system according to the manufacturer s instructions (roche diagnostics gmbh, mannheim germany). following rt - pcr, the samples were treated with exosap - it to remove excess primer and deoxyribonucleotides (dntps ; usb), as described by the manufacturer. a portion of the rt - pcr product was used in the fast start dna master sybr green i kit. this reaction was run on the light - cycler (roche diagnostics gmbh) until a sample reached plateau stage. pcr products were run on 1% agarose gel, and relative quantification was performed by spot densitometry comparisons with gapdh (glyceraldehyde-3-phosphate dehydrogenase) bands. primer sequences were as follows : for gapdh, forward 5-accacagtccatgccatcac-3 and reverse 5-tccaccaccctgttgcttgta-3 ; for vegf - a, forward 5-gcccactgaggagtccaacatc-3 and reverse 5-tttttgcaggaacatttacacg-3. melting curve analysis of the pcr products was performed to confirm the presence of a single specific amplicon. differences between patients and controls, as well as between different patient subsets, were evaluated using the mann - whitney u test. we studied 109 consecutive patients, of whom 55 were newly diagnosed and symptomatic with mm (n=55), and 54 asymptomatic mm patients (n=54). we also evaluated 27 patients with mgus at diagnosis and, as an internal control group, 22 healthy controls of similar age and gender (12m/10f ; median age : 67 years, range : 3983 years). the study was conducted after approval from the institutional ethical committee and under the guidelines of the declaration of helsinki. the medical history of all subgroups was reviewed to ensure that there was no disease (e.g., cardiovascular disorder, inflammatory disease, renal impairment, or infection) or drug administration that could alter angiogenesis at the time of sampling. serum samples were collected at the time of diagnosis from all patients, before the initiation of any antimyeloma treatment, including supportive treatment (e.g., bisphosphonate administration) and stored at 80c until the day of measurement. patient and control group characteristics are depicted in table 1. peripheral blood mononuclear cells (pbmcs) and plasma cells from bone marrow aspirates used in gene expression study briefly, pbmcs were isolated by using ficoll - plaque density sedimentation from freshly drawn peripheral blood as previously described. mm plasma cells from bone marrow aspirates were purified by positive selection with anti - cd138 magnetic activated cell - sorting (macs) separation microbeads, as described by the manufacturer (macs, miltenyi biotec, auburn, ca). purity, as confirmed by flow cytometry cd38 and cd45 staining (facscalibur cytometer), was above 95% in all mgus and mm cases, and above 90% in all mm patients. the gene expression of vegf - a in pbmcs and in mm plasma cells was estimated in a total of 10 patients with mgus, 10 patients with smm, and 10 patients with symptomatic mm. the gene expression of vegf - a in pbmcs was also studied in 10 healthy volunteers. circulating levels of ang-1, ang-2, vegf, and angiogenin were evaluated in all patients, using an elisa methodology (r&d systems, minneapolis, mn,), according to the manufacturer s instructions. total rna was extracted with trizol according to the manufacturer s instructions (invitrogen) and purified from salts and residual dna using the rnease mini kit (qiagen). quantity of rna in each sample was measured by spectroscopy and integrity was determined by gel electrophoresis. only rnas with clear 18s and 28s peaks were used. vegf - a expression was studied using semi - quantitative rt - pcr as described previously. briefly, rt - pcr was performed as a multiplex using the titan one tube rt - pcr system according to the manufacturer s instructions (roche diagnostics gmbh, mannheim germany). following rt - pcr, the samples were treated with exosap - it to remove excess primer and deoxyribonucleotides (dntps ; usb), as described by the manufacturer. a portion of the rt - pcr product was used in the fast start dna master sybr green i kit. this reaction was run on the light - cycler (roche diagnostics gmbh) until a sample reached plateau stage. pcr products were run on 1% agarose gel, and relative quantification was performed by spot densitometry comparisons with gapdh (glyceraldehyde-3-phosphate dehydrogenase) bands. primer sequences were as follows : for gapdh, forward 5-accacagtccatgccatcac-3 and reverse 5-tccaccaccctgttgcttgta-3 ; for vegf - a, forward 5-gcccactgaggagtccaacatc-3 and reverse 5-tttttgcaggaacatttacacg-3. melting curve analysis of the pcr products was performed to confirm the presence of a single specific amplicon. differences between patients and controls, as well as between different patient subsets, were evaluated using the mann - whitney u test. symptomatic mm patients at diagnosis showed increased levels of serum ang-2 (mean value sd : 52713370 pg / ml) compared with smm patients (20431881pg / ml ; p<0.001), mgus patients (18511516pg / ml ; p<0.001), and healthy controls (21491756 pg / ml, p<0.001). there was no statistically significant difference between asymptomatic patients, mgus patients, and controls (figure 1a). there was no difference in circulating levels of ang-1 between patients with mm, smm, mgus, and healthy controls (table 2). consequently, the ang-1/ang-2 ratio was reduced in symptomatic mm patients (mean value sd : 8.07.3) compared to smm patients (19.812.3 ; p<0.001), mgus patients (22.215.3 ; p<0.001), and healthy controls (18.016.6 ; p<0.001), but there was no significant difference between asymptomatic patients, mgus patients, and controls (figure 1b). serum levels of vegf were increased in patients with mgus (399188 pg / ml ; p=0.02), smm (403273 pg / ml ; p=0.04), and mm (613408 pg / ml ; p=0.001) compared to healthy controls (286267 pg / ml). patients with mm had increased vegf serum levels compared to mgus and smm patients (p=0.039 and p=0.009, respectively (figure 2a). patients with mgus, smm, and symptomatic mm had increased levels of serum angiogenin (379.9155.6 ng / ml, 377.1137.8 ng / ml and 255.6137.2 pg / ml, respectively) compared with the healthy controls (165.134.3 ng / ml ; p<0.001 for all comparisons). patients with mm had decreased angiogenin serum levels compared to mgus and smm patients (p<0.001) for both comparisons. there was no statistically significant difference between smm and mgus patients (figure 2b). a trend was observed in the correlation of angiogenin serum levels with ang-2 (r=0.250, p=0.008) and ang-1/ang-2 ratio (r=0.217, p=0.022). we found that vegf - a is expressed in pbmcs and mm plasma cells from all patients in all groups analyzed. in pbmcs, we observed no significant difference in the levels of vegf - a mrna expression between healthy volunteers, mgus, smm, and mm patients, with median sd vegf - a / gapdh ratios of 1.0190.081, 1.0870.093, 1.1160.091 and 1.1190.109, respectively. similarly, no difference in the levels of vegf - a expression was observed in mm plasma cells among the 3 patients groups, with median sd vegf - a / gapdh ratios of 0.9330.1322, 1.0280.1279, and 0.9730.1406, respectively (figure 3). symptomatic mm patients at diagnosis showed increased levels of serum ang-2 (mean value sd : 52713370 pg / ml) compared with smm patients (20431881pg / ml ; p<0.001), mgus patients (18511516pg / ml ; p<0.001), and healthy controls (21491756 pg / ml, p<0.001). there was no statistically significant difference between asymptomatic patients, mgus patients, and controls (figure 1a). there was no difference in circulating levels of ang-1 between patients with mm, smm, mgus, and healthy controls (table 2). consequently, the ang-1/ang-2 ratio was reduced in symptomatic mm patients (mean value sd : 8.07.3) compared to smm patients (19.812.3 ; p<0.001), mgus patients (22.215.3 ; p<0.001), and healthy controls (18.016.6 ; p<0.001), but there was no significant difference between asymptomatic patients, mgus patients, and controls (figure 1b). serum levels of vegf were increased in patients with mgus (399188 pg / ml ; p=0.02), smm (403273 pg / ml ; p=0.04), and mm (613408 pg / ml ; p=0.001) compared to healthy controls (286267 pg / ml). patients with mm had increased vegf serum levels compared to mgus and smm patients (p=0.039 and p=0.009, respectively (figure 2a). patients with mgus, smm, and symptomatic mm had increased levels of serum angiogenin (379.9155.6 ng / ml, 377.1137.8 ng / ml and 255.6137.2 pg / ml, respectively) compared with the healthy controls (165.134.3 ng / ml ; p<0.001 for all comparisons). patients with mm had decreased angiogenin serum levels compared to mgus and smm patients (p<0.001) for both comparisons. there was no statistically significant difference between smm and mgus patients (figure 2b). a trend was observed in the correlation of angiogenin serum levels with ang-2 (r=0.250, p=0.008) and ang-1/ang-2 ratio (r=0.217, p=0.022). we found that vegf - a is expressed in pbmcs and mm plasma cells from all patients in all groups analyzed. in pbmcs, we observed no significant difference in the levels of vegf - a mrna expression between healthy volunteers, mgus, smm, and mm patients, with median sd vegf - a / gapdh ratios of 1.0190.081, 1.0870.093, 1.1160.091 and 1.1190.109, respectively. similarly, no difference in the levels of vegf - a expression was observed in mm plasma cells among the 3 patients groups, with median sd vegf - a / gapdh ratios of 0.9330.1322, 1.0280.1279, and 0.9730.1406, respectively (figure 3). several cytokines are implicated in the angiogenesis process. the cross - talk among angiogenic and inflammatory cytokines plays an important role in the modulation of blood vessel growth in several pathological conditions, including cancer. in the abnormal tumor microenvironment, pro - angiogenic cytokines and inflammatory mediators prevail and promote structurally and functionally aberrant vessel formation. during tumor angiogenesis, quiescent endothelial cells become sensitive to vegf, then proliferate and migrate to form new vessels. bone marrow - derived endothelial cell progenitors are found in the peripheral blood and are recruited by angiogenin at sites of angiogenesis [2023 ]. mm was the first hematologic malignancy in which angiogenesis were defined as a prognostic factor. first demonstrated that tumor growth is angiogenesis - dependent, by reporting a high correlation between the extent of bm angiogenesis (microvessel area) and the proliferating fraction of bm plasma cells (labeling index) in mgus and mm patients. angiogenesis itself was significantly associated with active, as opposed to non - active, mm and mgus. angiogenesis is possibly important in the progression of mgus to mm and in the progression of early - stage myeloma to advanced, refractory disease. previous reports have shown that bone marrow angiogenesis progressively increases along the spectrum of plasma cell disorders, from the more benign stage of mgus to advanced myeloma, indicating that angiogenesis may be related to disease progression. a proposed mechanism is that angiogenesis contributes to myeloma pathogenesis and progression by ensuring an adequate tumor oxygen and nutrient supply and by paracrine stimulation of tumor cell migration and proliferation. however, the precise mechanism underlying the observed alterations of angiogenic cytokines in the evolution of mm has not been fully investigated ; our study aimed to answer this critical question. vegf was increased in the serum of patients with mm as well as in patients with mgus and smm compared with healthy control samples, indicating that myeloma genesis, the transition process to symptomatic disease, occurs via mechanisms that are less vegf - dependent. nevertheless, patients with symptomatic disease had higher vegf serum levels than other patients, perhaps due to greater tumor burden. however, post - transcriptional modifications such as stabilization of mrna, induction of translation and stabilization of final protein could be responsible, since we found no significant difference in the levels of vegf - a mrna expression from plasma cells of mgus, smm, and mm patients. reduced angiopoietin-1/angiopoietin-2 ratio has been previously correlated with advanced disease features, including international staging system (iss)-3 stage, renal impairment, 2-microglobulin levels, and extensive bone disease, as well as with inferior survival in newly diagnosed patients with mm who received therapy with novel antimyeloma agents. in the present study, symptomatic mm patients, but not mgus or smm patients, had increased serum levels of ang-2 and decreased ang-1/ang-2 ratio compared with healthy controls. interestingly, the patients with symptomatic mm exhibit decreased serum levels of angiogenin compared to mgus and smm patients, but had increased levels compared to healthy controls. furthermore, it is well established that there is a widespread expression pattern of angiogenin, suggesting a physiological function that is not restricted to the neovascularization process. further investigation is needed to unravel the complex network of angiogenic cytokines and their precise role in the biology of mm. patients with smm have similar levels of circulating angiopoietins, angiogenin, and vegf compared with mgus patients, but these levels are altered compared to symptomatic mm patients. ang-1/ang-2 and angiogenin is reduced in mm patients with symptomatic disease compared to mgus or smm. these results suggest that in the progression of mgus to smm, circulating angiogenic molecules appear to be the same. in contrast, in symptomatic disease the alterations of angiopoietins pathway along with vegf contribute to myeloma cell growth. because there is great need for novel drugs active against myeloma cells, our data support that the ang-1/ang-2/tie-2 axis may be an effective target for the development of novel antimyeloma agents.	backgroundsymptomatic multiple myeloma (mm) evolves from an asymptomatic precursor state termed monoclonal gammopathy of undetermined significance (mgus) and smoldering myeloma (smm). angiogenesis plays a key role in the pathogenesis of mm but there are very limited data for angiogenesis in smm.material/methodswe measured the circulating levels of angiopoietin-1 (ang-1), angiopoietin-2 (ang-2), vascular endothelial growth factor (vegf), and angiogenin in 54 patients with smm. the results were compared with those of 27 mgus patients, 55 mm patients, and 22 healthy controls. the expression of vegf - a gene was also evaluated in 10 patients with smm, 10 with symptomatic mm, and 10 with mgus.resultsthe ratio of circulating ang-1/ang-2 was reduced in mm patients with symptomatic disease due to a dramatic increase of ang-2 (p<0.001), but not in patients with smm or mgus, in whom it did not differ compared to controls. vegf and angiogenin were increased in all patients compared to controls. however, circulating vegf was higher in symptomatic mm compared to smm and mgus, while angiogenin was reduced. there were no differences in the expression of vegf - a among the 3 patients categories.conclusionssmm has a circulating angiogenic cytokine profile similar to that of mgus, but has altered profile compared to symptomatic mm. thus, in the progression of mgus to smm, circulating angiogenic cytokines seem to be the same. on the contrary, in symptomatic myeloma, the alterations of angiopoietins along with vegf contribute to myeloma cell growth, supporting the target of these molecules for the development of novel anti - myeloma agents.
the pneumococcal polysaccharide conjugated vaccine (13-valent, adsorbed ; pcv13, prevnar 13) is approved in adults 50 years for the prevention of pneumonia in the usa [13 ]. lichenoid dermatitis describes a skin condition that is microscopically characterized by band - like lymphocytic inflammation with alteration of the epidermal basal layer. the case of a man who developed a lichenoid dermatitis at the site of immunization within 2 weeks after receiving the pcv13 vaccine is described, and cutaneous conditions occurring at vaccinations sites are reviewed. a 70-year - old male presented for evaluation of a new, asymptomatic, red and scaly lesion on his left arm. he had received a vaccination, pneumococcal polysaccharide conjugated vaccine (13-valent, adsorbed ; pcv13, prevnar 13) at the site 5 weeks earlier. within 2 weeks after being immunized, he noticed the skin lesion. he had been vaccinated with the pneumococcal polysaccharide vaccine (ppsv23, pneumovax) 6 years earlier. cutaneous examination showed a 12 5-mm linear, focally crusted, erythematous nodule on the deltoid area of his left arm (fig. 1distant (a) and closer (b) views of the pneumococcal vaccination site on the left deltoid area show a 12 5 mm linear, focally crusted, erythematous nodule distant (a) and closer (b) views of the pneumococcal vaccination site on the left deltoid area show a 12 5 mm linear, focally crusted, erythematous nodule microscopic evaluation of a 3-mm punch biopsy showed orthokeratosis, acanthosis, and a prominent granular layer. dyskeratotic keratinocytes were present in the epidermis, and there was a vacuolar change of the basal cells at the dermoepidermal interface. in the upper dermis there was dense, band - like infiltration of lymphocytes with occasional exocytosis of the inflammatory cells into the overlying spongiotic epidermis (fig. (a) and closer (b, c) views of the skin biopsy show orthokeratosis, acanthosis, and a prominent granular layer (a, b). there is mild spongiosis (c), dyskeratotic keratinocytes (a, b), and vacuolar change of the basal cells at the dermoepidermal interface (a c). lymphocytes are present in a dense, band - like infiltrate in the upper dermis (a, b), and there is exocytosis of the inflammatory cells into the overlying epidermis (a, c) (hematoxylin and eosin, a = 10 ; b = 20 ; c = 20) distant (a) and closer (b, c) views of the skin biopsy show orthokeratosis, acanthosis, and a prominent granular layer (a, b). there is mild spongiosis (c), dyskeratotic keratinocytes (a, b), and vacuolar change of the basal cells at the dermoepidermal interface (a c). lymphocytes are present in a dense, band - like infiltrate in the upper dermis (a, b), and there is exocytosis of the inflammatory cells into the overlying epidermis (a, c) (hematoxylin and eosin, a = 10 ; b = 20 ; c = 20) correlation of the history, clinical morphology, and pathologic changes established the diagnosis of a lichenoid dermatitis occurring at the vaccination site of the pcv13 vaccine. the residual dermatosis resolved spontaneously within 4 weeks after the biopsy, corresponding to 9 weeks after immunization. the 7-valent pneumococcal conjugated vaccine (pcv, known by the trade name prevnar) was licensed by the food and drug administration (fda) on 17 february 2000. pcv13 consists of 13 serotype - specific polysaccharides of s. pneumoniae (1, 3, 4, 5, 6a, 7f, 9v, 14, 18c, 19a, 19f, and 23f) conjugated individually to non - toxic diphtheria crd197 carrier protein and adsorbed on aluminum phosphate. the us advisory committee on immunization practices (acip) has recommended pcv13 [in series with 23-valent pneumococcal polysaccharide vaccine (ppvs23, pneumovax) ] for all adults aged 65 years. a single 0.5-mm dose is given as an intramuscular injection in the deltoid muscle [3, 59 ]. pcv13 has been associated with not only systemic adverse events, but also local reactions of only mild or moderate severity ; no vaccine - related serious events were reported. systemic adverse events associated with the vaccine include arm movement limitation, arthralgia, chills, decreased appetite, diarrhea, fatigue, fever, headache, myalgia, rash, and vomiting. in general, these reactions were less common in older persons than in younger individuals [13, 7 ]. skin and subcutaneous tissue disorders accounted for 20% of total adverse events following immunization in a 10-year retrospective analysis of spontaneous reports following pediatric immunizations. cutaneous reactions to vaccinations can be nonspecific or related to the live attenuated virus ; they can also be localized to the injection site or associated with generalized hypersensitivity reactions. investigators have proposed not only the case definition, but also the guidelines for collection, analysis, and presentation of immunization safety data for local reactions at or near vaccine injection sites. cutaneous reactions at the site of pcv13 are summarized in table 1 [14, 1317 ]. local reactions such as erythema, pain and swelling at the injection site were common ; they were more severe in younger aged patients than in older individuals [13 ]. the reported patient is the first individual with a lichenoid dermatitis that appeared at the vaccine injection site within 2 weeks and spontaneously resolved in 9 weeks.table 1pneumococcal vaccination site reactionsinjection site reactions : abscess or cellulitis erythema, pain, and swelling [14]itching granuloma keratoacanthoma lichenoid dermatitis [current report]sweet s syndrome delayed maturation of the antibody response to the pneumococcal vaccine was observed in children with atopic eczema who received pneumovax ii specifically, in comparison to controls (of whom 57% responded to the vaccine), only 17% of children with atopic dermatitis aged 38 years responded to pneumovax ii injection site reactions following vaccination with the 7-valent pneumococcal conjugated vaccine (pcv, trade name prevnar, wyeth pharmaceuticals, philadelphia, pa) were described in 54% of 4154 reports of events after immunization ; 8 serious reports described abscess or cellulitis itching granuloma occurred in 38 of 4758 children (0.83%) who received infanrix or pentavac alone or concomitant with pneumococcal conjugate (prevnar). accompanying clinical features at the injection site in some of the patients also included bluish discoloration, dermatitis, excoriations, hyperpigmentation, hypertrichosis, and scar. contact allergy to aluminum was verified in 29 of the 34 children (85%) who were evaluated by epicutaneous testing with aluminum sweet s syndrome was also triggered by pneumococcal vaccination that had taken place 15 days earlier ; however, the patient did not have any lesions at the injection site pneumococcal vaccination site reactions delayed maturation of the antibody response to the pneumococcal vaccine was observed in children with atopic eczema who received pneumovax ii specifically, in comparison to controls (of whom 57% responded to the vaccine), only 17% of children with atopic dermatitis aged 38 years responded to pneumovax ii injection site reactions following vaccination with the 7-valent pneumococcal conjugated vaccine (pcv, trade name prevnar, wyeth pharmaceuticals, philadelphia, pa) were described in 54% of 4154 reports of events after immunization ; 8 serious reports described abscess or cellulitis itching granuloma occurred in 38 of 4758 children (0.83%) who received infanrix or pentavac alone or concomitant with pneumococcal conjugate (prevnar). accompanying clinical features at the injection site in some of the patients also included bluish discoloration, dermatitis, excoriations, hyperpigmentation, hypertrichosis, and scar. contact allergy to aluminum was verified in 29 of the 34 children (85%) who were evaluated by epicutaneous testing with aluminum sweet s syndrome was also triggered by pneumococcal vaccination that had taken place 15 days earlier ; however, the patient did not have any lesions at the injection site cutaneous dermatoses and malignancies have occurred at the sites of other vaccines, including bacillus calmette - guerin (table 2) [1844 ], hepatitis b (table 3) [28, 4553 ], smallpox (table 4) [28, 5479 ], tetanus (table 5) [14, 8086 ], and others (table 6) [36, 51, 55, 73, 8797 ]. granuloma annulare is one of the more common skin conditions to subsequently occur at the site of an immunization [25, 26, 46, 98, 99 ]. the most frequently observed vaccination site - associated malignancy is basal cell carcinoma [33, 38, 5557, 73 ], followed by squamous cell carcinoma [33, 38, 54, 55, 57, 73 ]. however, the prevalence of melanoma [55, 57, 73 ] and sarcomas (dermatofibrosarcoma protuberans, fibrosarcoma, and malignant fibrous histiocytoma) [55, 57, 6062, 72 ] in smallpox vaccination scars as compared to the scars of other vaccines is greater than expected and may be secondary to a unique characteristic of the vaccine (table 7) [15, 33, 38, 5457, 6062, 69, 72, 73, 86, 90, 9496].table 2bacillus calmette - guerin (bcg) vaccination site reactionsabscess [1820]basal cell carcinoma blistering [18, 19, 22]epithelial cysts [18, 19, 22]erythema [18, 19, 22]erythematous nodule (necrotizing granulomatous reaction) fixed drug eruption foreign body granuloma (non - necrotizing) granuloma annulare [25, 26]granuloma (delayed) isotopic response to patch testing keloid [7, 19, 22, 28]lupus vulgaris (cutaneous tuberculosis) [2933]lymphadenopathy (suppurative) [1820, 34]papular tuberculids pilomatricoma psoriasis sarcoidosis (juvenile) squamous cell carcinoma [33, 38]sweet s syndrome [39, 40]tufted angioma ulceration [18, 19, 22]ulceration during kawasaki disease [42, 43]vasculitis (ulcerating) table 3hepatitis b vaccination site reactionschurg strauss vasculitis granuloma annulare injection site reactions : edema, erythema, induration, and pain keloid mastocytoma necrobiotic granuloma nodules papulonodular lichenoid and pseudolymphomatous reaction subcutaneous nodule (cutaneous b - cell pseudolymphoma) generalized lichenoid reactions and lichen planus have occurred following hepatitis b vaccination ; however, the initial or individual lesion was not localized to the site of vaccination [52, 53]table 4smallpox vaccination site reactionsallergic contact dermatitis basal cell carcinoma [5557]dermatitis, chronic dermatofibroma [58, 59]dermatofibrosarcoma protuberans [55, 57, 60, 61]fibrosarcoma herpes simplex virus infection inflammatory reaction, localized keloid (exaggerated scarring) [28, 55, 6468]keratoacanthoma lupus erythematosus (discoid) [70, 71]malignant fibrohistiocytoma [55, 57, 72]melanoma [55, 57, 73]myxedematous infiltration, diffuse (graves disease) nevus sebaceous pigmentation [76, 77]post scab lesions progressive vaccinia pyogenic infections robust take scar response (normal) [57, 78]squamous cell carcinoma [54, 55, 57]sweet s syndrome a localized inflammatory reaction at the vaccination site heals with a slightly depressed smooth scar that slowly fades and rarely requires treatment the morphology of the lesion includes (in order of frequency) erythema, papule, pustule, vesicle, induration, and scab ; all except induration and scab may recur in some patients after the original lesions spontaneously resolve. lesion biopsies (in 4 patients) showed allergic contact dermatitis (2), chronic dermatitis (1), and squamous cell carcinoma (1) progressive vaccinia is also referred to as disseminated vaccinia, prolonged vaccinia, vaccinia gangrenosum, and vaccinia necrosum. the vaccination site does not heal ; there is painless progressive necrosis that develops into an ulcerative lesion. additional lesions may or may not appear at distant sites, such as skin, bone, and viscera robust take is a non - progressive cutaneous reaction at the vaccination site of > 7.5 cm with swelling, warmth, and joint pain ; the symptoms peak at 810 days post vaccination, and there is improvement within 2472 h the normal scar response at the site of vaccination is the following sequence : papule at day 4 post vaccination, pustule at day 714, and scab at day 21table 5tetanus vaccination site reactionstetanus vaccine angiolymphoid hyperplasia with eosinophils indurated erythematous plaque (cutaneous b - cell pseudolymphoma) granuloma annulare tetanus and diphtheria vaccine granuloma annulare tetanus, diphtheria, and pertussis vaccine abscess (mycobacterium tuberculosis) deep reactive nodular infiltrates of mixed inflammation necrotizing granuloma tetanus, diphtheria, pertussis, and polio vaccine abscess (mycobacterium chelonei) tetanus, diphtheria, pertussis, polio, and haemophilus influenza type b vaccine itching granuloma subcutaneous nodule (sterile abscess) tetanus, plague, and yellow fever vaccine dermatofibrosarcoma protuberans itching granuloma occurred in 38 of 4758 children (0.83%) who received infanrix or pentavac alone or concomitant with pneumococcal conjugate (prevnar). accompanying clinical features at the injection site in some of the patients also included bluish discoloration, dermatitis, excoriations, hyperpigmentation, hypertrichosis, and scar. contact allergy to aluminum was verified in 29 of the 34 children (85%) who were evaluated by epicutaneous testing with aluminum the patient received the pentacel vaccinetable 6other vaccination site reactionsearly summer meningitis subcutaneous nodule (cutaneous b - cell pseudolymphoma) influenza vaccine psoriasis sweet s syndrome [8789]leishmaniasis vaccine dermatofibrosarcoma protuberans varicella - zoster virus vaccine burning erythema pruritus subcutaneous nodule (pseudolymphoma) zosteriform eruption vaccine not specified basal cell carcinoma [55, 73 ] dermatofibrosarcoma protuberans [55, 94, 95 ] lentigo maligna lichen sclerosus et atrophicus melanoma squamous cell carcinoma [55, 73 ] includes pigmented dermatofibrosarcoma also referred to as bednar tumor [55, 94]table 7cancers at vaccination sitesbasal cell carcinoma bacillus calmette - guerin vaccine [33, 38 ] smallpox vaccine [5557 ] not specified [55, 72]dermatofibrosarcoma protuberans leishmaniasis immunization smallpox vaccine [55, 57, 60, 61 ] tetanus, plague, and yellow fever vaccines travel immunization not specified [55, 95]fibrosarcoma smallpox vaccine [57, 62]keratoacanthoma pneumococcal vaccine smallpox vaccine malignant fibrous histiocytoma smallpox vaccine [55, 57, 72]melanoma smallpox vaccine [55, 57, 73 ] not specified [55, 96]squamous cell carcinoma bacillus calmette - guerin vaccine [33, 38 ] smallpox vaccine [54, 55, 57 ] not specified [55, 73 ] these also include pigmented dermatofibrosarcoma protuberans (bednar tumors) [55, 94 ] bacillus calmette - guerin (bcg) vaccination site reactions hepatitis b vaccination site reactions generalized lichenoid reactions and lichen planus have occurred following hepatitis b vaccination ; however, the initial or individual lesion was not localized to the site of vaccination [52, 53 ] smallpox vaccination site reactions a localized inflammatory reaction at the vaccination site heals with a slightly depressed smooth scar that slowly fades and rarely requires treatment the morphology of the lesion includes (in order of frequency) erythema, papule, pustule, vesicle, induration, and scab ; all except induration and scab may recur in some patients after the original lesions spontaneously resolve. lesion biopsies (in 4 patients) showed allergic contact dermatitis (2), chronic dermatitis (1), and squamous cell carcinoma (1) progressive vaccinia is also referred to as disseminated vaccinia, prolonged vaccinia, vaccinia gangrenosum, and vaccinia necrosum. the vaccination site does not heal ; there is painless progressive necrosis that develops into an ulcerative lesion. additional lesions may or may not appear at distant sites, such as skin, bone, and viscera robust take is a non - progressive cutaneous reaction at the vaccination site of > 7.5 cm with swelling, warmth, and joint pain ; the symptoms peak at 810 days post vaccination, and there is improvement within 2472 h the normal scar response at the site of vaccination is the following sequence : papule at day 4 post vaccination, pustule at day 714, and scab at day 21 tetanus vaccination site reactions itching granuloma occurred in 38 of 4758 children (0.83%) who received infanrix or pentavac alone or concomitant with pneumococcal conjugate (prevnar). accompanying clinical features at the injection site in some of the patients also included bluish discoloration, dermatitis, excoriations, hyperpigmentation, hypertrichosis, and scar. contact allergy to aluminum was verified in 29 of the 34 children (85%) who were evaluated by epicutaneous testing with aluminum the patient received the pentacel vaccine other vaccination site reactions includes pigmented dermatofibrosarcoma also referred to as bednar tumor [55, 94 ] cancers at vaccination sites these also include pigmented dermatofibrosarcoma protuberans (bednar tumors) [55, 94 ] vaccine - associated adverse effects at the site of injection may, in part, be secondary to the the polysaccharides and bases it contains. however, there are no predictors as to which individuals will develop these side effects. it remains to be determined why some patients experienced inflammatory dermatoses whereas others developed neoplasms at their vaccination sites. proposed the immunocompromised district as a unifying concept for the development of skin disorders and cancer at lymphoedematous, herpes - infected, and otherwise damaged cutaneous sites in 2009. the concept has subsequently been expanded with regards to not only the factors responsible for the regional immune dysregulation (which also include burns, ionizing and ultraviolet radiation, neurologic disorders such as paralytic stroke and poliomyelitis, tattooing, and trauma such as amputation), but also the skin diseases arising in the affected sites (such as granulomatous reactions, immunity - related disorders, infections, and tumors) [100102 ]. the occurrence of cutaneous dermatoses or skin cancers at vaccination sites is another example of an immunocompromised cutaneous district that has been created by local immunologic changes induced by the vaccine. vaccination site reactions have been observed following immunization with several vaccines including bacillus calmette - guerin, hepatitis b, influenza, leishmaniasis, meningitis, pneumococcal, smallpox, tetanus (alone and in combination with diphtheria, pertussis, polio, haemophilus influenza type b or plague and yellow fever), and varicella - zoster. the reactions at the vaccine injection sites are either cutaneous dermatoses or neoplasms. in this report, a man is described who developed a lichenoid dermatitis that occurred at the pneumococcal vaccine injection site within 2 weeks after pcv13 vaccination ; the erythematous nodule resolved spontaneously within 9 weeks following immunization. dermatoses at the injection sites of vaccines can be granulomatous, immunity - related conditions, infections, lichenoid, neutrophilic, or pseudolymphomatous. vaccination site - associated malignancies most commonly are basal cell carcinoma or squamous cell carcinoma ; however, smallpox vaccine - related site neoplasms also include melanoma and sarcomas (dermatofibrosarcoma protuberans, fibrosarcoma, and malignant fibrous histiocytoma). the pathogenesis of vaccination site reactions is hypothesized to be the result of a cutaneous immunocompromised district created by vaccine - induced local immunologic changes. this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made.	backgroundcutaneous dermatoses and malignancies have occurred at the sites of vaccines.purposeto describe a man who developed a lichenoid dermatitis at the pneumococcal vaccine injection site and to review cutaneous dermatoses and malignancies occurring at vaccination sites.methodspubmed was used to search the following terms, separately and in combination : adverse, condition, cutaneous, dermatosis, dermatitis, injection, pcv13, pneumococcal, pneumonia, prevnar, reaction, skin, site, vaccination, and vaccine. all papers were reviewed, and relevant manuscripts, along with their reference citations, were evaluated.resultsseveral vaccines including bacillus calmette - guerin, hepatitis b, influenza, leishmaniasis, meningitis, pneumococcal, smallpox, tetanus (alone and in combination with diphtheria, pertussis, polio, haemophilus influenza type b or plague and yellow fever), and varicella - zoster have been associated with post - vaccination site reactions. a 70-year - old male developed a lichenoid dermatitis that occurred at the pneumococcal vaccine injection site within 2 weeks after pcv13 vaccination ; the erythematous nodule resolved spontaneously within 9 weeks following immunization.conclusionsdermatoses at the injection sites of vaccines can be granulomatous, immunity - related conditions, infections, lichenoid, neutrophilic, or pseudolymphomatous. basal cell carcinoma and squamous cell carcinoma are the most common vaccination site - associated malignancies ; however, melanoma and sarcomas (dermatofibrosarcoma protuberans, fibrosarcoma, and malignant fibrous histiocytoma) are also smallpox vaccine - related site neoplasms. a cutaneous immunocompromised district that is created by vaccine - induced local immunologic changes is hypothesized to be the pathogenesis of vaccination site reactions.
substance use on american college campuses is a significant public health problem with adverse effects including but not limited to impaired academic performance, interpersonal violence, acute injuries, and increased risk of mortality. cigarette smoking is an established risk factor for coronary heart disease, among other disease states. clinical researchers have accordingly devoted extensive effort to developing and refining methods of treatment or means of secondary prevention to reduce substance use and its negative consequences. no matter how well such intervention programs work, they can only help those who enroll in them. anyone choosing to participate in such a program must have identified substance use as a problem on which they would like to work. this conclusion could be prompted by advice from a parent, teacher, or physician, the concern of a roommate, mandate from a campus authority (e.g., in the wake of violation of an alcohol policy), or simply the burgeoning awareness that substance use has become excessive and is interfering in one 's life. unfortunately, this awareness, and the motivation to reduce substance use, may emerge only after an extensive period of problematic substance use. therefore, a potentially important contribution could be made by identifying an intervention method that has the effect of reducing substance use among college students but does not require that participants be motivated (at least initially) to make changes in this regard. theory and past research suggest that the transcendental meditation (tm) technique may comprise such an intervention. it is described as a simple, natural, and effortless practice whereby a person experiences a state of restful alertness. mantra specifically chosen for its facilitation of the process of settling the mind and a precise technique for using it. thinking the sound leads the meditator to experience quieter and quieter aspects of his or her awareness, eventually experiencing complete silence. tm instruction does not directly target changes in substance use, but it has been hypothesized that the technique should nonetheless lead to reduced substance use. in particular,... these effects occur naturally as a by - product of neutralizing stress and experiencing greater inner fulfillment, with no attempt made to manipulate behavior, beliefs, or attitudes. as inner contentment and fulfillment grow, the need to seek gratification outside of one 's self naturally subsides and addictive behavior falls off. [4, page 36 ]. both qualitative and quantitative reviews of effects of tm practice on substance use have yielded strikingly positive outcomes. for instance, a meta - analysis of 19 studies showed, at an average followup of 10 months, mean effect sizes (relative to control conditions) of.55 for alcohol consumption,.87 for cigarette smoking, and.83 for illicit drug consumption. in each case these effects compared favorably to effect sizes from meta - analyses of a range of other interventions. nevertheless, the empirical case for the tm program as a useful intervention in reduction of substance use on college campuses remains uncertain. of the 19 studies included in a systematic review and meta - analysis, for example, 13 either lacked control conditions or used control conditions not involving random assignment (e.g., yoked peer controls, in which a meditating participant recruits a nonmeditating friend to serve in the comparison condition). only six studies included random assignment to conditions, making it possible to ascribe effects with confidence to the tm program itself rather than preexisting characteristics of those who elect to learn the method. of the six randomized experiments, two were conducted in college samples [6, 7 ], only one of which was a published study. in the published study, tm participants significantly decreased their rate of cigarette consumption over a 10-week period relative to a no - treatment control. however, sample size was small (n = 18 per condition), and no information was provided on the number of participants achieving complete abstinence from smoking. additionally, the project was presented to participants as a study of smoking reduction, and smoking rates were self - monitored, so results are inconclusive with respect to a standard tm program in which smoking is not a content focus. finally, this study did not provide information on alcohol or other drug use. in short, tm practice appears to have the potential to fill a niche in the effort to reduce substance use on college campuses by decreasing use even among those not initially seeking a substance abuse treatment program, but the evidence base for such an effect is limited. therefore, in the context of a randomized controlled trial of the tm program for college students, researching the effects on various health and lifestyle parameters, we examined data on cigarette smoking, illicit drug use, and alcohol use. we hypothesized that students assigned to the tm condition would reduce substance use over a three - month period compared to students assigned to a wait - list control condition. participants were 295 students from universities in the washington, dc area who responded to advertisements, newspaper stories, flyers posted on campuses, or public presentations concerning a study of the effects of the transcendental meditation program on brain functioning, cognitive development, and health. effects on these other measures, aside from substance use, will be reported separately. the study was conducted at american university in collaboration with maharishi university of management research institute and was approved by institutional review boards at both institutions. participants had to meet the inclusion criteria of (a) being an undergraduate or graduate student and (b) being enrolled in school through august 2006 (study enrollment began in january 2006). forty - eight prospective participants were excluded for one or more of the following reasons : (a) systolic blood pressure over 140 or below 90 mm hg, (b) diastolic blood pressure over 90 or below 60 mm hg, (c) a history of hypertension, (d) history of coronary heart disease, (e) history of hypoglycemia, or (f) history of chronic fainting. of the 295 participants who provided informed consent and were randomized to a treatment condition and completed pretesting, 178 (60%) were women, and 117 (40%) were men. about one - sixth (16%) were african - american, while 7% were asian - american, 7% other race, 4% hispanic, and 4% declined to report race / ethnicity. substance use was measured once at pretreatment and once again at posttreatment by way of a questionnaire embedded in several other self report measures (not relevant to this report)., do you use non prescribed drugs (e.g., marijuana, amphetamines, cocaine / crack), and do you drink alcohol ?. anyone answering yes to any of these questions was directed to additional questions about quantity and frequency of recent consumption (cigarettes / day in past week, number of times using nonprescribed drugs in the past month, beers / glasses of wine / shots of hard liquor over the past week). adherence to the tm technique was measured via self - report at posttesting among those who had received tm instruction. participants were asked how many minutes in each session they usually practiced the tm technique. they were also asked to describe the regularity of their practice of the tm technique over the preceding week (not at all, less than once / day, once a day, or twice a day). students who expressed interest in participating in the overall study were scheduled for small group informational and testing sessions at baseline. at this session, a member of the research staff gave an overview of what the study would involve. a member of the tm instructional staff gave a brief description of the tm technique and answered students ' questions. students interested in enrolling then were given detailed information about study procedures, risks, and benefits and completed individual written informed consent with a member of the research staff. after a 5-minute rest period, prospective participants had three blood pressure measurements taken one minute apart. participants also completed the substance use inventory, demographic and medical history forms, and several other self - report instruments and cognitive ability tests not relevant to this report. participants who met the study criteria were subsequently randomized within blocks to (a) immediate tm instruction or (b) wait list / delayed start condition. the blocks used in random assignment were men versus women, crossed with presence versus absence of a diagnosis of attention deficit hyperactivity disorder (adhd) per self - report. the adhd stratification was for the purposes of a separate substudy ; 25 participants indicated that they had been diagnosed with adhd. participants paid a reduced course fee of $ 50 at the beginning of tm instruction and were paid $ 25 for each assessment session completed. assessments were conducted at baseline (pretest) and again approximately 3 months later (posttest) (the initial design plan for this project, the grant funding period, and the consent forms signed by participants were all based on including only the pretest and immediate posttest three months later. subsequently, an additional year of funding was obtained, and we attempted to conduct a third assessment of the same sample of participants. a separate informed consent procedure was used, and the third assessment constituted a follow - up / maintenance evaluation for the original immediate - tm group, a postinstruction evaluation for the original wait - listed group. however, only 83 participants completed the third assessment (i.e., 28% of the randomized sample, 39% of the subsample who had completed the second assessment). some of the original participants had graduated by the time we were recruiting for a third assessment. others had left the country for study abroad programs, and others simply lost interest and declined further participation in the study.). all assessments were administered by research staff who were masked to the participant 's treatment condition assignment. the transcendental meditation (tm) technique is described as a simple, natural, and effortless technique practiced for about 20 minutes, twice a day, while sitting comfortably with the eyes closed. tm practice is intended to take the mind from active levels of thinking to the state of least mental activity. this experience of restful alertness - full self - awareness devoid of customary mental content - is called transcendental consciousness [911 ]. tm practice is not intended simply as a relaxation method - it also promotes increased alertness. the primary areas of brain activation during tm practice have been localized to prefrontal executive circuits and anterior cingulate attention circuits in an meg study and eeg coherence studies [13, 14 ] and in frontal and parietal areas as demonstrated in a preliminary pet study. the preparatory steps include an introductory lecture, which offers a review of previous scientific research on the tm program and a vision of possible benefits through regular practice, a preparatory lecture, which offers a review of the mechanics and origin of the tm technique, and a personal interview, in which a qualified teacher of the tm program gathers basic information from the student to aid with the personalization of instruction. the introductory and preparatory lectures last about 90 minutes and take place in a group setting. the one - on - one personal interview lasts 10 minutes. the next step in learning the tm technique is personal instruction. in this study, personal instruction was conducted by a male teacher for male students and by a female teacher for female participants. it was believed that having a same - sex instructor would be more comfortable for the one - on - one personal instruction meeting, which lasts about 90 minutes. it is followed by three 90-minute group checking sessions. on the first day of checking, verification of the correctness of tm practice and further instruction the goal of the second day of checking is the understanding of the mechanics of the transcendental meditation technique from personal experiences. finally, the goal of the third day of checking is the understanding of the mechanics of development of higher states of wellness and health through the tm program. once the third checking session was completed, the student was invited to attend individual meetings with the tm instructor to check on the mechanics of the tm practice. these meetings, of about 30 minutes each, are held weekly for the first month after the basic course, then monthly thereafter. finally, weekly (group) knowledge meetings were available for those who were interested. two very experienced instructors (one female, one male) taught the tm technique to participants. each had been practicing the tm technique for over 35 years at the time the study was conducted. each was initially certified as a tm teacher in the early 1970s by maharishi mahesh yogi after a 6-month in - residence, fulltime training period with him. both instructors have completed numerous advanced training courses for tm teachers and were recertified as tm teachers in 2005. treatment effects were tested for each substance separately (cigarette smoking, illicit drug use, alcohol use). in each case, the subsamples analyzed were those who (a) were using that substance at pretest and (b) completed both pre- and posttest assessments. chi - squared tests were used to compare the tm and wait - list groups on the frequency with which pretest substance users reported abstinence at posttest. when sample size permitted, sex differences were evaluated, in view of previous research indicating sex differences in the extent of drug use among college students, in the correlates of alcohol use among college students, and in response to substance use interventions. participants were excluded, based on a priori decision rules, if they reported at baseline any of the following : (a) more than 80 cigarettes / day, (b) illicit drug use on more than 90 occasions in the past month, or (c) more than 42 drinks / week, as these reports were considered not believable. one had claimed illicit drug use 100 times in the past month, another 49 drinks / week and 260 occasions / month of illicit drug use, and the third 92 drinks / week and 210 occasions / month of illicit drug use. all subsequent analyses are based on the remaining 292 participants after this exclusion. to evaluate the success of random assignment, the 147 participants assigned to immediate tm instruction and the 145 participants assigned to wait list were compared on demographic and substance use variables (chi - square tests for discrete measures and t tests for continuous measures). none of these tests approached significance, suggesting that randomization was successful in generating substantially equivalent participant groups at the outset of the study. attritionof the 292 participants, 218 (75%) completed the second assessment, after the tm group had received instruction. as can be seen in the table, attriters were more likely to have reported at baseline use of illicit drugs in the previous month. gender, age, and prevalence of smoking and drinking did not differ, nor did rates of use of any substance among those who used at all. of the 292 participants, 218 (75%) as can be seen in the table, attriters were more likely to have reported at baseline use of illicit drugs in the previous month. gender, age, and prevalence of smoking and drinking did not differ, nor did rates of use of any substance among those who used at all. the sample for this analysis was completers only (i.e., participants with posttest data available), who had reported smoking at the baseline assessment. only 37 (13%) of our participants smoked cigarettes at baseline, and of these 25 (68%) completed the posttest assessment. of the 11 smokers in the immediate - tm condition, 3 (27%) reported abstinence at posttest. similarly, of the 14 baseline smokers in the wait - list condition, 4 (29%) reported abstinence at posttest, a nonsignificant difference between groups, chi square (df = 1, n = 25) = 0.01, ns. self - reported smoking rate at posttest was not normally distributed (kolmogorov - smirnov z = 1.56, p <.05, with skewness = 2.53 and kurtosis = 5.81). for the purposes of inferential statistical analysis, we therefore used transformed data. taking the natural log of (# posttest cigarettes / day + 1) reduced skewness to 0.66 and kurtosis to 0.42. treatment condition effects were evaluated in an ancova with condition (tm instruction versus wait - list) as the independent variable, log - transformed pretest cigarettes / day as the covariate, and log - transformed posttest cigarettes per day as the dependent variable. the treatment condition effect was nonsignificant, f (1, 22) = 0.05, ns. table 2 shows the (untransformed) average cigarettes per day at pretest and posttest among baseline smokers in each condition. of the 60 participants who reported illicit drug use at baseline, 37 (62%) provided posttest data, 15 in the tm condition and 22 in the wait - list condition. of the 15 in the tm condition, 33% (n = 5) reported abstinence from illicit drug use at posttest, compared to 27% (6 of 22) posttest abstinence in the wait - list condition, a nonsignificant difference, chi square (df = 1, n = 37) = 0.16, ns. posttest self - reported frequency (over the preceding month) of illicit drug use, among those using at all at baseline, was not normally distributed (kolmogorov - smirnov z = 1.67, p <.05, skewness = 2.43, kurtosis = 5.62). taking the natural log of (posttest frequency of use in past month + 1) reduced skewness to 0.54 and kurtosis to 0.80. treatment condition effects were evaluated in an ancova with condition (tm instruction versus wait - list) as the independent variable, log - transformed pretest drug use occasions in the past month as the covariate, and log - transformed posttest drug use frequency as the dependent variable. the treatment condition effect was nonsignificant, f (1, 33) = 2.73, ns. table 2 shows the (untransformed) average frequency of use of illicit drugs in the past month at pretest and posttest among baseline drug users in each condition. almost three - fourths (74%) of our participants reported drinking alcohol at pretest, consistent with other studies of american college samples. of the 217 participants who reported drinking alcohol at baseline, 161 (74%) completed posttesting. thus, alcohol was the one substance for which we had enough pretest substance users to conduct meaningful analysis of sex differences in treatment response. there was a significant three - way interaction of treatment condition x sex x posttreatment abstinence, chi square (df = 1, n = 161) = 4.71, p <.05. observed cell frequencies appear in table 3. among men, 17% of those who drank at baseline and were assigned to the tm condition reported abstinence at posttest, a nonsignificantly greater proportion than in the wait - list condition (6%), chi square (df = 1, n = 65) = 2.28, ns. among women, on the other hand, there was a nonsignificant effect in the other direction. of those who reported drinking at pretest, 4% reported abstinence at posttest in the tm condition, compared to 14% in the wait - list condition, chi square (df = 1, n = 96) = 2.42, ns. combining men and women, in each treatment condition 10% reported abstinence from alcohol at posttest, chi square (df = 1, n = 161) = 0.04, ns. descriptive statistics for (untransformed) drinks per week at pretest and posttest, separately by gender and by treatment condition, appear in table 4. the distribution of self - reported drinks per week (m = 5.44, sd = 6.97) at posttest was significantly nonnormal, kolmogorov - smirnov z = 2.75, p <.05, with skewness = 2.15 and kurtosis = 5.38. taking the natural log of (# posttest drinks / week + 1) reduced skewness to.13 and kurtosis to 1.03. analysis of covariance, with transformed drinks / week at posttest as the dependent measure and transformed drinks / week at pretest as the covariate, yielded significant main effects for treatment condition, with tm participants drinking less than did wait - list participants, f (1, 154) = 3.93, p <.05, and for sex, with women drinking less than did men, f (1, 154) = 4.16, there was also a significant sex x treatment condition interaction, f (1, 154) = 5.20, p <.05. we therefore examined treatment condition effects in analyses of covariance conducted separately by sex. for women there was no significant difference between treatment conditions, f (1, 91) = 0.13, ns. for men, however, there was a significant effect of treatment condition, f (1, 62) = 7.10, p < as can be seen in table 4, the direction of this effect was in favor of the tm condition ; drinks per week decreased from pretest to posttest among men in the tm condition but increased among men in the wait - list condition. the recommended practice schedule for tm participants is twice per day, but as noted in the method section we opted for a standard of adherence a bit more lenient than this ideal scenario. in particular, participants were considered adherent if they reported at posttest having practiced the tm technique at least once a day over the preceding week. by this standard, about two - thirds (65%) of participants in the tm condition were adherent. a series of secondary analyses (details of these analyses are available from the corresponding author) showed no significant differences in posttest substance use, either in rate of use or in likelihood of abstinence, for cigarettes, alcohol, or illicit drugs, between adherent and nonadherent participants in the tm condition. in a pre - post (three - month) randomized wait - list - controlled trial of the effects of the transcendental meditation (tm) program on substance use among university students, results differed by substance and by gender. there were no significant differences between tm instruction and a wait - list control condition in leading to cessation or reduction of cigarette smoking or use of illicit drugs. with respect to alcohol use, gender was a significant moderator of tm effects on abstinence as well as average intake. tm instruction did not differ from wait - list control for women, but tm instruction reduced drinking among men. reviews of earlier research indicating favorable effects of tm practice in lowering substance use [4, 5 ] have not reported sex differences in response to tm instruction. similarly, a well - developed area of tm research concerns its impact in reducing blood pressure, and blood pressure reductions have been obtained in both sexes. it is therefore not entirely clear why men were more responsive to tm instruction in our study with respect to reducing or eliminating alcohol consumption. as indicated in the method section, individual tm instruction was completed by same - sex instructors, so it is possible that an instructor effect is showing up in our results as a sex difference in response to the tm program. alternatively, male participants may simply have had more room to change their drinking habits, for they drank more at the outset (see table 4). given these uncertainties, we believe that future research on tm instruction and substance use should report results separately by sex in order to determine the replicability of our findings. a further question, aside from the sex differences in intervention effects on drinking, is why the effects of tm instruction were not more favorable across the board. earlier research had led us to hypothesize that tm instruction would alter cigarette smoking, illicit drugs, and alcohol use relative to wait - list control. we believe that several explanations for the lack of more consistent, robust effects of tm instruction in this study deserve consideration. first, the tm program may in general have the effect of reducing substance use, but this effect might be weaker in university settings. as noted in the introduction, a majority of prior studies reporting lowered drug use after tm instruction were not conducted in college settings, were not randomized trials, or both. the influence of a peer culture conducive to substance use might offset to some extent the benefits of tm practice to the individual student 's psychological well - being. second, tm effects on substance use may be minimized in the university context because there are other environmental mechanisms operating to reduce substance use. in other words, the university context, or at least the one studied here, might actually tend to create floor effects such that detecting any impact of tm practice is more difficult. for example, the campus on which this study was conducted is dry, such that possession or use of alcohol on campus is a violation of university policy. moreover, cigarette smoking is banned in most places on campus (e.g., residence halls, classroom buildings). illicit drug use is of course illegal anywhere, but it is perhaps more likely to be detected in a setting in which one lives and works among large numbers of peers as well as authority figures such as dormitory ras. consistent with this explanation, cigarette smoking and illicit drug use rates were low in our sample, detracting from statistical power for showing intervention effects on these variables. this feature was a constant across experimental conditions, and participants were assured of the confidentiality of their responses. however, in view of the sensitive nature of some of the variables (illegal drugs and for participants under the legal drinking age alcohol use), it is possible that participants would be inclined to underreport use. moreover, posttest self - reports of substance use were collected with respect to the past week (cigarettes, alcohol) or past month (illicit drugs) only. we did not incorporate detailed interviews or self - monitoring methods covering the entire three - month intervention period. self - monitoring might be obtrusive and therefore reactive in a study of an intervention such as the tm program that does not directly address substance use. future studies, however, might do well to use timeline follow - back interviews at posttest to obtain a more thorough, fine - grained analysis of substance use rates and patterns of use. for example, the health implications of 7 to 9 drinks per week for a young man (our preintervention means ; see table 4) are quite different if the male student is having one drink each day than if he is having one drinking binge on the weekend. fourth, perhaps tm practice would show consistent effects in reducing substance use only among those who are highly motivated to seek out instruction. we did not measure participants ' motivation, either in general or with specific reference to the possibility that some might have wanted to learn tm in order to reduce substance use. participation in our project was of course voluntary, but the volunteers were solicited through conspicuous advertisements and other promotional materials. instruction took place within walking distance of where most participants lived, and fees were drastically reduced - indeed, a participant who took part in both assessments received compensation equal to the cost of tm instruction for the study. future studies could address this possibility by enrolling participants only from among those who learn about and initiate involvement in the tm program through customary referral sources and by measuring motivation directly. in the meantime, though, psychotherapy outcome data suggest that recruitment methods are unlikely to play a large role in determining results of clinical trials. studies of solicited volunteers and studies of patients referred through routine clinical channels appear to obtain similar results. finally, it could be that the dose of tm practice in our project was insufficient to show significant reductions for both sexes in all substance use categories. simply being instructed in the tm program would not be expected to lower drug use if the student does not continue to practice. only about two - thirds of tm participants reported practicing at least once per day. secondary analyses did not show a relation between adherence and substance use, but it remains possible that full adherence (100% of those in the tm condition practicing twice per day) would have yielded stronger effects of the tm program on substance use. another aspect of the dose issue is the relatively brief (3 months) duration of our study. both retrospective and prospective studies of substance use among tm practitioners have indicated that its effects may be gradual and cumulative, with cessation occurring as long as two years after initiation of tm practice. only a longer - term randomized controlled trial of the tm program with college students could fully evaluate this possible explanation of our results. in conclusion, we found no evidence of an effect of transcendental meditation (tm) instruction on cigarette smoking or illicit drug use among university students, but tm instruction did lower alcohol use among male participants. given that male students are more likely than female students to have alcohol use disorders, the impact of tm practice on alcohol intake among men is a very encouraging result. additional studies with larger samples of baseline substance users (particularly for cigarettes and illicit drugs), multimethod assessments of substance use, higher adherence to the recommended twice daily practice, and a longer period of tm practice would be needed before concluding definitively that the tm program does not also reduce drinking among female students or illicit drug use or cigarette smoking in college student samples of either sex.	a randomized wait - list controlled trial (n = 295 university students) of the effects of the transcendental meditation program was conducted in an urban setting. substance use was assessed by self - report at baseline and 3 months later. for smoking and illicit drug use, there were no significant differences between conditions. for alcohol use, sex x intervention condition interactions were significant ; tm instruction lowered drinking rates among male but not female students. tm instruction could play a valuable role in reducing alcohol use among male university students. limitations are noted, along with suggestions for further research.
rheumatic heart disease (rhd), the primarily autoimmune sequelae of acute rheumatic fever (arf), is an autoimmune disease that arises following infection by s. pyogenes in people ages 319 years. the disease remains a major cause of cardiovascular disability in the young, and imposes a heavy burden on public health in the developing world. rhd, the most serious complication, occurs in 3045% of rf patients and leads to chronic valvular lesions. the pathogenesis of rf / rhd is complex and autoimmune reactions triggered by pathogen infection are thought to be the main factor. genetic factors that predispose a person to the development of autoimmune reactions were also considered to be related to rhd. in recent years, much attention has been paid to toll - like receptors (tlr) and their roles in autoimmune diseases. tlrs are a class of proteins that play a key role in the innate immune system. tlrs are a type of pattern recognition receptor (prr) ; they recognize molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen - associated molecular patterns (pamps). currently, 11 mammalian tlrs have been identified, each of which responds to a specific class of pamp. tlr2 can be activated by a variety of microbial components, including lipoproteins, peptidoglycan, lipoteichoic acid, and, especially, antigens of gram - positive bacteria, including streptococci. microrna (mirna) is a short non - coding rnas that suppress the expression through targeting the 3 untranslated regions (utrs) of messenger rnas (mrnas). according to the predicted results of bioinformatics, there may be more than 60% of protein coding genes with the expression regulated by mirna. in the present study, we detected 11 selected mirnas expression in clinical samples to investigate the relations between mirnas and rhd pathogenesis. the inclusion criteria for the experimental group were : (i) every patient diagnosed with rheumatic mitral valve insufficiency with or without mitral stenosis, and scheduled for mitral valve replacement ; (ii) normal preoperative erythrocyte sedimentation rate and anti - streptolysin o to eliminate rheumatism in active stage ; (iii) all patients in new york heart association (nyha) functional class ii iii ; and (iv) no other complications (patients with acute heart failure were excluded). the inclusion criteria of the control group were : (i) every patient diagnosed with mitral valve prolapse because of mitral chordae tendineae fracture and mitral insufficiency, and scheduled for mitral valve replacement. the other conditions are the same as criteria iii v of the experimental group. rhd cases and their controls were well matched based on the following details : (i) same sex, (ii) difference of age 0.05). these data indicate that mir-101 may suppress tlr2 expression through targeting the seed sequence at the 3-utr of tlr2. to further examine whether endogenous tlr2 expression is suppressed by mir-101 as shown in figure 2c, the level of tlr2 protein level was significantly suppressed by mir-101 mimics and up - regulated by mir-101inhibitor compared with corresponding controls in a549 cells. the tlr2 mrna levels were not significantly changed in these 4 groups (figure 2d). these results indicate that mir-101 repressed endogenous tlr2 expression by directly suppressing tlr2 mrna translation. thp-1 cells were seeded in a 24-well plate and then transfected with mir-101 inhibitor or corresponding control. at 48 h after transfection, cells were stimulated with various doses of pgn (0.011 g / ml) for 24 h. the cell culture supernatants were collected for cytokines detection. as shown in figure 3, the concentration of tnf-, il-1, and il-6 displayed a dose - dependent relationship with pgn. meanwhile, the mir-101 knockdown cells showed a significantly more radical response when stimulated by 0.5 and 1 g / ml than the control groups, with higher cytokines concentration. these results are in accordance with the data from clinical samples. as shown in figure 4a, the serum cytokines (tnf-, il-1, and il-6) level was significantly higher than the controls, and cardiac tissue samples also have higher level cytokines (figure 4b). we also detected the tlr2 expression in cardiac tissue samples in both protein and mrna levels. as shown in figure 4c, almost all the rhd clinical tissue samples had a higher tlr2 expression in protein levels. however, the mrna level of tlr2 was not significantly changed in rhd patient samples (figure 4d). rhd is an autoimmune disease that arises following infection by s. pyogenes in young people. nt - probnp is a well - known biomarker for congenital and acquired heart disease, which is also confirmed to be significantly elevated in children with acute rhd, but many limitations reduced its accuracy. research indicates that soluble st2 (suppression of tumorigenicity 2) is a potential early marker for cardiovascular disease, but the relationship between st2 and rhd is not understood yet. recently, reports indicated that mirnas participate in the pathogenesis of many kinds of cardiovascular disease. but to date, only the work of 1 group has shown that disturbed mirnas expression contributes to rhd. in the present study, to investigate the roles of mirna in the pathogenesis of rhd, we first detected 11 selected mirnas expression in cardiac tissue samples of rhd patients. we found mir-101 had a significantly down - regulated expression in patients, which may play a role in modulating the immune system by disturbing tlr2 expression. subsequently, by employing dual - luciferase assay and western blot, we first identified that tlr2 is a direct target gene of tlr2 and mir-101 knock - down is related to over - stimulated immune response in pgn - activated thp-1 cells. finally, we detected the concentration and mrna level of tnf-, il-1, and il-6 in patient serum samples and found these cytokines levels were significantly higher in patients. furthermore, the mrna and protein levels of tlr2 were also detected in cardiac tissue samples. we found that tlr2 had a higher expression in patients, mainly in the protein level rather than the mrna level, meaning that post - transcriptional regulation factors like mirna play a crucial role in regulating tlr2 expression. tlr2 was considered to be activated by streptococci, which is the major pathogen for rheumatic fever, which further contributed to rhd. although arg753gln polymorphism of the tlr2 gene was confirmed to be related to acute rheumatic fever susceptibility in child, 2 research groups have found that there were no relations between this arg753gln polymorphism and rhd pathogenesis. this study may provide clues for genetics researchers. since nucleotides variations can alter mature mirnas expression or disturb the mirna - mrna interaction, mutation sites in the mirna coding region may contribute to susceptibility to rhd [1113 ]. herein, we showed that mir-101 functions as an immune inhibitor through targeting tlr2 in rhd patients. however, zhu. indicated that mir-101 acted as an immune response activator by suppressing mkp1, which is a negative regulatory factor of the tlr4 pathway. this contradiction may be caused by the complexity of mirna function pattern and tissue specificity. since a single mirna may have hundreds of targeting genes and a single gene may be regulated by tens of mirnas in at the same time, the function of mir-101 in the immune system needs to be fully studied in the future. in conclusion, the present study confirmed for the first time that mir-101 targeted tlr2 3utr and repressed tlr2 expression. this work also found an association between down - regulated mir-101 and rheumatic heart disease, which sheds light on the mechanism of rhd pathogenesis and may create an opportunity to approach the diagnosis and treatment of rhd.	backgroundrhd is an autoimmune disease that arises following infection by s. pyogenes and imposes a heavy burden on public health.material/methodswe detected 11 selected mirnas expressed in the cardiac tissues of 11 rhd patients and 11 controls. by employing dual - luciferase assay and western blot, we identified the relationship between tlr2 and mir-101 and mir-101. we used elisa to test the concentration of tnf-, il-1, and il-6.resultsin cardiac tissue of rhd patients, mir-101 was significantly down - regulated (p=0.011). ectopically expressed mir-101 repressed the luciferase activity by 27% through targeting tlr2 3utr. combined with the results of western blot, we confirmed that tlr2 is a direct target gene of mir-101. mir-101 knock - down is related to over - stimulated immune response in pgn - activated thp-1 cells. we detected a significantly higher concentration of tnf- (p=0.0017), il-1 (p=0.015), and il-6 (p=0.014) in serum samples. tlr2 had a higher expression in patients in the protein level rather than the mrna level, indicating that post - transcriptional regulation factors play a crucial role in regulating tlr2 expression.conclusionsthe present study confirmed that mir-101 targets tlr2 3utr and represses tlr2 expression. this work also found an association between down - regulated mir-101 and rheumatic heart disease.
a 72-year - old male patient visited smg - snu boramae medical center with exertional chest pain, which had been aggravated during the most recent 3 months. the initial electrocardiogram revealed sinus bradycardia with a heart rate of 50 beat per minute, and there was no st segment elevation. the serum cardiac enzyme level was within the normal range, and there were no abnormal findings except mild functional mitral regurgitation in transthoracic echocardiography, which showed a left ventricular ejection fraction of 67% and no regional wall motion abnormality. myocardial single - photon emission computed tomography showed a reversible perfusion decrease at the basal inferior wall. preoperative coronary angiography showed 70% to 80% stenosis of the left anterior descending coronary artery (lad), 90% stenosis of the proximal ramus artery, 50% stenosis of the proximal left circumflex artery, 95% stenosis of the distal right coronary artery, and 90% stenosis of the posterolateral branch artery (plb). according to the patient s past medical history in addition, arteriosclerosis obliterans of both the lower extremities had been treated medically for 6 years. diagnosed with unstable angina with three - vessel disease, the patient underwent off - pump coronary artery bypass graft surgery (cabg). under general anesthesia, an advanced venous access catheter and a swan - ganz (sg) catheter several premature ventricular contractions (pvcs) occurred during the insertion of the sg catheter. we used the left internal mammary artery as a pedicled graft and anastomosed it to the mid - lad. we also harvested the saphenous vein from the left lower leg and anastomosed it to the left internal mammary artery to make a y - shaped composite graft. subsequently, the vein graft was anastomosed to the ramus artery and plb sequentially. during the operation as soon as the patient was transferred to the intensive care unit, frequent ventricular tachyarrhythmia began to occur and the hemodynamics became unstable. we tried our best to control the unstable ventricular arrhythmia with the administration of antiarrhythmic drugs like lidocaine and amiodar - one as well as a meticulous correction of the fluid and electrolyte imbalance, but none of the medical measures worked. furthermore, ventricular fibrillation (vf) occurred three times at intervals of about 1 hour (fig., we inserted an intra - aortic balloon pump to provide hemodynamic support, but failed to stop recurrent vf, and the patient s condition worsened considerably. in fact, we did not notice the severely angulated sg catheter demonstrated by the serial chest radiographs until around 6 hours after the patient was brought to the intensive care unit. we found that the sg catheter was located too deep in the right ventricle and relocated the catheter to remove the acute angulation (fig. however, the catheter still seemed to readily stimulate the ventricle ; thus, we decided to remove the catheter. during the removal of the sg catheter, vf occurred once again. the removal of the catheter finally resulted in prompt and complete cessation of recurrent ventricular arrhythmia. there were, in total, 5 events of vf and defibrillation was performed 9 times. the myocardial contractility, which was shown to be good by trans - esophageal echocardiography at the end of the operation, worsened and led to a left ventricular ejection fraction of 43%. the serum troponin - i level was also markedly elevated from an immediate postoperative value of 2.0 ng / ml to a peak value of 45.7 ng / ml on postoperative day 1. despite this event, the patient recovered and was transferred to the general ward on postoperative day 6 without any significant complications. myocardial function was fully recovered without any regional wall motion abnormality, but the saphenous vein harvest site was dehisced and the wound problem had to be addressed. the first presentation of sustained ventricular arrhythmia in the recovery period after cabg is uncommon, and its incidence in the previous studies ranged from 1.6% to 3.1%. the occurrence of ventricular tachycardia (vt) or vf after cabg has been reported to have a strong impact on in - hospital mortality, which ranged from 21.7% to 25%. recurrent ventricular arrhythmia was not infrequent, observed in 33% of the patients, and was associated with a high mortality ; 75% of the patients with these characteristics did not survive to hospital discharge. the known risk factors for ventricular arrhythmia after cabg are age under 65 years, female gender, body mass index under 25 kg / m, unstable angina, moderate or poor ejection fraction, and a need for pre - operative inotropic support or an intra - aortic balloon pump. as an sg catheter allows the measurement of hemodynamic variables that can not be assessed reliably or continuously by less invasive means, it is widely used in the perioperative monitoring of cardiac surgical procedures in many centers. however, a significant number of patients who undergo pulmonary artery catheterization experience catheter - related complications such as cardiac arrhythmia, pulmonary infarction, catheter knotting, catheter entanglement with other endovascular structures, pulmonary artery rupture, thrombosis, and tricuspid valve rupture. various types of arrhythmia related to the sg catheter placement have been described and include premature atrial contraction, atrial and ventricular tachycardia, pvc, and conduction abnormalities. the incidence of arrhythmia has been reported to range from 12.5% to more than 70% during sg catheter insertion. some cite a 52% to 68% incidence of pvc, the most commonly seen type of arrhythmia. these resolve either with the sg catheter advancement from the right ventricle into the pulmonary artery, or with the prompt sg catheter withdrawal into the right atrium or the superior vena cava. fortunately, clinically significant vt or vf requiring treatment occurs in < 1% of the patients, although fatal vt has been observed. the risk factors for ventricular arrhythmia during the insertion of an sg catheter include prolonged catheterization time, a predisposition to pvc, liver transplantation, myocardial infarction or ischemia, shock, acidosis, sepsis, electrolyte disturbances, and increased sympathetic tone. baldwin and heland reported a 2% incidence of vt associated with the removal of the sg catheter. in this report, the onset of vt was associated with a brief period of hypotension, and the duration of vt and hypotension was longer than the period of catheter removal, which implies that the activity is triggered by the procedure and has the potential to be prolonged beyond an initial stimulus. in patients with the wolff - parkinson - white (wpw) syndrome, pre - excited atrial fibrillation with a rapid ventricular response can induce potentially life - threatening arrhythmia. a previous prospective study reported that the incidence of vf with cardiac arrest among the wpw syndrome patients was 1.6% (3 of 184 patients). although it is not relevant to our case, the wpw syndrome is thought to be one of the predisposing factors of vf. in our case there was no risk factor for ventricular arrhythmia except unstable angina, and vf was thought to be triggered by the malpositioned sg catheter. the inserted length of the sg catheter was within the usual range because the kinking of the catheter masked the malposition. checking the pulmonary capillary wedge pressure could be helpful in detecting the malposition of the catheter, but we did not try it until we realized the cause of the vf. in conclusion, it is basic and imperative to check the position of the sg catheter and to keep in mind that life - threatening and otherwise intractable recurrent ventricular arrhythmia can be triggered by a malpositioned catheter.	recurrent ventricular arrhythmia can be fatal and cause serious complications, particularly when it is caused immediately after an operation. incorrect placement of a swan - ganz catheter can trigger life - threatening ventricular arrhythmia, but even intensive care specialists tend to miss this fact. here, we report a case of recurrent ventricular arrhythmia causing a severe hemodynamic compromise ; the arrhythmia was induced by a severely angulated swan - ganz catheter. the recurrent ventricular arrhythmia was not controlled by any measures including repositioning of the catheter, until the complete removal of the swan - ganz catheter. it is necessary to keep in mind that the position of the pulmonary artery catheter should be promptly checked if there is intractable recurrent ventricular arrhythmia.
	we report a novel method for the measurement of lipid nanotube radii. membrane translocation is monitored between two nanotube - connected vesicles, during the expansion of a receiving vesicle, by observing a photobleached region of the nanotube. we elucidate nanotube radii, extracted from spe vesicles, enabling quantification of membrane composition and lamellarity. variances of nanotube radii were measured, showing a growth of 4056 nm, upon increasing cholesterol content from 0 to 20%.
since the first report on the use of the combination of gonadotropin - releasing hormone agonists (gnrha) and gonadotropins for in vitro fertilization (ivf) in 1984 (1), gnrha has widely been used in controlled ovarian stimulation cycles for assisted reproduction. one known complication of pituitary down - regulation using gnrha in ivf treatment cycles is the formation of functional ovarian cysts (2). several case reports have indicated that a very small subgroup of patients may experience ovarian hyperstimulation following the administration of gnrha without gonadotropins (3 - 6). however, since very few case reports have been published on this topic, it is unclear what course to follow in subsequent cycles after ovarian hyperstimulation using only gnrha in the first cycle. in the present report, a depot preparation (3.75 mg) of tryptorelin without gonadotropins induced ovarian multifollicular enlargement with high estradiol level, and was followed by human chorionic gonadotropin (hcg) administration and oocyte retrieval. in a subsequent cycle of the same patient, a low dose of triptorelin (0.05 mg) did not induce ovarian hyperstimulation, and resulted in clinical pregnancy. our patient was a 33-yr - old nulliparous korean woman undergoing oocyte donation for her sister on march 2010. the patient reported irregular menstrual cycles, with only four to six periods each year. her body mass index was 21.5 kg / m, with a gynecoid fat distribution. she had mild acne on her face, but no hirsutism, virilization, or acanthosis nigricans. her exam results for the 53rd day of her cycle were as follows : negative u - hcg, follicle - stimulating hormone (fsh) 5.39 iu / l, luteinizing hormone (lh) 11.29 iu / l, estradiol 45 pg / ml, progesterone 0.66 ng / ml, and total testosterone 1.09 ng / ml. transvaginal ultrasound showed that the right ovary measured 3.9 3.5 2.8 cm (19 cm) and the left ovary 3.0 2.8 2.5 (10 cm). we scheduled a gnrha long protocol for controlled ovarian stimulation. on the 54th day of the cycle, a depot preparation of triptorelin (3.75 mg decapeptyl cr ; ferring, malmo, sweden) was administered subcutaneously. an ultrasound revealed multiple follicles ranging from 15 to 28 mm in diameter (fig. her exam results were : estradiol 2,560 pg / ml, lh 9.83 iu / l, fsh 2.24 iu / l, and progesterone 4.18 ng / ml. since serum estradiol concentrations were considered appropriate for the number of follicles present, a decision was made to continue the oocyte donation cycle. (fsh 5.96 iu / l, lh 10.78 iu / l, estradiol 41 pg / ml, progesterone 0.54 ng / ml), a short - acting preparation of triptorelin (decapeptyl ; ferring) was administered s.c. at a dose of 0.05 mg per day, which is half of the conventional dosage. seven and ten days after the initiation of gnrha, an ultrasound revealed no evidence of functional ovarian cyst. rockland, ma, usa) was started, which she received daily for 7 days. the dosage was later increased to 225 iu for an additional 5 days. on day 13, a total of 11 oocytes were retrieved, and three embryos were transferred to the recipient. a single gestational sac and fetal pulse one possible explanation is the initial flare - up caused by gnrha, which induced the release of gonadotropins from the pituitary thereby stimulating the growth of ovarian follicles. these follicles acquired fsh - independent growth dynamics, which finally led to mature follicles by relatively abundant lh. it is accepted that in the late stages of follicle development, granulosa cells become receptive to lh stimulation and lh becomes active (7). in pcos, lh secretion is characterized by greater sensitivity to gnrh, as compared to women without pcos. these responses to gnrh are more prominent in lean - pcos (8) ; for this reason, we speculate that the serum lh concentration remained high for ten days following the initiation of gnrha due to an extremely high initial flare - up. alternatively, previous researchers have suggested a direct effect of gnrha at the ovarian level (3, 5, 9). however, during treatment with gnrha, high concentration of gnrha circulate in the central and peripheral tissue. many studies have investigated the effects of gnrha on ovarian cell proliferation and steroidogenesis. in bussenot. (10), 5 different gnrh agonists were compared based on their effect on estradiol secretion in human granulosa lutein cell cultures. the results showed that buserelin and leuprolein significantly enhanced estradiol secretion. however, triptorelin, which was used in our case, had no effect on estradiol secretion in their study. recently, azem. (11) reported a live birth following the administration of gnrha without gonadotropins. they scheduled a conventional flare - up regimen on day 1 of the cycle (triptorelin 0.1 mg) and daily injections of gonadotropins from day 3 and on. contrary to their expectation, they observed that continued gnrha injection without gonadotropins induced follicular development with high estradiol levels until oocyte retrieval. however, it is unclear what course to follow in subsequent cycles, after ovarian hyperstimulation using only gnrha in the first cycle, as very few case reports have been published on this topic. weissman. (3) reported two cases of ovarian hyperstimulation following the administration of gnrha without gonadotropins. they also reported that recurrent ovarian hyperstimulation occurred in subsequent cycles of the same patients. in the present report the main difference between their cases and ours is the dose of gnrha. while weissman. (3) used a depot preparation of triptorelin (first cycle) and short acting preparation 0.1 mg / day in (subsequent cycle), we used triptorelin at 0.05 mg / day. however, we can not say that the dose of gnrha is only responsible for different results. some differences regarding patient 's characteristics and the timing of initiation of gnrha should be considered. in summary, a small subgroup of patients may develop ovarian hyperstimulation following the administration of gnrha without gonadotropins.	several case reports have indicated that a small subgroup of patients may develop ovarian hyperstimulation following the administration of gonadotropin - releasing hormone agonists (gnrha) without gonadotropins. however, since only few such cases have been published, it is unclear what course to follow in subsequent cycles after ovarian hyperstimulation in the first cycle using only gnrha. a 33-yr - old woman was referred to in vitro fertilization for oocyte donation. a depot preparation (3.75 mg) of tryptorelin without gonadotropins induced ovarian multifollicular enlargement with high estradiol level, and was followed by human chorionic gonadotropin administration and oocyte retrieval. in a subsequent cycle of the same patient, a low dose of tryptorelin (0.05 mg) did not induce ovarian hyperstimulation, and resulted in clinical pregnancy. this report shows potential management of ovarian hyperstimulation following the administration of gnrha without gonadotropins.
asthma is a chronic inflammatory disease of the airways characterized by bronchial hyperresponsiveness, variable airflow limitation, and symptoms such as dyspnea, wheezing, and cough, which improve spontaneously or with treatment. the primary goals of asthma treatment include complete symptom control, optimal management of limitations in activities of daily living, and reducing future risks. 1 2 most asthma patients have mild to moderate asthma, which is easily controlled with the use of inhaled corticosteroids (ics) alone or in association with a long - acting 2 agonist (laba). 3 however, among patients with severe asthma, there is a small subset of patients who remain symptomatic despite good adherence to treatment, correct use of inhalers, appropriate management of comorbidities, and use of high - dose ics in association with labas, oral corticosteroids, omalizumab, or any combination of the three. 4 5 such patients are considered to have uncontrolled severe asthma (ucsa), which, according to current asthma guidelines, should be treated at specialized centers. 4 5 patients with ucsa require high - dose ics alone or in association with oral corticosteroids, the adverse effects of which include decreased protein synthesis and increased protein degradation and contribute to muscle atrophy. 6 in addition, increased airway resistance and lung hyperinflation also contribute to reducing inspiratory muscle efficiency, resulting in increased muscle work and energy expenditure to overcome airflow limitation. 7 the aforementioned factors have yet to be well studied in patients with ucsa. the six - minute walk test (6mwt) is a submaximal exercise test that evaluates the overall and integrated responses of all body systems involved in physical exercise, including the pulmonary system, the cardiovascular system, peripheral and systemic circulation, blood, neuromuscular units, and muscle metabolism. 8 9 respiratory muscle strength can be estimated from airway pressures generated by respiratory muscle contraction. maximal static respiratory pressures are usually measured at the mouth during inhalation and exhalation, the former being designated mip and the latter being designated mep. 10 the role of the 6mwt in the evaluation and follow - up of patients with copd, interstitial lung disease, pulmonary arterial hypertension, and heart failure is well established. 8 however, the only study evaluating the impact of severe asthma on the six - minute walk distance (6mwd) in adults was a study conducted by canuto., 11 who found that the 6mwd was significantly shorter in patients with difficult - to - control asthma (dca) using oral corticosteroids than in healthy controls. in addition, although respiratory muscle strength (particularly mip) has been reported to be reduced in several studies involving asthma patients, it has yet to be evaluated in patients with ucsa. 12 the primary objective of the present study was to evaluate respiratory muscle strength and 6mwt variables in patients with ucsa. secondary objectives were to evaluate the impact of oral corticosteroid use on respiratory muscle strength and to determine the correlation of the level of physical activity with the 6mwd and the level of asthma control in such patients. this was a cross - sectional study of ucsa patients over 18 years of age treated at the difficult - to - control asthma outpatient clinic of the federal university of minas gerais hospital das clnicas, in the city of belo horizonte, brazil, between september and december of 2012. the research project was approved by the research ethics committee of the federal university of minas gerais (etic ruling no. the diagnosis of severe asthma was based on the american thoracic society / european respiratory society (ats / ers) criteria, 13 as follows : major criteria - use of oral corticosteroids for at least six months per year and continuous use of high - dose ics (1,600 g of budesonide or equivalent) in association with labasminor criteria - fev1 of 20% ; daily use of short - acting 2 agonists ; use of more than three courses of oral corticosteroids per year ; history of near - fatal asthma ; one or more emergency room visits in the previous year ; and rapid decline in lung function after a decrease in the dose of corticosteroid therapy major criteria - use of oral corticosteroids for at least six months per year and continuous use of high - dose ics (1,600 g of budesonide or equivalent) in association with labas minor criteria - fev1 of 20% ; daily use of short - acting 2 agonists ; use of more than three courses of oral corticosteroids per year ; history of near - fatal asthma ; one or more emergency room visits in the previous year ; and rapid decline in lung function after a decrease in the dose of corticosteroid therapy the patients who were included in the present study received outpatient treatment for at least six months in order to control environmental factors and comorbidities, as well as to improve inhaler technique and treatment adherence. in addition, we included only patients assigned to global initiative for asthma (gina) and brazilian thoracic association (bta) asthma treatment step 4 or 5 (labas / high - dose ics plus oral corticosteroids, anti - ige treatment, or both) 1 2 and meeting two major ats / ers criteria or one major and two minor ats / ers criteria. 13 we excluded patients with severe heart disease ; current smokers ; former smokers who had a smoking history 10 pack - years or who had quit smoking less than one year prior ; and patients who had had exacerbations requiring emergency room treatment / hospitalization, use of prednisone or equivalent at doses above 20 mg, use of antibiotics, or any combination of the three in the last four weeks. the 6mwt was performed in a 25.6-m corridor with the use of a portable oximeter (nonin medical, inc., plymouth, mn, usa), in accordance with the recommendations of the ats. 14 each of the participants underwent two 6mwts, at least 30 min apart. we evaluated the following parameters : spo2, as measured by pulse oximetry ; hr ; rr ; and patient perception of dyspnea and leg fatigue, as assessed by borg scale scores at the beginning and end of each test. in addition, we determined the percentage of the predicted maximal hr (% hrmax) for adults, hr recovery at one minute after completion of the 6mwt (hrr1), and the 6mwd. values of desaturation 4%, 8 14 % hrmax > 85% of predicted, 8 14 and hrr1 > 12 bpm were considered significant. 15 17 the 6mwt during which the longest 6mwd was covered was considered valid ; absolute and percent predicted 6mwd values were calculated by the reference equation for the 6mwd in the brazilian population. 18 spirometry was performed with a koko spirometer (pds instrumentation inc., louisville, co, usa). the tests were performed and the results were interpreted in accordance with the bta guidelines. 10 all post - bronchodilator test results were reported as absolute values and as percentages of predicted values, in accordance with pereira. 19 respiratory muscle strength was evaluated in accordance with the bta guidelines, 10 with a digital manometer (warren e collins inc., braintree, ma, usa), the signals being read and recorded through individual ducts. a 2-mm hole in the circuit prevented false measurements as a result of involuntary contractions of the cheeks. 20 the level of asthma control was assessed with the asthma control test (act), previously validated for use in brazil, 21 23 a score of 25 points indicating clinical remission of symptoms or totally controlled asthma, a score of 20 - 24 points indicating adequately controlled asthma, and a score of 20% ; uso dirio de 2-agonista de curta durao ; uso de mais de trs cursos de corticoide oral no ano ; histria de episdio de asma quase fatal ; uma ou mais consultas em servios de urgncia no ano anterior ; e deteriorao rpida da funo pulmonar ao diminuir a dose do tratamento com corticoide. os pacientes includos tiveram acompanhamento ambulatorial por, no mnimo, seis meses, visando o controle de fatores ambientais e de comorbidades, a melhora da tcnica de uso dos dispositivos inalatrios e o aumento da adeso ao tratamento. alm disso, foram includos apenas os pacientes nas etapas 4 e 5 de tratamento da asma (laba / ci em dose elevada mais corticoide oral e / ou anti - ige) segundo as diretrizes da global initiative for asthma (gina) 1 e da sociedade brasileira de pneumologia e tisiologia (sbpt), 2 e com dois critrios maiores ou um critrio maior e dois menores dos critrios da ats / ers. 13 foram excludos os pacientes com cardiopatia grave ; fumantes ativos ; ex - fumantes com carga tabgica 10 maos - ano ou que cessaram tabagismo h menos de um ano ; e pacientes que tiveram exacerbao necessitando de atendimento em pronto - socorro / internao, uso de prednisona ou equivalente acima de 20 mg e / ou de antibiticos nas ltimas quatro semanas. o tc6 foi realizado em um corredor de 25,6 m utilizando um oxmetro porttil (nonin medical, inc., plymouth, mn, eua), seguindo as recomendaes da ats. 14 todos os pacientes fizeram dois tc6, com um intervalo mnimo de 30 min entre eles. foram avaliados os seguintes parmetros : spo2 medida por oximetria de pulso, fc, fr, e percepo da intensidade de dispneia e de fadiga nos membros inferiores pela escala de borg ao incio e ao final dos testes. foram verificados tambm o valor percentual da fc em relao fc mxima prevista para adultos (% fcmx), a fc de recuperao no primeiro minuto aps o trmino do teste (fcr1) e a dtc6. foram considerados significativos valores de dessaturao 4%, % fcmx > 85% do previsto 8 14 e fcr1 > 12 bpm. 15 17 considerou - se vlido o tc6 com o maior valor para dtc6 ; os valores absolutos e percentuais do previsto para a dtc6 foram calculados atravs da equao de referncia para tc6 da populao brasileira. 18 a espirometria foi realizada utilizando um espirmetro da marca koko (pds instrumentation inc., louisville, co, eua). 10 os valores encontrados no teste ps - broncodilatador foram relatados como valores absolutos e percentuais em relao aos valores previstos de acordo com pereira. 19 a fora dos msculos respiratrios foi avaliada atravs de um manovacumetro digital (warren e collins inc., braintree, ma, eua) seguindo as diretrizes nacionais da sbpt, 10 com leitura e armazenamento dos sinais atravs de ductos individuais. um orifcio de 2 mm no circuito impedia falsas medidas por contrao involuntria das bochechas. a pimx foi mensurada ao nvel do vr, e a pemx foi mensurada ao nvel da cpt. os resultados foram expressos em cmh2o. 20 o nvel de controle da asma foi avaliado atravs do asthma control test (act, teste de controle da asma), no qual 25 pontos representam remisso clnica dos sintomas ou controle total da asma ; de 20 a 24 pontos, controle adequado da doena ; e abaixo de 20 pontos, asma no controlada ; foi utilizada a verso validada para uso no brasil. 21 23 o nvel de atividade fsica foi medido de acordo com international physical activity questionnaire (ipaq, questionrio internacional de atividade fsica), verso curta, validado para uso no brasil. 24 25 o instrumento contm sete questes relacionadas frequncia, em dias por semana, e durao, em minutos por dia, das atividades fsicas vigorosas, moderadas e de caminhada. o escore classifica os indivduos em muito ativo, ativo, irregularmente ativo e sedentrio. agrupamos as classificaes em dois grupos : muito ativo ou ativo e irregularmente ativo ou sedentrio. 24 25 todos os pacientes do estudo realizaram tcar de trax para o diagnstico diferencial de outras doenas associadas, como dpoc, bronquiectasias e aspergilose broncopulmonar alrgica. a anlise dos dados foi realizada no statistical package for the social sciences, verso 18 (spss inc., chicago, il, eua) e minitab, verso 16 (minitab inc., state college, ma, eua). o teste de shapiro - wilk foi utilizado para avaliar a distribuio dos dados. as variveis categricas foram apresentadas em frequncia e proporo, e as variveis contnuas foram expressas como mdia e desvio - padro (distribuio paramtrica) ou como mediana e variao (distribuio no paramtrica). para avaliar a diferena entre a dtc6 nos pacientes com agnc e o valor previsto da dtc6 em indivduos normais, assim como para verificar a associao da prtica de atividade fsica (ipaq) com as variveis dtc6, escore do act, % fcmx e fcr1, foi utilizado o teste de mann - whitney. a possvel associao entre o uso de corticoide oral com pimx e pemx foi avaliada atravs do teste de correlao de spearman e teste de kruskal - wallis. com base na maior dtc6 encontrada na literatura, considerando - se um desvio - padro de 90,44 m, nvel de significncia de 0,05, usando - se o teste t de student bicaudal para uma amostra nica, o tamanho da amostra foi estimado em 26 pacientes para se verificar uma diferena de trs indivduos foram excludos por se tratarem de pacientes com provvel sndrome de sobreposio asma / dpoc, e outro foi excludo devido a exacerbao da asma durante o ms da coleta dos dados. participaram 25 pacientes asmticos (18 do sexo feminino e 7 do sexo masculino), com mdia de idade de 49 anos e mdia de ndice de massa corprea de 28,9 7,9 todos os pacientes apresentaram escores do act < 15 pontos, 14 pacientes (56%) eram irregularmente ativos / sedentrios, e 20 (80%) tinham limitaes para exerccios no perodo intercrise. dez pacientes (40%) tinham histria de exacerbao com internao em terapia intensiva, e 7 (28%), com necessidade de ventilao mecnica. tabela 1.caractersticas basais dos pacientes com asma grave no controlada.acaractersticasn = 25idade, anos 49,8 12,6sexo feminino18 (72)imc, kg / m 28,9 7,9ex - fumantes 4 (16)limitaes das atividades na intercrisesem limitaes0 (0)no exerccio20 (80)nas atividades dirias3 (12)nos cuidados pessoais2 (8)em repouso0 (0)presena de crises no ltimo ano 5 (0 - 23)uso de ct sistmico nas crises (4 ciclos / ano)9 (36)uso de ct oral contnuo6 (24)total de internaes 15 (0 - 100)internaes em uti10 (40)necessidade de ventilao mecnica7 (28)histria de pcr1 (4)comorbidades21 (84)rinossinusite crnica22 (88)polipose nasal2 (8)drge13 (52)obesidade11 (44)has2 (8)diabetes mellitus4 (16)distrbio psiquitrico4 (16)dpoc2 (8)sequela de tuberculose1 (4)abpa1 (4)act 14,0 3,9atividades fsicas - ipaq ativo ou muito ativo11 (44)irregularmente ativo ou sedentrio14 (56)mc : ndice de massa corprea ; ct : corticosteroide ; pcr : parada cardiorrespiratria ; drge : doena do refluxo gastroesofgico ; has : hipertenso arterial sistmica ; abpa : aspergilose broncopulmonar alrgica ; act : asthma control test ; e ipaq : international physical activity questionnaire.. cconsiderado como o indivduo que cessou o tabagismo com carga tabgica = 10 anosmao ou que cessou o tabagismo h pelo menos um ano. mc : ndice de massa corprea ; ct : corticosteroide ; pcr : parada cardiorrespiratria ; drge : doena do refluxo gastroesofgico ; has : hipertenso arterial sistmica ; abpa : aspergilose broncopulmonar alrgica ; act : asthma control test ; e ipaq : international physical activity questionnaire. cconsiderado como o indivduo que cessou o tabagismo com carga tabgica = 10 anosmao ou que cessou o tabagismo h pelo menos um ano. todos os pacientes estavam em uso de laba e dose alta de ci (1.600 g de budesonida ou equivalente), 6 (24%) usavam pelo menos 10 mg / dia de prednisona h mais de um ano, e 3 (12%) utilizavam omalizumabe. as principais alteraes tomogrficas foram bronquiectasias mnimas / moderadas com predomnio central, em 3 pacientes ; pequenas reas de enfisema centrolobular, em 11 ; fibroatelectasia discreta do lobo superior direito, em 1 ; e aprisionamento areo, em 1. houve predomnio de distrbio ventilatrio obstrutivo com mdia de vef1 de 58,8 21,8% do previsto, e 17 pacientes (68%) tiveram prova broncodilatadora positiva. as mdias da pimx e pemx foram normais ; entretanto, 7 (28%) e 11 (44%) dos pacientes apresentaram pimx e pemx reduzidas (< 65% do previsto), respectivamente (tabela 2). dos 7 pacientes que tinham pimx reduzidas, somente 2 apresentaram sinais espiromtricos de hiperinsuflao pulmonar. tabela 2.resultados de espirometria e presses respiratrias mximas nos pacientes com asma grave no controlada (n = 25).avariveisresultadospr - bdps - bdvef1,% predito58,8 21,8 66 22,4vef1, l1,61 0,63 1,81 0,62cvf, % predito83,4 20,7 91,0 19,1cvf, l2,79 0,71 3,05 0,65vef1/cvf56,3 12,0pimx, cmh2o87,2 21,0pimx, % predito72,9 15,2pemx, cmh2o108,3 no houve diferena significativa (p = 0,14) entre os valores medianos da dtc6 - 512 m (variao : 307,2 - 597,3 m) - e da dtc6 prevista para indivduos normais - 534 m (variao : 382,6 - 621,3 m) - (figura 1). figura 1.distribuio da distncia percorrida no teste de caminhada de seis minutos no grupo de pacientes (n = 25) com asma grave no controlada (dtc6-agnc) em comparao com a mesma distncia prevista para adultos brasileiros saudveis (dtc6-as).(18)a valores discrepantes. nenhum paciente utilizou oxignio suplementar e no houve queda da spo2 mdia durante o tc6 ; porm, 5 pacientes apresentaram dessaturao durante o mesmo. dentre esses, 2 apresentaram broncoespasmo, que foi revertido com a utilizao de 2-agonista de curta durao, 2 tinham comorbidades (aspergilose broncopulmonar alrgica e sequela de tuberculose pulmonar), e 1 apresentara enfisema e aprisionamento areo na tc de trax. ao final do tc6, a intensidade da percepo da fadiga de membros inferiores foi leve e a de dispneia foi de leve a moderada. tabela 3.teste de caminhada de seis minutos nos pacientes com asma grave no controlada (n = 25).avariveis antes do tc6aps o tc6spo2, % 96,1 1,193,7 3,5fc, bpm81,8 16,7117,8 20,3fr, ciclos / min18,5 2,630,4 5,5escala de borg (fadiga membros inferiores)0,1 0,42,9 2,6escala de borg (dispneia)0,9 1,24,7 1,9dtc6, m-512 4,2fcr1, bpm-21,7 8,3fcmx, % -71 11tc6 : teste de caminhada de seis minutos ; dtc6 : distncia percorrida no tc6 ; fcr1 : queda da fc no primeiro minuto aps o trmino do tc6 ; e fcmx : fc mxima prevista. tc6 : teste de caminhada de seis minutos ; dtc6 : distncia percorrida no tc6 ; fcr1 : queda da fc no primeiro minuto aps o trmino do tc6 ; e fcmx : fc mxima prevista. ao compararmos o subgrupo de pacientes em uso continuo de corticoide oral ou com quatro ou mais ciclos por ano (n = 15) com o subgrupo de pacientes com trs ou menos ciclos por ano (n = 10) a fim de investigar a associao entre a frequncia de uso de corticoide oral e a fora muscular respiratria, no encontramos diferenas estatisticamente significativas entre os valores medianos de pimx - 74,5% (variao : 59,0 - 104,0%) e 72,5% (variao : 43,0 - 104,0% ; p = 0,15), respectivamente - e pemx - 67,5% (variao : 29,0 - 121,0%) e 63,5% (variao : 34,0 - 121,0% ; p = 0,45), respectivamente. os 11 pacientes mais ativos fisicamente e os 14 menos ativos apresentaram resultados semelhantes em relao a act, dtc6, fcr1 e % fcmx no tc6 (tabela 4). tabela 4.associao do nvel de atividade fsica dos pacientes com asma grave no controlada (n = 25) com o nvel de controle de asma e variveis do teste de caminhada de seis minutos.avariveisnvel de atividade fsica (ipaq)pmuito ativo ou ativoirregularmente ativo ou sedentrio(n = 11)(n = 14)dtc6, m537,6 (320,0 - 597,3)490,0 (307,2 - 588,6)0,308fcr1, bpm19,0 (11,0 - 37,0)19,5 (10,0 - 36,0)0,935fcmx, % 72 (53 - 89)73 (50 - 84)0,621escore act15 (7 - 17)13 (6 - 24)0,144ipaq : international physical activity questionnaire ; dtc6 : distncia percorrida no teste de caminhada de seis minutos ; fcr1 : queda da fc no primeiro minuto aps o trmino do teste de caminhada de seis minutos ; fcmx : fc mxima prevista ; e act : asthma control test. ipaq : international physical activity questionnaire ; dtc6 : distncia percorrida no teste de caminhada de seis minutos ; fcr1 : queda da fc no primeiro minuto aps o trmino do teste de caminhada de seis minutos ; fcmx : fc mxima prevista ; e act : asthma control test. historicamente, os pacientes que no atingem o controle da asma, apesar do tratamento nas etapas 4 ou 5 segundo o gina, so classificados como portadores de adc. 1 entretanto, h mais de duas dcadas persistem muitas discusses sobre a terminologia mais apropriada devido aos diferentes critrios de definio utilizados para asma grave, adc, asma refratria ou resistente ao tratamento e, mais recentemente, agnc. em 2000, uma publicao da ats sugeriu critrios de definio de adc, que ajudaram a reduzir parte dessas controvrsias. 13 no entanto, somente aps a publicao da fora tarefa da ats / ers em 2013, ratificada e citada na diretriz gina de 2014, que essa classificao tornou - se mais clara. 4 5 de acordo com aquelas publicaes, o termo " asma difcil de tratar " deve ser reservado para os pacientes que melhoram o controle da asma aps medidas de higiene ambiental, melhora da tcnica de uso dos dispositivos, melhora da adeso ao tratamento ou manejo adequado de comorbidades. por outro lado, as mesmas publicaes definiram " asma grave " como o grupo de pacientes que, apesar das medidas citadas anteriormente, necessitaram de medicamentos nas etapas 4 e 5 segundo gina nos ltimos 12 meses ou de corticoides sistmicos por, no mnimo, 6 meses nos ltimos 12 meses para manter o controle da asma ou que permaneceram sem um controle adequado apesar do tratamento. 4 5 cabe destacar ainda que os pacientes definidos como agnc seriam aqueles que mantiveram pelo menos um dos critrios alm do baixo controle de sintomas 4 5 : act < 20 pontos ou resultados equivalentes em outro escore validado para a verificao do controle da asma ; duas ou mais exacerbaes, necessitando de corticoide sistmico no ltimo ano ; exacerbao grave com necessidade de internao no ltimo ano ; e persistncia de limitao do fluxo areo apesar do tratamento nas etapas 4 ou 5 segundo gina. 4 5 portanto, torna - se cada vez mais importante a caracterizao de pacientes com agnc, bem como a identificao de desfechos que auxiliem na avaliao do impacto de intervenes nesses pacientes. nesse contexto, inclui - se a capacidade de exerccio e a fora muscular respiratria. no nosso estudo, os pacientes com agnc percorreram uma dtc6 semelhante quela prevista para a populao brasileira. alm disso, eles apresentaram comportamento normal da spo2 mdia ao final do teste, reduo adequada da fc um minuto aps o trmino do tc6 e fora muscular respiratria normal. como se sabe, o tc6 fornece indicadores da capacidade funcional, por meio da dtc6, da integridade da troca gasosa intrapulmonar pela spo2, do estresse cardiovascular pela fc, da automao cardaca pela fcr1 e do estresse sensorial pelos escores de dispneia. 8 em nosso estudo, os pacientes percorreram, em mdia, 512 72 m no tc6, apesar de apresentarem padro obstrutivo moderado, serem irregularmente ativos ou sedentrios e apresentarem asma no controlada. adicionalmente, no se observou dessaturao mdia significativa durante o teste (queda da spo2 < 4% em relao aos valores basais). aproximadamente 71% dos participantes atingiram a % fcmx, denotando uma troca gasosa eficiente e estresse cardiovascular satisfatrio. a mdia de fcr1 (21,7 8,3 bpm) foi significativa, sugerindo automao cardaca normal, o se que contrape com o achado de percepo da fadiga moderada e de dispneia (um pouco forte) ao final do teste. aps revisarmos a literatura, encontramos apenas um estudo em adultos sobre o comportamento de pacientes com asma grave durante o tc6. 11 naquele estudo, a dtc6 nos pacientes com adc foi significativamente menor em relao aos seus controles. 11 entretanto, a mdia da dtc6 no grupo com adc foi de 435 m, 11 aproximadamente 112 m inferior dtc6 em nossa amostra. cabe ressaltar que os pacientes daquele estudo eram mais velhos que os do presente estudo (52,3 8,3 anos vs. 49,8 14,4 anos), com maior grau de obstruo, indicado pelo vef1 em % do previsto (44,0 15,9% vs. 58,8 21,8%) e faziam uso de mais medicamentos potentes, como corticoide oral (64% vs. 24%) e omalizumabe (47% vs. 12%). em 2 pacientes essa queda foi atribuda a broncoespasmo, revertido rapidamente com repouso e uso de 2-agonistas de curta durao. os outros 3 pacientes tinham comorbidades (bronquiectasias devido a sequela de tuberculose, aspergilose broncopulmonar alrgica e enfisema pulmonar com aprisionamento areo, respectivamente). esses resultados sugerem que, na ausncia de broncoespasmo, a presena de dessaturao durante o tc6 em pacientes com agnc deve alertar para a possibilidade de presena de comorbidades. para um grupo de autores, 27 a limitao ao exerccio em pacientes com agnc deve ser investigada quanto aos possveis fatores, como a prpria asma, hiperventilao alveolar, broncoconstrio induzida pelo exerccio, descondicionamento fsico, teste submximo e isquemia cardaca. os autores observaram que, ao se identificarem os fatores limitantes, pode - se rever o tratamento, em especial o uso de altas doses de corticoide naqueles pacientes nos quais no foi encontrada limitao pulmonar. 27 a medida da fora muscular respiratria (pimx e pemx) dos pacientes no presente estudo, em mdia, no foi diferente da observada em indivduos normais. o aumento na capacidade residual funcional, causado pela hiperinsuflao pulmonar, retifica o diafragma e altera a mecnica respiratria, resultando em desvantagem mecnica, que pode ser inferida pela reduo da fora dos msculos respiratrios. 7 28 entretanto, os asmticos com distrbio ventilatrio obstrutivo leve ou moderado podem no apresentar hiperinsuflao pulmonar significativa que altere a posio do diafragma. 29 30 como os pacientes da presente casustica apresentavam obstruo moderada das vias areas, possvel que esse fato justifique a similaridade de seus valores de pimx e pemx com a de indivduos normais. no encontramos diferenas significativas entre as mdias de pimx e pemx dos asmticos com uso frequente de corticoide oral com aqueles com uso infrequente (trs ou menos ciclos por ano). um grupo de autores relatou reduo da fora muscular inspiratria em asmticos dependentes de corticoides orais quando comparados com pacientes em uso de ci em altas doses e com graus semelhantes de hiperinsuflao pulmonar. 28 deve - se ressaltar que 28% e 48% dos pacientes tiveram pimx e pemx reduzidas, respectivamente. a fora muscular respiratria pode estar reduzida principalmente em mulheres obesas em consequncia de uma disfuno muscular respiratria. a atividade muscular ventilatria pode ser tambm comprometida pelo aumento da resistncia elstica causada pelo excesso de tecido adiposo na caixa torcica e no abdome, o qual acarreta desvantagem mecnica aos msculos. 31 32 em nosso estudo, 72% das pacientes eram do sexo feminino, com mdia de ndice de massa corprea de 28 kg / m. embora os resultados do presente estudo devam ser avaliados com cautela, principalmente devido falta de um grupo controle, pequena casustica, ao delineamento transversal e por ter sido conduzido em um nico centro, os mesmos so relevantes devido escassez e s dificuldades de pesquisa da capacidade de exerccio em pacientes com asma grave, assim como por representar uma amostra de um centro de referncia de um hospital universitrio e pelo alto poder estatstico observado (poder do teste de 81%). em concluso, de acordo com a presente casustica, a dtc6 nos pacientes com agnc foi semelhante aos valores previstos para a dtc6 em indivduos normais no brasil. a queda da spo2 em 5 pacientes pode ser explicada pela presena de broncoespasmo e de comorbidades. a mdia das foras musculares respiratrias foi acima do limite inferior previsto, independente do uso de corticoide oral. so necessrios novos estudos envolvendo um maior nmero de pacientes e com a incluso de um grupo controle para um maior entendimento do comportamento da capacidade de esforo em pacientes com asma grave e para a definio do seu valor no manejo dessa doena.	objective : to evaluate respiratory muscle strength and six - minute walk test (6mwt) variables in patients with uncontrolled severe asthma (ucsa). methods : this was a cross - sectional study involving ucsa patients followed at a university hospital. the patients underwent 6mwt, spirometry, and measurements of respiratory muscle strength, as well as completing the asthma control test (act). the mann - whitney test was used in order to analyze 6mwt variables, whereas the kruskal - wallis test was used to determine whether there was an association between the use of oral corticosteroids and respiratory muscle strength. results : we included 25 patients. mean fev1 was 58.8 21.8% of predicted, and mean act score was 14.0 3.9 points. no significant difference was found between the median six - minute walk distance recorded for the ucsa patients and that predicted for healthy brazilians (512 m and 534 m, respectively ; p = 0.14). during the 6mwt, there was no significant drop in spo2. mean mip and mep were normal (72.9 15.2% and 67.6 22.2%, respectively). comparing the patients treated with at least four courses of oral corticosteroids per year and those treated with three or fewer, we found no significant differences in mip (p = 0.15) or mep (p = 0.45). conclusions : our findings suggest that ucsa patients are similar to normal subjects in terms of 6mwt variables and respiratory muscle strength. the use of oral corticosteroids has no apparent impact on respiratory muscle strength.
mucus extravasation cysts or mucoceles are an extremely common lesion of the minor salivary glands. we report upon an unusual case of a submandibular gland mucocele presenting as a lateral neck swelling. the term plunging or cervical ranula refers to mucoceles that extend below the mylohyoid muscle, beyond the sublingual space and invariably are associated with the sublingual salivary gland. differentiation from submandibular gland mucoceles is potentially difficult, though plunging ranula is usually characterised by a so called tail sign on imaging. in our case a 54 year old woman presented with a large asymptomatic swelling of the right lateral neck. the swelling had been present for three months but had attained large dimension and was still felt to be increasing in size. she denied any history of trauma, surgery, infection, dysphagia, voice change or weight loss. palpation of the neck mass revealed a soft, fluctuant, non - tender swelling approximately 6 cm in diameter arising superficially within the right submandibular region. a magnetic resonance imaging (mri) scan revealed a large cystic structure in the right lateral neck involving the submandibular space (fig 1). the sample proved inadequate for cytological assessment, however was found to have a raised amylase and protein content suggesting salivary origin. mri scan showing cystic lesion within lateral neck under general anaesthesia the cystic mass was meticulously excised intact via a low submandibular incision. it was found to be in continuity with the ipsilateral submandibular gland (fig 2). histolopathology of the specimen confirmed a fibrous - walled retention cyst associated with a submandibular gland showing chronic obstructive changes. the patient made an uneventful post - operative recovery with no evidence of recurrence at six months. the presentation of a submandibular gland mucocele is uncommon with only a few reported cases in the literature (1 - 3). it has been postulated that this rarity might be due to the anatomical location of the gland and the protective nature of the mandible1. furthermore the physiology of the gland is such that unlike the sublingual gland, which exhibits continuous secretion, the submandibular gland secretes saliva only following stimulation (4). nevertheless, despite the true aetiology being unknown it is thought likely that trauma, obstruction and congenital anomalies are most likely causes (5). the differential diagnosis of a submandibular gland mucocele should include the numerous inflammatory, infective, developmental and neoplastic processes affecting the lateral neck. in particular, a solitary cystic mass should be thoroughly investigated to exclude the possibility of cystic degeneration of a lymph node secondary to metastatic squamous cell carcinoma. fine needle aspiration biopsy for cytological and biochemical assessment can confirm the nature of the cyst and is usually found to have a high amylase and protein content (6). the management of a submandibular gland mucocele is potentially controversial and includes a number of treatment modalities that have been advocated in the management of the cervical or plunging ranula. injection of scelorosing agents have been used to induce inflammation and fibrosis from within the lumen of the cyst thus sealing further extravasation. aspiration is a simple technique but yields a high recurrence rate and as such its use has been largely restricted to the conservative management in the paediatric population. marsupialisation has gained popularity not least because of its relative simplicity, but also because it involves only limited dissection and therefore a reduced risk of damage to adjacent structures. recurrence however is unpredictable leading some authors to advocate a modified technique incorporating packing the cyst cavity (2). contemporary opinion suggests that because mucoceles are pseudocysts there is no imperative to remove the cyst lining. appropriate management should focus on identification and elimination of the salivary gland responsible for the source of mucus secretion (1). removal of the stimulus for secretion should cause involution of the cystic cavity, which seals itself, naturally (7). we would advocate that in cases of a submandibular gland mucocele the lesion should be excised via a cervical approach in conjunction with the submandibular gland. if any doubt exists as to the close involvement of the sublingual gland then it would be advisable to remove this gland as well.	mucus extravasation cysts or mucoceles are an extremely rare occurrence in the major salivary glands. we report upon an unusual case of a submandibular gland mucocele presenting as a neck lump. it should therefore be considered in the differential diagnosis of swellings in the lateral neck. diagnosis and management are complicated by their similarity to the plunging or cervical ranula and differentiation may be potentially difficult. detailed imaging often reveals the plunging ranula as being characterised by a so called tail sign. in our case this sign was absent and subsequent excision confirmed origin from the submandibular gland. we discuss potential treatment modalities and propose a rationale for definitive management.
increasing evidence from animal and human studies points to a nonnegligible role for the innate and adaptive immune system in the development of atherosclerosis, still the leading cause of cardiovascular morbidity and mortality in our western society [14 ]. in fact, it is estimated that approximately 30% of all deaths worldwide can be attributed to cardiovascular disease. therefore, there is a great need for the discovery of new biomarkers that may help in the early detection of patients at risk as well as the development of new therapies leading to the stabilization or regression of atherosclerotic plaques. recent findings suggest that a mismatch in the distribution, phenotype, and/or function of dendritic cells (dc), the main orchestrators of the immune response, contributes to the susceptibility to and the course of atherosclerosis [610 ]. there are two major subpopulations of dc, namely, conventional dc (cdc) and plasmacytoid dc (pdc). in mice, cdc subtypes comprise cdc type 1 (cdc1), encompassing lymphoid - tissue - resident cd8 cdc and their migratory counterparts cd103 cdc, and cd11b cdc2 [8, 10, 11 ]. the specific and highly conserved transcription factor zbtb46 mouse atherosclerotic plaques contain both subtypes of cdc, but cd11b cdc are most abundant and have been shown to rapidly increase during atherogenesis. in contrast, cd103 cdc protect against atherosclerosis as they have been shown to support the homeostasis of regulatory t cells (tregs) in a mouse model of atherosclerosis. in humans, cdc are also segregated into two subtypes, namely, bdca-3 cdc (cdc1) and bdca-1 cdc (cdc2) [8, 10 ]. in addition to cdc, few pdc can be detected in murine and human atherosclerotic lesions, though their exact role in the disease process is still a subject of discussion [1619 ]. murine and human unstable plaques are associated with increased levels of t cells. activated cd4 effector and memory t cells with a t helper (th) 1 profile are among the earliest cells to accumulate in atherosclerotic lesions. natural killer t (nkt) cells represent another subset of t cells that recognize glycolipid antigens presented on cd1d molecules (on antigen - presenting cells) and share surface receptors in common with nk cells. in the past few years nkt cells have become of great interest given the fact that lipid accumulation is a prominent aspect of atherogenesis. moreover, bidirectional interactions between nkt cells and dc ensure amplification and control of the subsequent innate and adaptive immune responses. most of the experimental data from animal models attribute a proatherogenic role to nkt cells [2123 ]. in humans, however, the pathophysiological role of these cells is less clear. although multiple immune cells are involved in atherosclerosis, most studies focus on a single cell type due to technical limitations. we previously described a protocol and a gating technique to identify and isolate immune cells from human atherosclerotic plaques using multiparametric flow cytometry. in this study, local and systemic immune cell distributions in murine and human atherosclerosis were characterized simultaneously using flow cytometry and real - time qpcr. the distribution of dc (subsets), nk(t) cells, t cells, and monocytes / macrophages was analyzed both in blood and plaques. previous research has shown that disturbed flow, caused by carotid ligation, induces rapid and dynamic leukocyte accumulation in the arterial wall. however, adaptive immunity may not be solely driven from within the plaque but may also be driven from plaque - draining lymph nodes or even the periphery (e.g., the spleen). additionally, we assessed the expression of different chemokine receptors during disease development to determine whether the homing functionality of immune cells correlates with changes in immune cell dynamics or plaque development. male and female apoe mice were fed a western - type diet (wd, 4021.90, ab diets) starting at an age of 6 weeks (wk). mice were sacrificed with sodium pentobarbital (250 mg / kg, i.p.) before onset of atherosclerosis (0 wk of wd) or after 6, 12, and 24 wk of wd. these time points represent healthy artery, fatty streak, fibroatheroma, and advanced atherosclerotic plaques in mice. analysis of total plasma cholesterol was performed by using a commercially available kit (randox) following the manufacturer 's instructions. age - matched nonatherosclerotic c57bl/6j control mice on chow feeding were used to adjust for changes related to ageing rather than atherosclerosis. the animals were housed in a temperature - controlled room with a 12-hour light / dark cycle and had free access to water and food. to characterize the immune cells in human atherosclerosis, 72 patients that were eligible for endarterectomy at the carotid (n = 35 ; 49%) and femoropopliteal level (n = 37 ; 51%) were recruited from the clinical departments of thoracic and vascular surgery of the antwerp university hospital and zna middelheim. from 57 (79%) of the included patients, high sensitivity c - reactive protein (hs - crp) levels were measured in serum from 35 patients by the clinical lab of the antwerp university hospital. the mouse protocols were approved by the antwerp university ethics committee on animal experiments (permit number : 2013 - 68). the animals received human care and were treated according to the national guidelines for animal protection, and the guide for the care and use of laboratory animals protocols involving patients were approved by the local ethics committee (number 12/25/212), and all research was based on written informed consent with proper arrangements for the protection of the confidentiality of personal data of the individuals concerned. after sacrificing the mice, single cell suspensions of the aorta draining mediastinal lymph nodes (ln) and the spleen were prepared by passage through a 40 m cell strainer. erythrocytes were lysed using a red blood cell lysing buffer (hybri - max, sigma - aldrich). remaining leukocytes were counted using a hemocytometer and labelled with anti - mouse monoclonal antibodies (supplemental table s1 in supplementary material available online at http://dx.doi.org/10.1155/2016/6467375) at 4c in facs buffer (pbs + 0.1% bsa (sigma - aldrich) + 0.05% nan3 (merck)) in the presence of cd16/32 fc - receptor blocker (biolegend). debris and dead cells were excluded based on forward scatter, side scatter, and positive staining for propidium iodide (life technologies). atherosclerotic plaques were collected in rpmi 1640 medium (life technologies) and kept at room temperature until processing. briefly, within 2 h after surgery the plaque specimens were dissected into small pieces, followed by an enzymatic digestion with 2.5 mg / ml collagenase iv (life technologies) and 0.2 mg / ml dnase i (roche) for 2 h at 37c. after digestion, pbmc were isolated from blood samples by ficoll (ge healthcare) density gradient centrifugation. after isolation, cells from plaque and blood were blocked with mouse gamma globulins and stained with an optimized 9-color panel of mouse anti - human monoclonal antibodies (table s1). to eliminate the abundance of cell debris and extracellular lipids in the digested plaque suspensions the aortic adventitia of mice was partially digested and removed from the rest of the vessel following incubation in an enzyme digestion solution composed of 781.25 u collagenase ii and 14.0625 u elastase (worthington) in 2.5 ml pbs for 10 minutes at 37c. total rna was extracted from the aorta, stripped from the adventitial layer, using a trizol - based rna isolation protocol (ambion). rna was reverse - transcribed with the superscript ii reverse transcriptase kit (life technologies). quantitative gene expression analysis was performed on a 7300 real - time pcr system (applied biosystems) using sybr green technology (sensimix, gc biotech). the parameters for pcr amplification were 50c for 2 min and 95c for 10 min, followed by 40 cycles of 95c for 15 s, 60c for 1 min, and 72c for 30 s. melting curves were checked for amplification of a single, specific product. used primer pairs are summarized in table s2. all data were analyzed using qbase+ 3.0 (biogazelle). after sacrificing apoe mice, the proximal ascending aorta and brachiocephalic artery were collected, embedded in neg-50 (thermo scientific), and snap - frozen in liquid nitrogen. atherosclerotic plaque size, stenosis, and necrotic core (acellular area with a threshold of 3000 m) were analyzed on haematoxylin - eosin (h - e) stained 5 m cryosections (table 2). all images were acquired with universal grab 6.1 software using an olympus bx40 microscope and were quantified with imagej software (national institutes of health). multiple comparisons of means were performed for the analysis of all mouse data using one - way anova followed by dunnett 's multiple comparison test or two - way anova followed by bonferroni 's multiple comparison test where appropriate. differences between human plaques derived from the carotid and femoral artery were tested with the independent student 's t - test. variables that failed normality were logarithmically transformed or analyzed with the nonparametric mann - whitney u test. correlations between local and circulating cells in atherosclerosis were described using spearman 's rank - order correlation coefficient. during atherosclerotic plaque development in apoe mice the percentage of total dc increased in the spleen but no distinct changes were observed in blood or ln (figure 2(a)). interestingly, at all locations and time points, cd11b cdc (or cdc2) represented the most predominant subset. furthermore, a significant drop of this cdc subset was seen at all locations after 12 wk of wd (figure 2(b), red bars). regarding the other cdc subset, cd103 cdc or cdc1, we found no significant difference over time (figure 2(c)). no significant changes were seen in the frequency of nk cells (data not shown). additionally, the percentage of ly-6c resident monocytes and their inflammatory counterparts, ly-6c monocytes, in blood is decreased after 12 wk of wd. the percentage of both subtypes increased between 12 and 24 wk of wd, which was most pronounced in the ly-6c subset (figure 2(e)). regarding cells of the adaptive immunity, the percentage of t cells gradually declined in all studied compartments. the reduction in t cells was most pronounced after 24 wk of wd in blood, spleen, and mediastinal ln compared with mice sacrificed before the onset of atherosclerosis (figures 2(e)2(g)). with regard to t cell subsets, the percentage of total cd4 th cells significantly decreased after 12 wk of wd in blood and spleen as compared to mice at 0 wk of wd (figures 2(e) and 2(f)). interestingly, percentages of nkt cells, known as t lymphocytes with innate effector functions, are increased after 12 wk (in blood and spleen) and 24 wk of wd (in blood, spleen, and ln) (figure 2(d)). all graphs indicate a clear turning point in immune cell dynamics at 12 wk of wd (figures 2(e)2(g), arrows). at this time point, a substantial increase was observed in the size and areas of the atherosclerotic plaques in the brachiocephalic and proximal aorta (table 2). between 12 and 24 wk of wd systemic immune activation is induced as evidenced by an increase in the majority of immune cells above baseline in all locations investigated. expression levels of dc (subset) genes within plaque - containing aortic tissues were measured with qpcr. the expression of zbtb46, a transcription factor used to distinguish the total cdc population from other immune cells, was increased after 6 wk of wd, returned to baseline after 12 wk of wd, and was reduced below baseline levels after 24 wk of wd (figure 2(h)). sirp and xcr1 gene expression was used to discriminate between cd11b cdc and cd103 cdc, respectively. the expression level of sirp increased 11-fold in mice after 12 wk of wd compared to mice sacrificed before the start of the wd (figure 2(i)). a significant increase (2.6-fold) was also observed in the expression of xcr1 after 6 wk of wd (figure 2(j)). the same is true for the amount of nkt cells, as detected by v14-j18 mrna (2-fold increase, figure 2(k)). in mouse plaques, strong features of inflammation could be detected, including the expression of t - bet, the main director of th1 lineage commitment. the relative mrna expression of t - bet was significantly increased (3-fold) after 6 wk of wd feeding compared to mice that had not yet received a wd (figure 3(a)). furthermore, the expression of different chemokine receptors, involved in homing of leukocytes to inflammatory sites or lymph nodes, was investigated. plaque mrna expression for c - c chemokine receptor type 5 (ccr5) and ccr7 was significantly higher (3-fold) after 6 wk of feeding on the wd and returned to baseline when lesions were progressing (figures 3(b) and 3(c)). in aortic plaques, the expression of t - bet is positively correlated with ccr5 and ccr7 expression at 6 wk and 12 wk of wd (table 3). furthermore, there is a strong correlation between the degree of stenosis in the proximal aorta and the mrna level of ccr7, v14-j18, zbtb46, and sirp in mice sacrificed at 6 wk of wd. no correlations were found between the expression levels of ccr5/7 and the numbers of circulating t cells, cd4 th cells, b cells, or nk cells after 6 wk on the wd. in mice from the 12 wk of wd group, v14-j18 expression is correlated to t - bet, ccr5, ccr7, zbtb46, sirp, and xcr1 (table 3). because blood provides a conduit between all organs and tissues, correlations between local and circulating cells in atherosclerosis were also described using spearman 's rank - order correlation coefficient (table 3). plaque development and inflammation were most pronounced in mice that had a low number of circulating nkt cells at 6 wk of wd. at this time point, a strong inverse correlation was determined between nkt cell numbers in blood and the expression of t - bet, ccr5, ccr7, zbtb46, and xcr1 in plaque - containing aortic tissue. in line with these findings, the degree of stenosis in the proximal aorta was inversely correlated to circulating nkt cell numbers. similar observations were seen for the number of circulating cd11b cdc and the expression of the same genes in the aorta at 12 wk of wd. to correct for changes related to age rather than atherosclerosis, we compared the number of cd11b cdc and nkt cells in apoe mice fed an atherosclerotic diet and age - matched healthy wild - type mice fed a chow diet at two time points which represent early and advanced atherosclerotic lesions. percentages of circulating cd11b cdc are higher in apoe mice as compared to healthy wild - type controls in blood, spleen, and mediastinal ln during early lesion (6 wk of diet) formation (figures 4(a)4(c)). in the case of advanced plaques (24 wk of diet), the cd11b cdc percentage is also significantly higher in the blood of apoe mice (figure 4(a)). the opposite is seen for the nkt cells : at the initiation of the disease (6 wk of diet) nkt cell numbers in blood (figure 4(d)) and mediastinal ln (figure 4(f)) of apoe mice are low as compared to healthy controls. there is no difference in the nkt cells percentage in the spleen between apoe and control mice at the onset of atherosclerosis but they increase with enhanced atherosclerosis (figure 4(e)). hence, high numbers of cd11b cdc and low numbers of nkt cells at 6 wk of diet are attributable to the induction of atherogenesis in apoe mice. to analyze different leukocyte subsets in plaque and blood samples from advanced atherosclerosis patients, as observed in mice and similar to our previous data we observed predominance of cd11b cdc within the cd45 population in the plaques, compared to the cd16 monocyte - derived (mo)dc subset. in contrast to the plaque, the cd16 modc was the predominant subset in the blood compared to the cd11b cdc (table 4). clec9a was used as a marker for the human equivalent of cd103 cdc in mice. however, due to their low numbers, we refrained from studying this cdc subset in subsequent analyses in this study. within the cd45 population, furthermore, relatively high mean percentages of nkt cells and t cells were also observed, both in blood and plaque, as compared to the dc (subsets) and monocytes / macrophages (table 4). to extend the evaluation of atherosclerotic plaque composition we compared the immune cell distribution in plaques from distinct anatomical locations (carotid versus femoral artery). to correct for size differences between plaques from femoral and carotid artery, comparing between plaque locations, the number of cells per gram was significantly higher in carotid plaques for the total dc population (311 70 versus 121 24 ; p = 0.014), cd16 modc subset (20 6 versus 5 1 ; p = 0.029), cd11b cdc subset (224 51 versus 85 19 ; p = 0.014), nkt cells (1652 623 versus 127 38 ; p = 0.01), and t cells (1551 556 versus 208 65 ; p = 0.029). in contrast, the macrophage (403 212 versus 10 3 ; p = 0.260) and nk cell numbers per gram (1344 221 versus 1092 271 ; p = 0.477) did not significantly differ between the two locations. we investigated whether correlations could be found between the immunological parameters in blood and advanced plaques collected from the same endarterectomy patient. we could find a strong predictive role for nkt cells which strengthens our observations in mice. remarkably, the percentage of nkt cells in blood correlates strongly with the percentage of total t cells in the plaque (= 0.744 ; p 0.6, to obtain multiple regression models with only the most significant predictors for each plaque variable. strikingly, for both the percentages of t cells and nkt cells in plaques, the nkt cell numbers in blood are the most significant predictors. in addition, partial r values were calculated to describe how strongly the cells in blood contribute to the prediction of cells in the plaque, based on all blood parameters and risk factors from a patient. the nkt cell numbers in blood strongly improve the prediction of the amount of both nkt cells (partial r = 0.36 ; p = 8.9 10) and t cells (partial r = 0.20 ; p = 1.1 10) in plaques, even if all other risk factors are accounted for. for the prediction of nk cells in plaques, the contribution of nkt cells in blood was not significant (partial r = 0.006 ; p = 0.34). c - reactive protein (hs - crp) has been endorsed by multiple guidelines as a biomarker of atherosclerotic cardiovascular disease risk [31, 32 ]. however, in this study, hs - crp levels in blood do not correlate with the percentages of nkt cells in blood or plaques from the same patient (data not shown), although this may be due to the fact that there were only few data points available for hs - crp (n = 35). to date, only a few studies reported the analysis and association of circulating inflammatory cells and advanced atherosclerosis. the aim of the present study was to analyze the frequency of immune cells in blood, plaque, and associated lymphoid tissues (i.e., mouse spleen and aorta - draining ln) and to investigate whether fluctuations in leukocytes are associated with or can be predictive for plaque growth and inflammation. the most pronounced changes during atherosclerosis in mice occur early in plaque development in cells of the innate immune system. early atherogenesis is marked by an elevation in plasma cholesterol levels followed by (oxidative) modification of low density lipoproteins, a well - known trigger of inflammation. antigen - presenting cells are needed at this time to encounter these foreign antigens ; hence more dc are present in the circulation and draining lymph nodes. as atherosclerosis progresses the number of cd11b cdc declines significantly at 12 wk of wd in all locations investigated, suggestive of massive recruitment to the growing lesions in the aortic wall. indeed, we observed an increase in the relative expression level of sirp in the aorta at the same time, together with a substantial increase in plaque size in the brachiocephalic artery and proximal ascending aorta. recruitment of immune cells to sites of inflammation, infection, or injury is stimulated by chemokines and their receptors [3437 ]. ccr5 directs recruitment of immune cells to inflammatory sites like atherosclerotic lesions, while ccr7 can mediate monocyte / macrophage egress from lesions and controls the subsequent migration of immune cells from the plaque to secondary lymphoid organs. we observed a strong inverse correlation between circulating cd11b cdc numbers and ccr5/7 expression in mouse aortic plaques at 12 wk of wd. accordingly, mice that have a low number of cd11b cdc in their circulation, as is the case at 12 wk of wd, have high expression levels of ccr5/7 in their plaques. in addition, we have also seen an inverse correlation between circulating cd11b cdc and the expression levels of t - bet, v14-j18, and zbtb46, which points to an increased inflammatory status in the plaque. additionally, this study revealed that plaque development and inflammation were most pronounced in mice that have a low number of circulating nkt cells at 6 wk of wd. at this time point, expression of inflammation markers, including t - bet, chemokines (ccr5/7), and cdc (zbtb46), as well as the degree of stenosis in the proximal ascending aorta, correlated with nkt cell numbers in blood, pointing to a very significant role of nkt cells in the initiation of atherosclerosis. in line with these findings, previous research demonstrated that the contribution of nkt cells on atherosclerosis is transient and limited to early fatty streak lesions [38, 39 ]., we detected v14-j18 mrna in early lesions (6 wk of wd) but found no accumulation of v14-j18 after the 6-week time point (12 and 24 wk of wd). consistent with results from a study by major., we found that nkt cell numbers are low in blood and ln of apoe mice compared with age - matched wild - type mice at an early stage of atherosclerosis development (6 wk of diet). as the lesion progresses to advanced atherosclerosis, the total dc number increased in the spleen after 12 wk and even more after 24 wk on the wd, due to systemic immune activation. when the bone marrow can no longer handle the production and differentiation of hematopoietic cells, it will outsource the production of circulating leukocytes, including dc. of all organs, the spleen is an ideal outsource destination in apoe mice [42, 43 ]. taken together, these data suggest that inflammatory processes, with an emphasis on cd11b cdc and nkt cells, are crucial in the early development of atherosclerosis before any morphological changes (plaque development) are visible. based on these data, we propose 12 wk as a preferred time point for intervention, especially when assessing the effects of immunomodulatory therapies for preventing the development and progression of atherosclerosis., who reported a peak at 12 wk of diet in inflammatory mediators icam-1, ccr2, il-6, il-12p40, and il-17. parallel to mice, we also observed predominance of the cd11b cdc subset in human plaques when compared with cd16 modc, while the latter is the main subset in blood. in a recent study, cd11b cdc were described to promote atherosclerosis development by limiting the expansion of tregs. in consonance with the drop in circulating cd11b cdc in mice, we and others have shown previously that circulating cd11b / bdca-1 cdc numbers are reduced in patients with coronary artery disease [45, 46 ]. in this study, we only enrolled patients with symptomatic advanced atherosclerosis and were therefore not able to draw a comparison with asymptomatic patients. however, to our knowledge, we are the first to report a direct correlation between nkt cell numbers in blood and the load of t cells (and nkt cells) in the atherosclerotic plaques. both cd1d expressing cells and nkt cells were previously shown to be present in advanced human atherosclerotic plaques. here, we demonstrated that the percentage of nkt cells in blood strongly improves the prediction of both t cells and nkt cells in the plaque, independent of all the other risk factors. in line with these findings, levula. applied gene set enrichment analysis and real - time qpcr to human advanced atherosclerotic plaques from carotid and femoral arteries as well as aortas. 26 genes, out of a total of 29 genes, of the nkt pathway were significantly upregulated in atherosclerotic plaques versus nonatherosclerotic controls. furthermore, in humans, it was reported that circulating nkt cell numbers are reduced in patients who experienced previous cardiovascular events compared with either asymptomatic atherosclerosis patients or young healthy individuals. unfortunately, data on leukocyte cell numbers in the arterial wall during early atherogenesis are virtually nonexistent as patients mostly present themselves in the clinic when serious blockages are already present. nevertheless, in the search for better or additional biomarkers that can alert physicians for the presence of inflammatory plaques, circulating nkt cells should be further explored, as also proposed for type 2 diabetes and cancer [49, 50 ]. nkt cell numbers can fluctuate substantially and nkt cell subsets may play different functional roles in atherosclerosis. future studies, including a higher number of patients and different stages of atherosclerosis, will need to clarify the true potential of nkt cells as biomarkers (or even cellular therapy) for inflammatory, and thus unstable, atherosclerotic plaques. finally, we could not find a correlation between circulating hs - crp levels and the percentages of nkt cells in blood or plaques, although this is most likely due to the small sample size and the associated large variation in hs - crp levels. crp is increased in individuals with an overlap to other risk factor pathways such as obesity, low social class, and smoking. studies on the added value of crp in risk prediction of cardiovascular disease show that hs - crp levels can confirm the presence of plaques but do not provide insight on the degree of stenosis or the inflammation in the plaque. to date, the value of circulating leukocyte profiles as biomarker of atherosclerosis is underappreciated. despite the fact that cdc and nkt cells are quantitatively minor components of the immune system, they do appear to play a major role in modulating the course of the disease. we believe that a profound analysis of circulating leukocytes, in particular cd11b cdc and nkt cells, may thus provide a helpful tool to assess the inflammatory and immune status of an atherosclerosis patient. we provide an extensive quantitative description of systemic and peripheral immune cell dynamics over the entire life span of atherosclerotic lesion development in apoe mice. based on the crucial shift in leukocyte trafficking at 12 wk of wd, we propose this to be a preferred time point for therapeutic intervention, aimed at targeting the dysregulated immune response in atherosclerosis. furthermore, our results show that circulating nkt cells may carry biomarker potential reflecting atherosclerotic lesion progression and/or inflammation, both in mice and humans. because of its predictive value, the dc - nkt cell axis in atherosclerosis could provide potential as a tool for better patient risk stratification and/or a target for plaque stabilization, especially when determining the optimal timing for therapy.	atherosclerosis remains the leading cause of death and disability in our western society. to investigate whether the dynamics of leukocyte (sub)populations could be predictive for plaque inflammation during atherosclerosis, we analyzed innate and adaptive immune cell distributions in blood, plaques, and lymphoid tissue reservoirs in apolipoprotein e - deficient (apoe/) mice and in blood and plaques from patients undergoing endarterectomy. firstly, there was predominance of the cd11b+ conventional dendritic cell (cdc) subset in the plaque. secondly, a strong inverse correlation was observed between cd11b+ cdc or natural killer t (nkt) cells in blood and markers of inflammation in the plaque (including cd3, t - bet, ccr5, and ccr7). this indicates that circulating cd11b+ cdc and nkt cells show great potential to reflect the inflammatory status in the atherosclerotic plaque. our results suggest that distinct changes in inflammatory cell dynamics may carry biomarker potential reflecting atherosclerotic lesion progression. this not only is crucial for a better understanding of the immunopathogenesis but also bares therapeutic potential, since immune cell - based therapies are emerging as a promising novel strategy in the battle against atherosclerosis and its associated comorbidities. the cdc - nkt cell interaction in atherosclerosis serves as a good candidate for future investigations.
' mesotherapy ' is a non - surgical, minimally invasive method of drug delivery that consists of multiple intradermal or subcutaneous injections of a mixture of compounds ' melange ' in minute doses. it has gained disrepute because of inadequate scientific evidence to prove its efficacy. in spite of this, many new substances such as platelet - rich plasma (prp), peptides, growth factors and collagen are finding their way to enter the field of mesotherapy. since small amounts of material have to be injected by multiple pricks rapidly over a large area, mesogun is used. mesogun is a motor - driven equipment that can be used to rapidly deliver the chemicals into the skin and subcutis. however, most mesoguns are expensive and tend to waste material due to dripping from the needle postulated to be due to rapid withdrawal [figure 1 ]. while this may not be significant in financial terms when inexpensive substances are injected, products such as prp or meso solutions for injection which are either expensive or too precious should not be wasted. the cause for dripping is possibly due to lack of minimal recoil, following the injection by the motor - driven plunger. on searching the web, we found a syringe with a spring attached to the piston which would provide the recoil needed to prevent dripping. since these syringes are not freely available, we modified the available disposable syringe to rapidly deliver small quantity of material into the skin, similar to a mesogun. a 5 ml syringe or an insulin syringe is taken and the piston is removed from the body and a spring which is sterilised using ethylene oxide is inserted onto the piston which is inserted back into the body of the syringe [figure 2 ]. the material to be injected is drawn into the syringe and the regular needle is replaced with the meso needle. the specifics of the spring provided for the purpose of duplication and standardisation used are as follows : wastage of injectable material from mesogun spring - loaded 5 cc syringes length 37 mm, thickness 1.21 mm, pitch 4 mm, outer diameter 15 mm, inner diameter 12.6 mm, spring constant (k) = 1300 n / m. length 71.30 mm, thickness 0.75 mm, spring outer diameter 5.7 mm, spring inner diameter 4.58 mm, spring constant (k) = 890 n / m. spring constant (k) is calculated as follows : the spring constant of a spring represents the force required to deform it by a certain amount. as most springs obey hooke 's law, the spring constant k is treated as a constant characteristic operational parameter for the spring. in the case of helical coiled springs, it represents the force required to extend (or compress) the spring by a certain length. experimentally, it is measured by fixing it at one end and applying a force ' f ' on the other end, along its axis, causing it to extend by a length x. the spring constant is then calculated by the formula : f is expressed in newton and x is in metres. small quantities of material can be injected by first piercing the needle into the tissue, pressing the piston using the thumb and removing the thumb which results in the recoil of the piston ; the syringe is withdrawn from the skin, following which the next site is injected [video 1 ]. before disposal of the syringe, the spring can be removed and retained for reuse after sterilisation. the approximate cost of the spring - loaded syringe would be 30 inr for the 5 cc syringe and 26 inr for the 1 cc syringe. thus the modified spring loaded syringe can be an efficient, cost effective method of drug delivery for mesotherapy and intralesional injections. length 37 mm, thickness 1.21 mm, pitch 4 mm, outer diameter 15 mm, inner diameter 12.6 mm, spring constant (k) = 1300 n / m. length 71.30 mm, thickness 0.75 mm, spring outer diameter 5.7 mm, spring inner diameter 4.58 mm, spring constant (k) = 890 n / m. spring constant (k) is calculated as follows : the spring constant of a spring represents the force required to deform it by a certain amount. as most springs obey hooke 's law, the spring constant k is treated as a constant characteristic operational parameter for the spring. in the case of helical coiled springs, it represents the force required to extend (or compress) the spring by a certain length. experimentally, it is measured by fixing it at one end and applying a force ' f ' on the other end, along its axis, causing it to extend by a length x. the spring constant is then calculated by the formula : f is expressed in newton and x is in metres. small quantities of material can be injected by first piercing the needle into the tissue, pressing the piston using the thumb and removing the thumb which results in the recoil of the piston ; the syringe is withdrawn from the skin, following which the next site is injected [video 1 ]. before disposal of the syringe, the spring can be removed and retained for reuse after sterilisation. the approximate cost of the spring - loaded syringe would be 30 inr for the 5 cc syringe and 26 inr for the 1 cc syringe. thus the modified spring loaded syringe can be an efficient, cost effective method of drug delivery for mesotherapy and intralesional injections.	mesotherapy refers to multiple injections of small quantity of the drug over a large area. the mesoguns available are expensive and the motor - driven models tends to waste the expensive material to be injected since the plunger stops after injecting without recoil. we searched for a less expensive device which would inject like the mesogun and still not waste the solution. on searching the web, we identified a spring - loaded syringe. we describe the assembly and use of this inexpensive syringe for delivering multiple injections with minimal wastage.
as antiretroviral therapy (art) coverage in sub - saharan african (ssa) populations continue to increase, deaths from hiv / aids has dramatically declined in the past decade. in ssa and other resource limited settings (rls), where stavudine - containing art regimens have been extensively used, a wide range of disfiguring body fat changes and metabolic abnormalities have been reported [35 ]. stavudine and zidovudine are both thymidine nucleoside analogues used in combination with lamivudine and either nevirapine or efavirenz as first - line therapy of hiv in most rls. after a recent who recommendation against use of stavudine - containing regimens, stavudine has been largely discontinued, but a large proportion of patients remain on zidovudine - containing regimens in uganda and other ssa due to cost considerations. the mechanisms of these metabolic complications remain largely unclear, but several studies suggest that hiv itself, cytokine disregulation [7, 8 ], and the thymidine nucleosides - induced mitochondrial toxicity contribute to these abnormalities. epidemiological data on these metabolic abnormalities and risk of cardiovascular morbidity are still limited in ssa. a few cross - sectional studies in the region have concentrated mainly on stavudine - containing regimens and little is reported on zidovudine - containing regimens [3, 5, 10 ]. in this study we describe the metabolic abnormalities observed in patients on first - line art in an urban outpatient infectious diseases clinic in kampala, uganda on art for at least 12 months. between may and august 2008, patients were enrolled for the study at the adult infectious disease clinic at the infectious diseases institute (idi) in kampala, uganda. we enrolled patients aged 18 years and above, on first line art for at least 12 months with an adherence level by self - report of 95% and who gave informed consent. after fasting for at least 8 hours, blood was drawn for glucose, lipid profile and cd4 count. persons were excluded if their art had ever been switched or substituted, they were diabetic prior to art initiation, had index dyslipidemias, or used lipid - lowering drugs, corticosteroids, or oral contraceptives pills. a trained research assistant obtained a medical history from the patient and abstracted clinical information from the patient 's chart including the date of art start, pre - art cd4 count, who clinical stage, body weight at art start, current cd4 count, and peak body weight while on art. a clinician, masked to the patient 's art regimen, performed a physical examination and anthropometric measurements, including the determination of blood pressure (bp) and waist / hip circumferences. a patient was considered hypertensive, when the systolic blood pressure was 140 mmhg or diastolic bp was 90 mmhg on more than two occasions. features suggestive of fat loss in the face included sunken eyes, prominent zygoma, sunken cheeks, and in the upper and lower limbs included prominent veins, subcutaneous fat wasting especially in the arms. features of fat accumulation were the presence of excessive abdominal fat compared to peripheral fat (after excluding organomegaly and intra - abdominal masses as assessed by palpation), buffalo humps and lipomata on any part of the body. cd4 cell count was done using bd facscalibur (becton dickinson, mountain view, ca, usa). serum lipid assays were batched and run by an automated enzyme - based chemistry analyzer (konelab 30 system, thermo electron corporation, finland), with quality controls done at the beginning of each batched run. ten duplicate samples from each batch were analyzed in a separate laboratory within the national referral hospital complex as an external quality check. finger prick blood for glucose was done on an automated one touch analyzer (lifescan. participants with fasting blood sugar 7 mm were asked to return for a repeat test to confirm diabetes mellitus (dm) at the next convenient date. those confirmed with dm were referred to a specialist at the diabetic clinic for further management. the international classification system recommended by the world health organization (who) and the national cholesterol education program (ncep) for glucose and lipid profiles [1113 ] were used (table 1). data was cleaned and entered in epidata version 3.1 and exported to stata version 10.0 for analysis. at univariate level all variables were compared according to art regimen (stavudine- or zidovudine - containing regimen). categorical variables were compared using a chi square test and a p value was obtained. continuous variables were compared using a wilcoxon rank - sum test, a nonparametric test. at bivariate level, all predictor variables including art regimen were compared with lipid and glucose levels : both were dichotomized into abnormal and normal levels. study participants were categorized as having abnormal glucose levels if they had 6.11 mmol / l, while those who were classified as having abnormal lipid levels had either total cholesterol 5.17 mmol / l or ldl - cholesterol 3.36 mmol / l or hdl - cholesterol 1.53 mmol / l or triglycerides 2.26 mmol / l. odds ratio was used as the measure of association, a 95% confidence interval (ci) was obtained, and a p value was used to show statistical significance. variables with a p value 6.11 mmol / l) was (69/423, 16.3%) of which 63.8% had borderline hyperglycemia, and 36.2% had overt diabetes mellitus (defined as > 6.94 the proportion of patients with borderline hyperglycemia and overt diabetes mellitus on stavudine compared to azt were similar (11%, 3% versus 18%, 5% resp.). at bivariate analysis, use of stavudine (odds ratio (or) 4.18, 95% ci 2.377.37, p 65 kgs was also more likely to be associated with dyslipidemia (or 1.35, 95% ci 1.031.75 p = 0.027) ; family history of hypertension and diabetes mellitus increased the odds of being diagnosed with dyslipidemia (or 1.75, 95% ci 1.022.98 ; p = 0.040 and or 2.29, 95% ci 1.054.97, p = 0.037), respectively (table 4). factors that were associated with hyperglycemia at bivariate level were a 5-year increase in age (or 1.32, 95% ci 1.141.54, p 65 kgs during art. the majority of our patients were from an urban setting, and compared with individuals from rural communities, they are more likely to live a sedentary life, and to eat unhealthy diets which may be confounders in this study. age and art duration which have been reported in another study, were not significantly associated with dyslipidemia possibly because we only enrolled patients who were already on therapy for at least one year. lipodystrophy was significantly associated with dyslipidemia at bivariate analysis, although it was not significant when adjusted for other factors. age over 40 years was independently associated with hyperglycemia. however, at subanalysis when patients with borderline glucose levels were excluded, being overweight at assessment and obese at enrollment (by bmi) were independently associated with overt diabetes mellitus whereas higher median cd4 (> 100 cells/l) at initiation of art was protective (data not shown). again, aging and urban lifestyle are likely to play a significant role in the development of insulin resistance and diabetes mellitus in hiv - infected patients on art. the consequences of these hiv - associated metabolic disturbances in an african population are still unclear. however from dad study in europe and north american populations, a significant risk of cardiovascular morbidity was reported. the major limitations of this study are its cross - sectional design, nonrandomized treatment allocations and a subjective clinical assessment of lipodystrophy. allocation to fixed regimen occurred in rls in sub - saharan africa where the relative risk of thymidine analogues compared to nnrti could not be separated. although increased access to relatively cheaper generic fixed - drug combinations of art have dramatically improved the outcome of patient with hiv infection in africa, the problem of the increasing prevalence of dyslipidemia, hyperglycemia and body fat changes associated with the commonly available art regimens should be addressed. coupled with the adoption of western diets and sedentary lifestyle in our urban population, we are likely to see a steady rise in atherosclerotic diseases in patients on long - term art. mutimura and colleagues in rwanda have shown in a clinical trial that simple but regular exercise can be beneficial in alleviating lipodystrophy and improving metabolic abnormalities, which can be adopted in an art package of hiv - infected patients in rls. our study justifies the ministry of health action in uganda to phase out stavudine - use from first - line art drug list but our study shows that zidovudine contributes to metabolic abnormalities as well. all hiv - infected patients on thymidine analogues should be screened for glucose and lipid abnormalities. furthermore, prospective studies to evaluate long term effect of these metabolic disturbances on cardiovascular health are needed.	introduction. while the introduction of highly active antiretroviral therapy decreased hiv - related morbidity and mortality rates in the sub - saharan africa, a subsequent increase in metabolic abnormalities has been observed. we sought to determine the prevalence of hiv - associated metabolic abnormalities among patients on first - line antiretroviral therapy (art) in an art clinic in kampala, uganda. methods. four hundred forty - two consecutive patients on first - line art for at least 12 months were screened for eligibility in a cross - sectional study, and 423 were enrolled. pre - art patient characteristics were abstracted from medical charts, examinations included anthropometric measurement and physical assessment for lipodystrophy. results. the prevalence of hyperglycemia and dyslipidemia was 16.3% (69/423) and 81.5% (345/423), respectively. prevalence of dyslipidemia between stavudine- and zidovudine - based regimens (91% versus 72% ; p < 0.001). being on stavudine (aor 4.79, 95%, 2.459.38) and peak body weight (aor 1.44, 95% ci 1.051.97) were independent risk factors for dylipidemia. stavudine (aor 0.50, 95% ci 0.270.93) use was associated with lower risk for hyperglycemia while, and older age (aor 1.31, 95% ci 1.111.56) and having a family history of dm (aor 2.18, 95% ci 1.104.34) were independent risk factors for hyperglycemia. conclusions. hiv - associated metabolic complications were prevalent among patients on thymidine analogue - containing art regimens. screening for lipid and glucose abnormalities should be considered in art patients because of cardiovascular risks.
for decades, critical care medicine and nephrology have been fighting but finally emerged altogether with the concept of critical care nephrology almost 20 years ago.1 emergency and critical care medicine have grown into robust self - supporting disciplines with an increasing demand for dedicated highly - skilled physicians. in the past, core specialists were asked to offer bedside advice in acute care wards. yet, patient often lacks the typical features of a particular pathology but rather displays a subacute, hyperacute or in order to further elaborate on this concept, we would like to present a case report highlighting this atypical presentation of a disease while the patient was in an intensive care unit (icu). a 51 year - old male was admitted to the icu after bilateral nephrectomy for invasive urothelial cancer in order to benefit from hemodynamic monitoring in the first 48 hours following the operation. after 48 hours, the patient developed high fever up to 40c with a typical distributive shock pattern. the patient s c - reactive protein was elevated (250 mg / l) and the intensivists were considering whether this patient was developing septic shock. after realizing all the cultures, the patient was taken for a computed tomography (ct) scan of the abdomen which did not reveal an abscess. so, although the patient was considered as a septic shock patient, they found no problematic source whatsoever. they started the patient on antibiotics (tazocin 4 g tds and amikacin 25 mg per kg). the patient was further adequately resuscitated with adequate volume loading and then started on a noradrenaline infusion at a dose of 0.1 mcg / kg / min. after 72 hours, the patient further deteriorated needing an increase in the nonadrenaline infusion up to 1.9 mcg / kg / min. the patient had received 5 l of fluids within 24 hours. he still had a high fever of 39.5c. the infectiologist and the microbiologist at his bed were thinking about enlarging the antimicrobial coverage in order to treat potentially drug - resistant gram - negative bacteria and also fungal infections, despite the fact that all the cultures remained negative. ultimately, before enlarging the microbiological cover, the intensivist decided to call a senior nephrologist - intensivist in order to obtain a second opinion. for the senior intensivist, it was relatively clear as he had seen a very similar case some years ago. for him, a bilateral nephrectomy means in the majority of the cases, a bilateral resection of the adrenal glands. while classical addison s disease can include severe fatigue and weakness, loss of weight, increased pigmentation of the skin, faintness and low blood pressure, nausea, vomiting, salt cravings, and painful muscles and joints under the acute setting, the situation is extremely different and this acute case of addison s disease may be mimicking septic shock with high fever. later the same day, the patient was given 100 mg of hydrocortisone in bolus followed by an infusion of 300 mg of hydrocortisone given through an electrical syringe pump. the patient s condition was completely normalized within 12 hours and all antimicrobials were stopped. this case illustrated that a specific disease may have a completely different presentation while the patient is in icu and therefore, need specific knowledge about atypical intensive care presentation of a disease. physician to discern the multitude of potential pitfalls that complicate diagnosis and treatment in an acute setting. from the case presented here, it is obvious that acute care departments need to be manned with specialized acute care physicians to ascertain timely diagnosis and adequate treatment of patients.2 the list of involved sub - specialities is steadily growing and has recently welcomed nephrology, infectious disease, pharmacology, and endocrinology.3,4 of course, no single intensive care or emergency physician is able to remain fully updated on the ever expanding knowledge and practice regarding these subspecialities.35 as a result of competent manpower, we underscore the need to have intensivists and acute care physicians who have been trained initially in another primary speciality for instance nephrology, cardiology, infectious disease, pharmacology, and endocrinology. in order to be successful, they need to work in a full time position in icu or in an acute care setting developing their primary speciality inside icu and acute care. if they return to their initial speciality, this will result in a complete failure regarding the development of competent manpower. as one may easily understand, this condition of competent manpower is extremely linked with the two other conditions (well - designed technical framework and sufficient financial support) that we shall expand on in other paragraphs. nephrology, for example, has evolved at such a pace over the past decades that it needed to be subdivided in to dialysis chronic care, kidney transplantation, peritoneal dialysis, and critical care nephrology, each focusing on a specific patient population.5,6 from its creation almost 20 years ago,7,8 critical care nephrology has progressively become a key component in acute care. many critical care physicians some of them even without a specific nephrological background have become highly qualified international experts in the management of acute kidney injury. when adapting currently accepted antimicrobial dosing guidelines in crrt, a majority of patients will be left underdosed.9 thanks to the outstanding pioneer work of several groups, dose adaptation regimens for antimicrobial drug therapy under crrt have been created and molded into workable bedside schemes.1012 this is unacceptable since it exposes patients to inadequate treatment and will induce resistance.13 optimal clinical care would need the use of loading doses followed by continuous infusion for administration of time - dependent antibiotics and the use of considerably higher bolus doses of concentration - dependent antibiotics.1013 another example is the recently re - discovered cardio - renal syndrome. hereupon, the associated viewpoints of critical care nephrologists, cardiologists, and intensive care physicians14 allowed for better insight into pathophysiology and a more efficient therapeutic approach.15 if we take the example of the cardio - renal type i syndrome,14,15 the cardiologist will give insight in order to improve the cardiac function not only systolic but also diastolic. a good example is the rediscovery of the use of nitroprusside16 for diastolic dysfunction which can lead also to cardio - renal syndrome.14,15 it is clear for every clinician that nitroprusside can only be safely used while the patient is in icu. the nephrologist will also play an important role here as fluid overload is extremely serious and if the patient does not respond adequately to loop diuretics, a slow continuous ultrafiltration will be necessary17 in order to remove fluid overload from the patient and get him back on the left side of the frank starling curve subsequently improving the systolic function of the left ventricle. importantly, the coordination of all logistic issues on intermittent and continuous dialysis in an icu setting8 enables the generation of sufficient financial resources to engage a critical care nephrologist.6,7,9 sub - specialities such as infectiology, pharmacology, and endocrinology also fulfill an indispensable role in daily icu management.18 critical care infectiology focuses (on a 24/7 basis) on antimicrobial treatment options in typical emergency and icu conditions (eg, reanimation, acute shock resuscitation, etc). in addition, advice from the infectious diseases specialist can be summoned in various challenging situations that may not or only partly be appreciated by a general infectiologist (eg, prevention and treatment of emerging multi - drug resistant icu pathogens, and1921 antibiotic dosing in patients undergoing complex therapies [eg, extracorporeal membrane oxygenation, crrt, burn treatment, etc]).11,12,20 by describing a well - designed technical framework, we underline the importance of specific technical needs for intensive care physicians or acute care physicians while they wish to continue to develop their initial speciality. for instance, an echocardiography tool should have specific functionalities while in icu and should be accessible 24 hours a day in icu. needless to say, crrt devices should have specific features like potential extracorporeal co removal as additional module. again, we can easily understand that the second requirement is tightly linked to the third one about financial support. life - threatening drug drug interactions have been described in approximately 15% of icu patients.22 also, up to 10% of icu admissions are due to drug overdosing or interactions occurring in general wards.23 as recently shown,24 the introduction of critical care pharmacology as a satellite of its clinical counterpart can dramatically reduce these harmful events and thus improve patient safety and outcomes.2325 protracted illness and its inherent metabolic and hormonal changes have paved the way for critical care endocrinology.26 by unravelling the intricate hormonal pathways and stress interactions that accompany a stay in the icu, this relatively new sub - speciality will probably contribute to lowering morbidity, length of icu stay, and mortality.27 the third requirement is looking at the financial support for the appointment of this new critical care physician, and is obviously a key requirement. in order to increase the financial support of a new colleague, it is important that a new technique brought in by this new colleague and which is generating financial benefits, could be directly added to the icu budget and not to any other department. therefore, agreements need to be made between icu and other departments regarding this financial rule : whoever is performing the technique is getting the money. also, the technique needs to benefit even in icu from the same type of reimbursement (so the technique should be reimbursed at 100% like in other departments, and not 50% or less). those two conditions are the two pillars needed in order to convince the hospital directors to invest the needed financial support into those subspecialties inside icu, and to finally appoint those physicians, knowing that they will be bringing a substantial amount of money by implementing their techniques in icu, broadening their scope of indications as they better master this pathology, the physiopathology, and the dedicated technique going along the same line. in conclusion, polyvalence is no longer a valid option in modern critical care. uniting forces between disciplines represents the only way to cope with the increasing complexity and cumulating knowledge in the critical care setting. for this reason, the wide array of upcoming acute care sub - specialities must be committed to unrestricted growth and development. this will require competent manpower, a well - designed technical framework, and sufficient financial support. it is time to say goodbye to the omniscient intensivist and to welcome constructive multidisciplinarity as we welcomed critical care nephrology 20 years ago, although at the time with a lot of skepticism, but not anymore today !	emergency and critical care medicine have grown into robust self - supporting disciplines with an increasing demand for dedicated highly - skilled physicians. in the past, core specialists were asked to offer bedside advice in acute care wards. in the same regard, critical care medicine and nephrology have been fighting but finally emerged altogether with the concept of critical care nephrology almost 20 years ago. indeed, polyvalence is no longer a valid option in modern critical care. uniting forces between disciplines represents the only way to cope with the increasing complexity and cumulating knowledge in the critical care setting. for this reason, the wide array of upcoming acute care sub - specialities must be committed to unrestricted growth and development. this will require competent manpower, a well - designed technical framework, and sufficient financial support. the worldwide success of critical care nephrology proves the feasibility for this concept.
many exanthemata initially present very similarly, even though caused by different organisms ranging from virus to bacteria as well as having different rates of morbidity and mortality. in the majority of cases however, in some cases accurate and prompt diagnosis is paramount, since therapy and appropriate hygiene measures prevent added morbidity and mortality. we present a case which demonstrates the importance of considering relatively rare conditions as the cause of a childhood exanthema. the one main differential diagnosis, staphylococcal scalded skin syndrome (ssss), also known as dermatitis exfoliativa neonatorum or staphylogenic lyell syndrome, is caused by group ii coagulase - positive staphylococci, usually phage type 71, which elaborate exfoliatin (also called epidermolysin), a toxin that splits the upper part of the epidermis just beneath the granular cell layer. the infection often begins during the first few days of life in the umbilical stump or diaper area ; in older children, the face is the typical site. the differential diagnosis includes drug hypersensitivity, viral exanthemas, scarlet fever, thermal burns, genetic bullous diseases (e.g. some types of epidermolysis bullosa), acquired bullous diseases (e.g. pemphigus vulgaris, bullous pemphigoid), toxic epidermal necrolysis and rare the kawasaki syndrome. kawasaki syndrome (ks) or kawasaki disease is an acute febrile illness of unknown etiology that primarily affects children younger than 5 years of age. ks is characterized by fever, rash, swelling of the hands and feet, irritation and redness of the whites of the eyes, swollen lymph glands in the neck, irritation and inflammation of the mouth, lips, and throat. the standard treatment with intravenous immunoglobulin and aspirin substantially decreases the development of these coronary artery abnormalities. for epidemiologic surveillance, cdc defines a case of ks as illness in a patient with fever of 5 or more days duration (or fever until the date of administration of intravenous immunoglobulin if it is given before the fifth day of fever), and the presence of at least 4 of the following 5 clinical signs : rash cervical lymphadenopathy (at least 1.5 cm in diameter) bilateral conjuctival injection oral mucosal changes peripheral extremity changes. cervical lymphadenopathy (at least 1.5 cm in diameter) bilateral conjuctival injection oral mucosal changes peripheral extremity changes. because ssss is, in contrast to kawasaki syndrome, highly transmissible to other paediatric patients via the hands of the staff, isolation of cases a 15 month old boy presented with generalised rash for two weeks, runny nose for one week and off feeds. one day prior to admission the family s general practitioner prescribed 250 mg flucloxacillin, 8 hourly, of which only one dose was taken. his asthma was treated with salbutamol 100 g inhalers for the last 4 months, his eczema with hydrocortisone cream. the little boy has had his pertussis, tetanus, diphtheria, haemophillus influenza b and oral polio vaccinations at 2, 3 and 4 months of age. the patient was irritable and crying, a generalised erythematous rash was present with mild exfoliation in the axilla, the back of the neck and ears. the pulse was 120 beats per minute, the systolic blood pressure 90 mm hg, the respiration rate 30 per minute, the temperature 37.0c, the capillary return was 1 second, the oxygen saturation 98% in air. his pupils were equally reacting to light, and normal fundi and a marked conjungtival injection were noted. one of the differential diagnoses was ssss because of generalized erythematous rash, exfoliation of the skin and conjunctival injection. in ssss a scarlatiniform erythema develops diffusely and is accentuated in flexural and periorificial areas. the inflamed conjunctivae occasionally become purulent. characteristically, circumoral erythema is prominent as is radial crusting and fissuring around the eyes, mouth and nose. at this stage ssss occurs predominantly in children under 5 years of age and includes a range of different skin manifestations, ranging from localized bullous impetigo to generalized cutaneous involvement with systemic illness (, p. 1891 - 2). even though no pyrexia was observed, there was a strong suspicion of ks, since four of the six criteria for diagnosing ks were present. the diagnosis of ks depends on the identification of clinical criteria, which are the following : (1) fever for at least 5 days, (2) nonexudative conjunctival injection, (3) erythema of the oropharynx and lips (with cracking), strawberry tongue, (4) acute no purulent lymphadenopathy (one or more nodes of at least 1.5 cm in diameter), (5) polymorphous erythematous exanthema, (6) at least one of the following : erythema of the palms and soles, oedema of the palms and soles, desquamation of the tips of the digits and around the nails. there are also some distinctive, however nondiagnostic, laboratory features in ks : elevated erythrocyte sedimentation rate (esr), leukocytosis, thrombocytosis, sterile pyuria, moderately elevated transaminase levels and elevated level of acute phase proteins. another quite common cause for a similar presentation is toxic epidermal necrolysis (ten), which appears to involve a hypersensitiviy reaction that results in damage to the basal cell layer of the epidermis. it is triggered by a variety of drugs and vaccinations such as diphtheria and measles. the criteria for diagnosing ten are : (1) widespread blister formation and morbilliform or confluent erythema, associated with skin tenderness, (2) absence of target lesions, (3) sudden onset and generalisation within 24 hours, (4) the histological finding of a full thickness epidermal necrosis and minimal to absent dermal infiltrate. in ten the nikolsky sign is only positive at the site of erythema, the desquamation does not occur over macroscopically unaffected skin (, p. 1852). while awaiting the results of the investigations, the patient s skin suddenly started to peel dramatically and developed a generalized positive nikolsky sign. due to the absence of pyrexia, a normal crp, a normal platelet count, no recent history of vaccination, a prolonged onset of more than 24 hours and the massive desquamation, the admitting team excluded ks and ten. the clinical diagnosis of ssss was established. as a consequence further investigations such as electro cardiogram (ecg) and echocardiography were not performed. the blood cultures were negative after 48 hours and the skin swabs grew heavy mixed commensal skin flora. in japan and the united states of america ks has become the most common cause of acquired heart disease in children. the incidence of ks in great britain is 3.4 per 100.000 children under 5 years as compared to 172.2 per 100.000 children under 5 years in japan. the serious complications of ks are systemic vasculitis resulting in coronary artery aneurysmata (1530%) and myocardial infarction or aneurysm rupture. the case fatality rate of 3.7% observed in ks patients in great britain in 1990 is unfavourably high compared to 0.1% in japan. these figures may be a direct reflection of the fact that british doctors are not that commonly exposed to patients with ks and hence do not include ks in their differential diagnosis. it is also very important to consider ks if less than 5 criteria are present, as occurring in the well described atypical ks. in these patients it is recommended to perform serial echocardiography including 2 examinations in the first 3 weeks after onset the treatment of choice is 30 mg / kg acetylsalicylic acid daily until the fever settles as well as intravenous immunoglobulins, 2 g / kg, as a single infusion within 10 days of onset. this significantly decreases the incidence and severity of aneurysm formation as well as relieving symptoms. there is no clear evidence of the infectious etiology of this condition and infection control measures do not appear to be necessary. however, therapy for ssss requires intravenous antibiotic treatment, directed towards eradication of the exfoliating toxin producing staphylococcus aureus, supportive skin care, and attention to the fluid and electrolyte balance. furthermore it is important not to hesitate in commencing analgesia, since the patients suffer from marked skin tenderness. from an infection control point of view, ssss is, in contrast to ks, highly transmittable to other paediatric patients via the hands of the staff. therefore correct hand hygiene as well as barrier nursing is important until the diagnosis is established. diagnosis is either clinical or via skin biopsy, which shows subcorneal granular layer split and as a characteristic feature the absence of inflammatory infiltrate. in ten it is paramount to appreciate etiologic factors, particularly when the symptoms are drug induced. in those circumstances management is directed towards correcting fluid and electrolyte imbalances as well as treating secondary skin infections. in this situation, strict application of infection control measures and the importance of prompt isolation of all infectious patients and those with presumed or possible infection until an infectious etiology has definitely ruled out can not be overemphasised. in staphylococcal infections the most important route of transmission is direct by contact from one patient to another and indirect by the hands of the staff. therefore, by application of general infection control measures and barrier nursing, as long as they are perfectly practised, strict isolation in a single room is not warranted. however, implementation of infection control in children is aggravated by the normal behaviour of the children themselves. hence, to facilitate infection control measures in paediatric patients infected or colonised with pathogenic organisms, contact isolation in a private room should be practised. when a private room is not available, special emphasis has to be placed on selecting the roommates for a possibly infectious patient. immunocompromised patients and patients who are about to undergo extensive surgery with insertion of prosthetic devices should not be chosen as roommates for children with suspected ssss. also, patients with impaired kidney function may also be at high risk of acquiring complications after colonisation with exotoxin producing strains of staphylococcus aureus, since the exotoxin, which is produced by a localised infection and released into the blood stream, is primarily eliminated through the kidneys. barriers should be used as needed when soiling of clothing, skin or mucous membranes is anticipated. gloves should be used for anticipated contact with mucous membranes, intact and non - intact skin and all secretions. meticulous hand disinfection has to be performed after gloves are removed and before taking care of another patient. gowns are worn only when patient contact is likely to lead to direct soiling of garments with infective material. the likelihood of contamination or soiling usually determines whether a health - care worker needs to wear a gown or other barriers, such as plastic aprons. the use of masks is eventually indicated for those who care in a close contact for the patient and droplet transmission by inducing aerosol during skin - manipulations can not be excluded. linen and contaminated dressings must not be handled with fingers but should immediately be placed, with forceps or gloves, into a container or bag which is immediately closed and sealed. in general, high standards of cleaning, avoiding overcrowding, minimising inter - ward transfers and maintaining adequate levels of trained ward staff should be maintained.	childhood exanthemata are caused by a broad spectrum of common pathogens. many exanthemata initially present very similarly, even though caused by different organisms, ranging from virus to bacteria and their respective toxins. in the majority of cases the diagnosis is only of academic value, since therapy does hardly differ. however, in some cases accurate and prompt diagnosis is paramount, since therapy and appropriate hygiene measures prevent morbidity and mortality. we present a case with two differential diagnoses, staphylococcal scalded skin syndrome and kawasaki syndrome, which demonstrates the importance of considering relatively rare conditions as the cause of a childhood exanthema and discuss differences in therapeutic and infection control management. from an infection control point of view, staphylococcal scalded skin syndrome is, in contrast to kawasaki syndrome, highly transmittable to other paediatric patients via the hands of the staff. therefore maintaining correct hand hygiene as well as other infection control measures are of importance until the final diagnosis is established.
the two most common forms of senile dementia, alzheimer s disease (ad) and vascular dementia (vad), share many of the same clinical signs and symptoms. both diseases are characterized by cognitive decline, functional deterioration and neuropsychiatric symptoms that may present as behavioral alterations.1 vascular disease risk factors such as hypertension, diabetes mellitus, obesity, hypercholesterolemia and vascular changes in the white matter of the brain may not only be present in vascular dementia, but also in alzheimer s disease.2,3 these shared features always cause difficulties for the differential diagnosis of these two diseases, especially for the insidious onset of subcortical vascular dementia (svad) due to small - vessel disease, which leads to lacunar infarcts and the demyelination of white matter and deep subcortical gray and white matter. although it is generally accepted that episodic memory impairment, dyscalculia, agnosia, apraxia, and aphasia are more prominent in ad and that executive / attentional processing, semantic memory and visuospatial / perceptual function are more impaired in vascular dementia, especially in patients with svad, there is a lack of a uniformly identified cognitive, clinical, imaging and pathological profile typical of svad.4,5 ideomotor limb apraxia, which is the most common subtype of apraxia, is the disturbance of planning and the execution of motor activity without any dysfunction of the motor or sensory nervous system or the lack of understanding and motivation.6 dysfunction has been conventionally localized on the left fronto - parieto - temporal cortex, which has been implicated in processing movements of relatively high complexity, planning and sequencing movements. bihemispheric lesions of the deep white matter, the commissural tracts or the basal ganglia have also been reported as a cause of ideomotor apraxia.710 these reports suggest that an interruption in the association or commissural tracts of relative cortical areas can also cause the symptom of ideomotor apraxia. it is still accepted as a diagnostically indicative sign for alzheimer s disease.11 a small study reported that the frequency of apraxia in mild cognitive impairment patients was not different from that of the age - matched healthy population. retrospective analyses have shown that only mci patients who subsequently proceeded to a clinical diagnosis of ad were significantly slower than controls in completing sequential movement tasks despite unimpaired performance.12 these results suggested that the absence of apraxia can be one of the clinical indicators for differentiating mci from the other types of dementia. in this study, we hypothesized that presence of apraxia may be an indicator to differentiate svad and mci patients from mild to moderate ad patients and we investigated the difference in ideomotor apraxia frequency amongst ad, svad and mci patients and the relationship between the dementia severity and the presence of ideomotor apraxia in each group of cognitive disease patients. the patients data were retrospectively gathered from two neurodegenerative diseases units (eskiehir osmangazi university medical faculty and marmara university medical faculty neurology clinics), which were participating in the turkuaz alzheimer working (ta) group. this turkish web - based dementia registry includes neuropsyhiatric and cognitive scales that are commonly used for dementia assessment and is currently being used in 18 dementia referral centers across turkey.13 in this registry, patients are recorded with their detailed neuropsychiatric assessments including the neuropsychiatric inventory (npi). ninety - six consecutive ad, 72 subcortical svad and 84 mci patients were recruited from the web - based data of the two centers. the ad diagnosis was based on the dsm iv - tr and ninds - adrda criteria.11 svad patients met the criteria for vascular dementia also proposed by the dsm iv - tr and ninds - airen. all of the patients showed at least two of the following focal neurological symptoms or signs : facial palsy, dysarthria, dysphagia, pathologic laughing or crying, weakness or sensory loss of limbs, hyperactive deep tendon reflexes, extensor plantar responses, rigidity of limbs, axial rigidity, bradykinesia, hemiplegic gait, stooped posture, short - step gait, festinating gait, shuffling gait, decreased arm swing while walking, and multi - step turning. these changes were defined by high signal intensity (hsi) on t2-weighted or flair images corresponding to grade 3 of the fazekas criteria.14 brain mri scans confirmed the absence of structural lesions such as intracranial hemorrhage, tumor, traumatic brain injury, or hydrocephalus. the diagnosis of mci was consistent with the criteria proposed by petersen and colleagues.15 all of the ad patients were in the mild to moderate stage of the disease according to the global deterioration scale / functional assessment staging system.16 apraxia was examined using the apraxia screening test of tulia (ast), which has high diagnostic accuracy (95% sensitivity and 100% specificity) in stroke.17 the ast requires the performance of 12 gestures in two domains : (1) imitation including one meaningless gesture, one intransitive (communicative) gesture and five transitive (tool - related) gestures and (2) pantomime including two intransitive gestures and three transitive gestures. the performance was dichotomously (fail : 0, pass : 1) scored by the investigators immediately after the patient s performance. the scoring method is described in detail elsewhere.17 a validated turkish version of the mini - mental status examination (mmse) was used to evaluate the cognitive status.18 the clinical dementia rating scale was applied to all of the patients for staging the severity of the dementia.19 spss 15 (spss inc, chicago, il, usa) was used for statistical analyses. continuous variables were expressed as mean sd and were compared between the patients with and without prior stress tests using the one - way anova test with the post - hoc tukey test. categorical variables were expressed as frequency percentages and were compared between the groups using the chi - square test or the fisher exact test as appropriate.20 statistically significant level was accepted as 0.05 and it was two - sided. no significant differences were found between the ad and svad groups for each demographic group. there were no significant differences amongst the three groups in terms of sex ratio and age. compared to the ad and svad groups, the mci group had significantly higher mmse scores and lower disease duration and cdr (p 0.05, for each variable). apraxia was significantly more frequent in ad disease patients (32.3%, n = 31) than both svad (16.7%, n = 12) (x = 4.787 at df = 1, p = 0.0287) and mci (4.8%, n = 4) (x = 18.778 at df = 1, p < 0.005) patients. the frequency of apraxia was also significantly higher in svad patients than the mci patients (x = 6.545 at df = 1, p = 0.0105). when we compared the groups according to the apraxia scores, we observed that ad patients had significantly lower apraxia scores than both of the svad and mci patients (odds ratio : 5.04 [95% confidence interval { ci } : 1.8411.73 ], p < 0.05 and odds ratio : 4.17 [95% ci : 2.679.87 ], p < 0.001, respectively). in addition, a significant difference was found between the svad and mci patients in terms of apraxia scores (odds ratio : 3.12 [% 95 ci : 1.125.71 ], p < 0.05) (figure 2). although a large number of studies have revealed many aspects of the pathophysiology of ad, no strongly specific biological or laboratory markers for this disease have been detected. this paucity is a possible reason for low diagnostic accuracy, especially in the early stages of the disease in which the disease is often confused with other forms of dementia, particularly vascular dementia.21,22 with the lack of a specific diagnostic marker, the diagnosis is still based primarily on clinical findings. the disease progression over time leads to involvement of other cognitive domains in addition to memory, including visuospatial function and praxis, language, and executive skills.23 in our study with ad and svad patients with similar disease duration and dementia severity scores, apraxia seems to be a significant differential marker between these diseases, but it must be noted that svad patients can also have significant ideomotor apraxia. therefore, apraxia, unaccompanied by other signs of dementia, is a weak differential diagnosis parameter for ad and svad. although apraxia is accepted as one of the diagnostic clinical variables for ad, there are a few studies about its diagnostic value.2428 in one of the early studies, kramer investigated the frequency of apraxia with agnosia and aphasia in groups of cortical (probable ad) dementias and a heterogeneous group of subcortical dementias (parkinson s disease and normal pressure hydrocephalus) ; they reported that only aphasia had been significantly more frequent in cortical dementias and had a very low diagnostic sensitivity, specificity and total predictive value for all three symptoms. these authors concluded that cortical and subcortical dementias can not be reliably dissociated on the basis of apraxia, aphasia or agnosia. in a similar recent report assessing the occurrence of ideomotor apraxia in ad and huntington disease patients, the authors concluded that ideomotor apraxia was a common sign in both groups of patients. furthermore, they reported that significantly more huntington disease patients had been classified as apraxic according to the hands and finger imitation and pantomimic subtypes than ad patients.26 however, our study found that apraxia was more frequent in ad than the svad patients. this inconsistency between our results and those of the other subcortical dementia studies could be the result of the difference in the pathogenesis and lesion localization of neurodegenerative and vascular subcortical dementias. for example, in huntington disease, prominent heterogeneously cortical dysfunction overlapping with the brain areas of praxis function, even in early stages of the disease, can be a different pathological process from svad.2931 as the authors of the huntington disease study concluded, the cortical degeneration during the disease process may contribute highly to the development of apraxia.26 previous studies have reported that thalamic and basal ganglia vascular lesions were more associated with apraxia.32 however, our svad group had heterogeneous subcortical lesion localization, and the basal ganglia lesions were not separately investigated in the present study. this approach could be the reason for the difference between our results and those of previous neurodegenerative dementia studies. significantly lower apraxia scores in ad patients than in svad patients with similar disease duration may suggest that praxis function is affected earlier and more severely in the neurodegenerative process. without any pathologically proven diagnosis, however, our study is insufficient to make this conclusion. in our study, the frequency of apraxia in svad patients (16.7%) was significantly higher than in mci (4.8%) patients. although there is insufficient data in the literature, the frequency of apraxia in subcortical stroke patients was reported to be 33.3% by a previous study.33 the authors of that study used a different apraxia test, the movement imitation test, to assess the patients, and they included all of the stroke patients regardless of the presence of dementia.34 this methodological difference could explain the frequency disparity between our results and those of that study. these findings may suggest a methodological problem in the assessment methods for apraxia or the difference between the assessed groups of patients between the studies. for example, apraxia is a well - recognized diagnostic sign in ad, but the exact frequency is still controversial. the frequency has a wide range, from 33% to 77%.35,36 the assessment tests used in the studies, the stage of illness, disease severity, and the psychological status of patients can influence the results we used a screening test called the apraxia screening of tulia (ast) that comprises 12 items extracted from the more comprehensive test of upper limb apraxia, tulia by item reduction analysis.17 the ast is a simple bedside test that was previously validated for stroke and parkinson s disease patients with high clinimetric standards.17,37 the validation for both vascular and neurodegenerative disease patients was the reason for selecting this test for our study. although a high diagnostic sensitivity and specificity for the presence of apraxia in stroke and parkinson s disease patients have been reported, the reliability of this test in dementia may be questionable. reliable results of screening for apraxia in multiple sclerosis have been reported in a recent study.38 however, it must be kept in mind that this test is only a fast screening test and does not allow for the subclassification of limb apraxia. this can also be a limitation for our results, but it should be noted that all the data have been recorded by specialists in cognitive disorders. besides the study gives data only from the turkish population and the results absence of a control group that could give information about the apraxia in a normal population, may be another limitation for the study. but we believe that the significant difference for mci patients seems to be an apperant indicator. in this study, we investigated the frequency and severity of ideomotor apraxia amongst three different cognitive problems (ad, svad and mci) in addition to whether it is a good clinical marker to differentiate these diseases. although ideomotor apraxia was observed more commonly and was more severe in the ad patients than in the svad and mci patients, this significant difference was also observed between the svad and mci patients. these results suggest that, contrary to widespread belief, it is not completely accurate to state that apraxia and cortical dementias are associated but do not include subcortical dementias. however, the significant difference between both of the dementia groups and mci patients suggests that the absence of apraxia can only be a predictor for the diagnosis of mci and that apraxia can be an additional supporting sign for the existing diagnostic criteria.	although ideomotor limb apraxia is considered to be a typical sign of cortical pathologies such as alzheimer s disease (ad), it has been also reported in subcortical neurodegenerative diseases and vascular lesions. we aimed to investigate the difference between ad, subcortical vascular dementia (svad) and mild cognitive impairment (mci) patients by means of ideomotor limb apraxia frequency and severity. ninety - six ad, 72 svad, and 84 mci patients were assessed with the mini - mental status examination (mmse), clinical dementia rating (cdr) and the apraxia screening test of tulia (ast). apraxia was significantly more frequent in the ad patients (32.3%) than in both of the svad (16.7%) and mci (4.8%) patients. the frequency of apraxia was also significantly higher in svad patients than in mci patients. ad patients had significantly lower apraxia scores than both svad and mci patients. in addition, a significant difference was found between svad and mci patients in terms of apraxia scores. these results suggest that the widespread belief of the association between apraxia and cortical dementias is not exactly correct. the significant difference between both of the dementia groups and the mci patients suggests that the absence of apraxia can be an indicator for mci diagnosis.
heidelberger and his postdoctoral fellow forrest kendall who had been rendered one handed (but, by all accounts, no less dextrous) by a farm threshing machine took advantage of purified bacterial polysaccharides (isolated during their earlier studies) for their quantitative experiments. they incubated varying proportions of purified antigen and antibody and determined the nitrogen content (i.e., antibody content) of the resulting precipitate. the data derived from these assays showed that the precipitin reaction could be expressed based on simple equations derived from the laws of mass action and that antibodies and antigens were multivalent. when they repeated the experiment with whole serum, their calculations held up, thus assuring them that their equations were not exclusively applicable to purified solutions (1,2). this was all very well for antibodies to polysaccharides, noted heidelberger in a 1979 article, but what about those elicited by the vast numbers of protein antigens ? (3). as n had not yet been discovered, heidelberger and kendall instead used a colorful trick to distinguish antigen - derived nitrogen from antibody - derived nitrogen. hen egg albumin against which they raised specific antibodies in rabbits (4). the amount of antigen - derived nitrogen in the redissolved precipitate could then be determined by comparing its color (by eye, as colorimeters did not yet exist) with solutions containing known concentrations of the dye ; the remainder of the total nitrogen content was attributed to the antibody (5). heidelberger later took on his first graduate student, elvin kabat. together, they helped settle a long - standing debate concerning whether serum precipitins and agglutinins (antibodies that agglutinate bacteria) were the same or different. at the time, many people thought that these distinct functional properties of antibacterial antiserum could be ascribed to separate entities. returning to the pneumococcal bacteria, the duo showed that precipitins and agglutinins were present in identical amounts in antipneumococcal serum. reduction of one activity by adsorbing the serum with a bacterial or polysaccharide solution resulted in an equivalent reduction in the other activity, suggesting that the two functions were properties of the same antibody molecules (6). kabat went on to show that antibodies in serum came in two sizes large and small (now known as igm and igg)based on their mass and sedimentation rate (7). heidelberger 's work, said former colleague herman eisner (massachusetts institute of technology) in a 2001 article, changed the concept of the antibody from an essentially ill - defined set of serum activities to a protein molecule, measurable in conventional chemical units whose recognition of antigens could be analyzed in molecular terms heidelberger, who was also a talented musician and linguist, worked in the lab until his death at age 103. at his 100th birthday party, he was reportedly asked how many papers he had published in his lifetime. three hundred and four, he answered, adding slyly, so far	having defined the protein nature of antibodies under the tutelage of oswald avery, michael heidelberger was the first to apply mathematics to the reaction of antibodies and their antigens (the precipitin reaction). heidelberger 's calculations launched decades of research that helped reveal the specificity, function, and origin of antibodies.
peripheral giant cell granuloma (pgcg) is the most common oral giant cell lesion appearing as a soft tissue extra - osseous purplish - red nodule consisting of multinucleated giant cells in a background of mononuclear stromal cells and extravasated red blood cells. this lesion is probably not present as a true neoplasm, but rather may be reactive in nature. the initiating stimulus has been believed to be due to local irritation or trauma, but the cause is not certainly known. it has been termed a peripheral giant cell reparative granuloma, but whether it is in fact reparative has not been established and its osteoclastic activity nature appears doubtful. its membrane receptors for calcitonin demonstrated by immunohistochemistry and its osteoclastic activity when cultured in vitro are evidences that the lesions are osteoclasts,[15 ] whereas other authors have suggested that the lesion is formed by cells of the mononuclear phagocyte system. the pgcg bears a close microscopic resemblance to the central giant cell granuloma, and some pathologists believe that it may represent a soft tissue counterpart of the central bony lesion. a 22-year - old female patient reported to the department of oral and maxillofacial surgery with the complaint of swelling in the left upper jaw since 1 year. history revealed that the swelling started as a small one and progressively increased to the present size over a period of 1 year. there was no history of trauma, neurological deficit, fever, loss of appetite, loss of weight. on extraoral examination, a single, diffuse swelling was seen on the left side of the face in the region of anterior maxilla. the surface of the swelling was lobulated and present in relation to 11 21 22. the swelling was firm in consistency and bluish in color, and the overlying mucus membrane was intact [figure 1 ]. the fine needle aspiration cytology (fnac) features showed numerous giant cells in a hemorrhagic background. preoperative intraoral presentation of lesion surgery (excisional biopsy) was planned under local anesthesia (la). lesion was separated from the adjacent tissue by blunt dissection and removed in one piece [figure 2 ]. there was no evidence of recurrence till 5 months of follow - up [figure 4 ]. immediate postoperative view postoperative view after 1 month histopathologic examination of biopsied specimen revealed it to be whitish in color, oval in shape, firm in consistency and measuring about 2 1 cm in dimension [figure 5 ]. numerous giant cells of various shapes and sizes, containing 815 nuclei, were seen with proliferating and dilated endothelial lined blood capillaries with extravasated red blood cells (rbcs). histopathologic examination of biopsied specimen revealed it to be whitish in color, oval in shape, firm in consistency and measuring about 2 1 cm in dimension [figure 5 ]. numerous giant cells of various shapes and sizes, containing 815 nuclei, were seen with proliferating and dilated endothelial lined blood capillaries with extravasated red blood cells (rbcs). the etiology and nature of pgcg (giant cell epulides) still remains undecided. in the past, several hypotheses had been proposed to explain the nature of multinucleated giant cells, including the explanation that they were osteoclasts left from physiological resorption of teeth or reaction to injury to periosteum. there is strong evidence that these cells are osteoclasts as they have been shown to possess receptors for calcitonin and were able to excavate bone in vitro. the pgcg occurs throughout life, with peaks in incidence during the mixed dentitional years and in the age group of 3040 years. the clinical appearance is similar to that of the more common pyogenic granuloma, although the pgcg often is more bluish - purple compared with the bright red color of a typical pyogenic granuloma. although the pgcg develops within soft tissue, cupping superficial resorption of the underlying alveolar bony crest is sometimes seen. at times, it may be difficult to determine whether the mass is a peripheral lesion or a central giant cell granuloma eroding through the cortical plate into the gingival soft tissues. the extra - osseous lesions of cherubism involving the gingiva appear very similar to giant cell epulides. however, the other distinctive clinical and radiographic features of cherubism will indicate the correct diagnosis. histologically, pgcg is composed of nodules of multinucleated giant cells in a background of plump ovoid and spindle - shaped mesenchymal cells and extravasated rbcs. the giant cells may contain only a few nuclei or up to several dozen of them. some of them are large, vesicular nuclei ; others demonstrate small, pyknotic nuclei. ultrastructural and immunological studies[26 ] have shown that the giant cells are derived from osteoclasts. there is also a growing body of opinion that giant cells may simply represent a reactionary component of the lesion and are derived via blood stream from bone marrow mononuclear cells and may be present only in response to an as yet unknown stimulus from the stroma. this concept is based on the results of some more recent studies using cell culture and transplantation, in which the giant cells have been found to be short lived and to disappear early in culture in contrast to the active proliferation of the stromal cells. a study by willing. revealed that the stromal cells secrete a variety of cytokines and differentiation factors, including monocyte chemoattractant protein-1 (mcp1), osteoclast differentiation factor (odf), and macrophage - colony stimulating factor (m - csf). these molecules are monocyte chemoattractants and are essential for osteoclast differentiation, suggesting that the stromal cell stimulates blood monocyte immigration into tumor tissue and enhances their fusion into osteoclast - like, multinucleated giant cells. furthermore, the recently identified membrane - bound protein family, a disintegrin and metalloprotease (adam), is considered to play a role in the multinucleation of osteoclasts and macrophage - derived giant cells from mononuclear precursor cells. in the most recent study by bo liu., in situ hybridization was carried out to detect the mrna expression of the newly identified receptor activator of nuclear factor (nf)-kappab ligand (rankl) that is shown to be essential in the osteoclastogenesis, its receptor, receptor activator of nf - kappab (rank), and its decoy receptor, osteoprotegerin (opg). they concluded that rankl, opg and rank expressed in these lesions may play important roles in the formation of multinucleated giant cells.	peripheral giant cell granuloma or the so - called giant cell epulis is the most common oral giant cell lesion. it normally presents as a soft tissue purplish - red nodule consisting of multinucleated giant cells in a background of mononuclear stromal cells and extravasated red blood cells. this lesion probably does not represent a true neoplasm, but rather may be reactive in nature, believed to be stimulated by local irritation or trauma, but the cause is not certainly known. this article reports a case of peripheral giant cell granuloma arising at the maxillary anterior region in a 22-year - old female patient. the lesion was completely excised to the periosteum level and there is no residual or recurrent swelling or bony defect apparent in the area of biopsy after a follow - up period of 6 months.
prepare the following solutions (see table 1 for compositions) : (a) 1 liter nfr (normal frog ringer), (b) 100 ml 10% saline, (c) 100 ml cmf (ca /mg - free solution), (d) 1 ml its (insulin - transferrin - selenium, sigma i1884), (e) 100 ml l-15 culture medium, (f) 10 ml hcg (human chorionic gonadotropin sigma cg-10), (g) 100 ml k - internal solution, (h) 100 ml k - internal solution for amphotericin b. the osmotic strength of solution 1 should be checked to ensure that it is approximately 260 mosm using a vapor pressure osmometer (e.g. wescor model # 5100c). solutions (a) (b) and (c) can be stored up to three months at 4 c. prepare solution (d) by adding 1 ml deionized h2o to the vial containing the lyophilized powder via a 25 gauge syringe needle and syringe filter. prepare solution (e) in a laminar flow hood by combining all components in a beaker or flask. prepare the hcg (solution (f)) by adding 10 ml deionized h20 to the vial containing the lyophilized powder via a 25 gauge syringe needle and syringe filter. fabricate at least two microdissection tools by gluing a minutien pin (26002 - 10, fine science tools) to the end of a glass pasteur pipette with cyanoacrylate glue. allow the sharp end of the pin to extend past the end of the pipette approximately 0.5 cm. identify a breeding - ready pair of xenopus by observing a prominent, reddish cloaca on the female and dark pigmentation on the planar surface of the front paws of the male. one at a time, net each frog and hold it ventral side down on a sink with the net so that it can not escape. inject 1 ml of solution (f) through the net and subcutaneously into one of the dorsal lymph sacs. place the breeding pair together in a covered ten gallon tank of water. to ensure that the animals will not trample the newly laid and fertilized eggs, install a screened floor with a mesh size of approximately " fitted approximately 1 - 2 inches above the bottom of the tank (figure 1a). leave frogs undisturbed for 12 - 48 hr until the animals are in amplexus and fertilized eggs are observed on the floor below the screen (figure 1b). remove the animals from the tank, but leave the eggs undisturbed for at least 24 loosen the embryos from the bottom of the tank and transfer them to four or five 60x15 mm culture dishes containing 10% saline (solution b). sort the embryos by stage according to the scheme of niewkoop and faber (ref. figure 2a shows an embryo at approximately stage 22 while figure 2b shows an embryo that is too far along in development to be useful (approximately stage 28). it is important to choose embryos that are healthy : those that are smooth in appearance with light brown and white mottling are ideal. embryos that have large black or white patches are generally unhealthy and unusable. inside a laminar flow hood, label and fill approximately halfway three 60x15 mm sterile culture dishes with 10 % saline ; and one with cmf. using a sterile, glass pasteur pipette transfer five to ten stage 22 - 24 embryos into one of the dishes containing 10% saline. with the aid of a stereo zoom dissecting microscope inside the hood (0.6 - 5x with 10 x eyepieces), remove the jelly coat and vitelline membrane from each embryo using two pairs of sterile # 5 forceps (11251 - 30, fine science tools). wash the bare embryos by passing them, one at a time, through the remaining two dishes of 10% saline and finally into the dish containing cmf. use a new, sterile pipette for each transfer and minimize the volume of solution transferred from dish to dish. in turn, hold each embryo gently but firmly with a pair of forceps and, using the microdissection tool fashioned in step 1.5, remove the neural tube and associated myotomes which are located at the most dorsal aspect of the animal. do this by making three slices, one at either end of the location of the neural tube and a third just ventral to it (figure 4a). move each dissected neural tube / myotome to a clean part of the dish away from the yolk granules and other debris (figure 4b). the dorsal - ventral axis, and the locations of the neural tube and myotomes are indicated in figure 4. after about fifteen minutes in this solution (cmf) use forceps to lift the pigmented skin free of the dissected tissue and discard. after an additional 30 - 60 min, the cells will form a " sand pile " as they become dissociated from one another (figure 4c). thaw a 10 ml tube of culture medium and aseptically add 70 l its and 35 ng / ml bdnf (sigma b3795). label and fill sterile 35 mm culture dishes (fd35 - 100 world precision instruments) approximately halfway with l-15 culture medium (solution (e)), one for each embryo dissected. fabricate a plating pipette by grasping each end of a glass pasteur pipette while holding the tapered portion over a flame. pull the ends apart to about 10 cm and then break off the end to yield a tip of approximately 0.2 mm. use the plating pipette to suck up the " sand pile " from one embryo minimizing the amount of solution drawn. expel the cells onto the bottom of a culture dish. leave the dishes of plated cells undisturbed for at least fifteen minutes to allow the cells time to attach to the dish. after 12 - 24 hr in culture, plated cells take on distinct morphological characteristics : muscle cells become spindle - shaped and spinal neurons extend long processes (figure 6). functional synaptic contact between neurite varicosities and muscle cells " m ", " s " and " v " refer respectively to the muscle cell, neuronal soma and presynaptic varicosity. prepare two patch electrodes for recording : one for the presynaptic varicosity and one for the muscle cell. for the presynaptic electrode, prepare an amphotericin - containing solution as follows : add 100 l dmso to a 1.5 ml microcentrifuge tube containing 5 mg amphotericin b (sigma a4888). next, add 10 l of this solution to a second microcentrifuge tube that contains 625 l k - internal solution for amphotericin b (solution (h)). fill the pre - synaptic electrode with the amphotericin - containing k - internal solution prepared in step 4.3 and the post - synaptic electrode with k - internal solution (solution (g)). replace media in the culture dish with nfr and relocate it to an inverted microscope fitted with phase contrast optics. obtain the whole - cell configuration with the muscle cell electrode before the perforated patch configuration with the varicosity electrode. to confirm functionality of the synapse, depolarize the varicosity with a patch clamp amplifier (e.g. axopatch 200b, molecular devices) under software control (e.g. by pclamp 9, molecular devices) and observe the simultaneous presynaptic and postsynaptic currents. figure 7 shows the currents seen in response to depolarizing steps of voltage given to the presynaptic varicosity in 10 mv increments from -30 mv to + 40 mv. the inward currents seen in the presynaptic cell are carried by na and ca and the outward currents by k. currents recorded in the postsynaptic cell are responses to neurotransmitter released from the varicosity and are carried mostly by na through nicotinic acetylcholine receptor channels. prepare the following solutions (see table 1 for compositions) : (a) 1 liter nfr (normal frog ringer), (b) 100 ml 10% saline, (c) 100 ml cmf (ca /mg - free solution), (d) 1 ml its (insulin - transferrin - selenium, sigma i1884), (e) 100 ml l-15 culture medium, (f) 10 ml hcg (human chorionic gonadotropin sigma cg-10), (g) 100 ml k - internal solution, (h) 100 ml k - internal solution for amphotericin b. the osmotic strength of solution 1 should be checked to ensure that it is approximately 260 mosm using a vapor pressure osmometer (e.g. wescor model # 5100c). solutions (a) (b) and (c) can be stored up to three months at 4 c. prepare solution (d) by adding 1 ml deionized h2o to the vial containing the lyophilized powder via a 25 gauge syringe needle and syringe filter. prepare solution (e) in a laminar flow hood by combining all components in a beaker or flask. prepare the hcg (solution (f)) by adding 10 ml deionized h20 to the vial containing the lyophilized powder via a 25 gauge syringe needle and syringe filter. fabricate at least two microdissection tools by gluing a minutien pin (26002 - 10, fine science tools) to the end of a glass pasteur pipette with cyanoacrylate glue. allow the sharp end of the pin to extend past the end of the pipette approximately 0.5 cm. identify a breeding - ready pair of xenopus by observing a prominent, reddish cloaca on the female and dark pigmentation on the planar surface of the front paws of the male. one at a time, net each frog and hold it ventral side down on a sink with the net so that it can not escape. inject 1 ml of solution (f) through the net and subcutaneously into one of the dorsal lymph sacs. place the breeding pair together in a covered ten gallon tank of water. to ensure that the animals will not trample the newly laid and fertilized eggs, install a screened floor with a mesh size of approximately " fitted approximately 1 - 2 inches above the bottom of the tank (figure 1a). leave frogs undisturbed for 12 - 48 hr until the animals are in amplexus and fertilized eggs are observed on the floor below the screen (figure 1b). remove the animals from the tank, but leave the eggs undisturbed for at least 24 loosen the embryos from the bottom of the tank and transfer them to four or five 60x15 mm culture dishes containing 10% saline (solution b). sort the embryos by stage according to the scheme of niewkoop and faber (ref. figure 2a shows an embryo at approximately stage 22 while figure 2b shows an embryo that is too far along in development to be useful (approximately stage 28). it is important to choose embryos that are healthy : those that are smooth in appearance with light brown and white mottling are ideal. inside a laminar flow hood, label and fill approximately halfway three 60x15 mm sterile culture dishes with 10 % saline ; and one with cmf. using a sterile, glass pasteur pipette transfer five to ten stage 22 - 24 embryos into one of the dishes containing 10% saline. with the aid of a stereo zoom dissecting microscope inside the hood (0.6 - 5x with 10 x eyepieces), remove the jelly coat and vitelline membrane from each embryo using two pairs of sterile # 5 forceps (11251 - 30, fine science tools). wash the bare embryos by passing them, one at a time, through the remaining two dishes of 10% saline and finally into the dish containing cmf. use a new, sterile pipette for each transfer and minimize the volume of solution transferred from dish to dish. in turn, hold each embryo gently but firmly with a pair of forceps and, using the microdissection tool fashioned in step 1.5, remove the neural tube and associated myotomes which are located at the most dorsal aspect of the animal. do this by making three slices, one at either end of the location of the neural tube and a third just ventral to it (figure 4a). move each dissected neural tube / myotome to a clean part of the dish away from the yolk granules and other debris (figure 4b). the dorsal - ventral axis, and the locations of the neural tube and myotomes are indicated in figure 4. after about fifteen minutes in this solution (cmf) use forceps to lift the pigmented skin free of the dissected tissue and discard. after an additional 30 - 60 min, the cells will form a " sand pile " as they become dissociated from one another (figure 4c). thaw a 10 ml tube of culture medium and aseptically add 70 l its and 35 ng / ml bdnf (sigma b3795). label and fill sterile 35 mm culture dishes (fd35 - 100 world precision instruments) approximately halfway with l-15 culture medium (solution (e)), one for each embryo dissected. fabricate a plating pipette by grasping each end of a glass pasteur pipette while holding the tapered portion over a flame. pull the ends apart to about 10 cm and then break off the end to yield a tip of approximately 0.2 mm. use the plating pipette to suck up the " sand pile " from one embryo minimizing the amount of solution drawn. expel the cells onto the bottom of a culture dish. leave the dishes of plated cells undisturbed for at least fifteen minutes to allow the cells time to attach to the dish. after 12 - 24 hr in culture, plated cells take on distinct morphological characteristics : muscle cells become spindle - shaped and spinal neurons extend long processes (figure 6). functional synaptic contact between neurite varicosities and muscle cells " m ", " s " and " v " refer respectively to the muscle cell, neuronal soma and presynaptic varicosity. prepare two patch electrodes for recording : one for the presynaptic varicosity and one for the muscle cell. for the presynaptic electrode, prepare an amphotericin - containing solution as follows : add 100 l dmso to a 1.5 ml microcentrifuge tube containing 5 mg amphotericin b (sigma a4888). next, add 10 l of this solution to a second microcentrifuge tube that contains 625 l k - internal solution for amphotericin b (solution (h)). fill the pre - synaptic electrode with the amphotericin - containing k - internal solution prepared in step 4.3 and the post - synaptic electrode with k - internal solution (solution (g)). replace media in the culture dish with nfr and relocate it to an inverted microscope fitted with phase contrast optics. obtain the whole - cell configuration with the muscle cell electrode before the perforated patch configuration with the varicosity electrode. to confirm functionality of the synapse, depolarize the varicosity with a patch clamp amplifier (e.g. axopatch 200b, molecular devices) under software control (e.g. by pclamp 9, molecular devices) and observe the simultaneous presynaptic and postsynaptic currents. figure 7 shows the currents seen in response to depolarizing steps of voltage given to the presynaptic varicosity in 10 mv increments from -30 mv to + 40 mv. the inward currents seen in the presynaptic cell are carried by na and ca and the outward currents by k. currents recorded in the postsynaptic cell are responses to neurotransmitter released from the varicosity and are carried mostly by na through nicotinic acetylcholine receptor channels. figure 4b shows the dorsal view of an isolated spinal cord / myotome immediately after its removal from a stage 22 xenopus embryo. figure 4c shows that, after skin removal and incubation in ca - mg - free solution (cmf, solution (c)), cells dissociate into a " sand pile " and are ready for plating. immediately after plating into culture medium cells exhibit little morphological variability (figure 5b) but take on distinct shapes after twenty - four hours in culture. muscle cells become spindle - shaped while neurons remain spherical whilst extending neurites that elaborate varicosites at synapses with muscle cells (figure 6). simultaneous paired patch recording from the presynaptic varicosity and postsynaptic muscle cell (figure 7) reveals the presynaptic inward and outward currents associated with the release of neurotransmitter and the resultant excitatory postsynaptic currents. figure 3. a : stage 22 embryo before removal of vitelline membrane and jelly coat. b : acutely isolated dorsal portion of embryo ; " d " and " v " refer to the dorsal and ventral aspects of the embryos, while " m " and " n " indicate the approximate locations of the mytomes and neural tube. scale bar represents 1 mm for a, and 0.5 mm for b and c. figure 5. scale bar represents 40 m for a, and 5 m for b. figure 6. neuromuscular junction in culture with identification of neuronal soma (s), presynaptic varicosity (v), and postsynaptic muscle cell (m). presynaptic (top) and postsynaptic (bottom) currents seen in response to application of 10 mv incremental voltage steps presented to the presynaptic varicosity from -30 mv to + 40 mv. key steps in the successful co - culturing of motoneurons and muscle cells are the use of appropriately staged embryos produced from the induced breeding of xenopus frogs, and the careful aseptic dissection of the undifferentiated spinal neurons and myoblasts. fertilized eggs should be left undisturbed until they reach approximately stage 10 or so as moving them sooner often halts their development. healthy embryos are identified by a smooth appearance and a brown and white mottled coloring. stage 22 - 24 embryos are the most useful because this is the point during development just after the neural tube closes and before the myocytes have differentiated significantly. care should be taken when removing the vitelline membrane as it adheres strongly to the embryo. the membrane should be torn apart using sharp forceps so that the embryo emerges intact. another important precaution is to carefully place the cells onto the bottom of the culture dish (rather than letting them settle there). this method is preferred as this increases the likelihood that the cells will adhere to the culture dish. functional neurotransmission between nerve and muscle can be determined with just postsynaptic recording and often can be ascertained by the observation of spontaneous muscle contraction after innervation. postsynaptic recording alone is useful for recording miniature endplate currents or potentials but measurements of evoked release requires presynaptic stimulation, and correlation of pre- and postsynaptic currents requires double patch - clamp. besides the paired - patch recording method described here, this preparation offers the opportunity to introduce a third pipette at the neuronal soma this allows the generation of an action potential that can propagate to the presynaptic varicosity and lead to the release of neurotransmitter. in addition, putative agents that are thought to mediate or modulate synaptic transmission can be introduced to either side of the synapse : via the muscle cell pipette or through diffusion from a third pipette placed at the soma.	much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. however, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. embryonic xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature ; they will form functional synapses within twelve to twenty - four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). some advantages of these co - cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2 - 4. the preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5 - 8. in addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9 - 12, molecular mechanisms of neurotransmitter release13 - 15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18 - 19.
central serous chorioretinopathy (cscr) is a condition characterized by the accumulation of subretinal fluid at the posterior pole of the fundus, causing single or multiple areas of serous retinal detachment. the pathophysiology of the disease is thought to involve retinal pigment epithelial (rpe) dysfunction, increased leakage of choriocapillaris vessels and bruch 's membrane dysfunction [1, 2 ]. the result of localized exudation is the formation of retinal edema and small rpe detachments at the macular and perimacular areas. although the aetiology and triggering factors of cscr are not clearly understood, the condition is commonly associated with the use of systemic or topical steroids [2, 3 ]. consequently, a critical therapeutic step is the withdrawal of any steroids the patient might be using. unfortunately, when steroids are indispensable for the treatment of a patient 's systemic disease, the management of cscr becomes challenging. a 40-year - old white male was referred to our clinic due to a 15-day history of bilaterally blurred vision and metamorphopsia. he had a history of myasthenia gravis for which he had been treated with oral prednisolone and azathioprine during the last 2 years. his best - corrected visual acuity (bcva) was 8/10 in the right eye (od) and 9/10 in the left eye (os). fundoscopy revealed a few small, yellow - whitish circumscribed macular elevations and one larger, irregularly shaped parafoveal elevation in od (fig. fluorescein angiography showed multiple areas of early hyperfluorescence and late leakage in od (fig. optical coherence tomography (oct) revealed rpe detachment, elevation of the neurosensory retina and highly reflective subretinal fluid mostly in od (fig. the diagnosis of multifocal cscr was made. as the patient 's myasthenia could not be optimally controlled at the time, unfortunately, we noticed further deterioration of the bcva and subfoveal expansion of the subretinal fluid in od, so it was decided to administer an intravitreal injection of ranibizumab in this eye. one month later, his bcva had deteriorated to 3/10 in od and 7/10 in os. fundoscopy revealed that the area below the fovea in od was enlarged with the deposition of material presumed to be fibrin in the subretinal space. oct demon - strated further elevation of the neurosensory retina and a more irregular subretinal accumulation of highly reflective material (fig. m over 10 min, waited for 5 min and then directed the diode laser at the larger lesion of leakage in od, using a wavelength of 689 nm and laser energy of 25 mj / cm. after the first application, the laser spot was directed at the second and the third lesion of leakage in od (fig. as the patient 's myasthenic symptoms improved, it became possible for the attending neurologist to reduce the dose of prednisolone, increase the dose of azathioprine and add neostigmine. one month later, and while undergoing prednisolone tapering, his bcva in od had improved to 6/10 with no subretinal fluid, while the area with the fibrin deposition had nearly disappeared (fig. the bcva in os improved to 10/10 over the next couple of months. to date, 1 year after treatment, the patient 's bcva remains stable at 6/10 in od and 10/10 in os with no signs of active disease. although the aetiology and triggering factors of cscr are not clearly understood, the condition has been associated with the use of steroids through virtually any route of administration. furthermore, type a personality, emotional stress, endogenous hypercorti - solism as well as an imbalance between sympathetic and parasympathetic activity have also been suggested as risk factors for cscr [2, 4, 5, 6 ]. corticosteroids may be implicated in these changes by virtue of their propensity to change rpe ionic pump function or alter the permeability of ocular barriers. in addition, steroids increase platelet aggregation and may favor conditions promoting microthrombus formation and increased blood viscosity, thus altering choroidal microcircu - lation. nevertheless, as shown in our case, the competing interests of continuing steroids for a systemic condition versus the necessity to withhold them in patients with cscr can lead to therapeutic dilemmas and may call for close cooperation between clinicians of different specialties. in our patient, pdt with verteporfin acts by decreasing choroidal permeability and tightening the blood - retinal barrier at the level of the retinal pigment epithelium. lately, it has been suggested that reducing the light dose (fluence) is safer because standard pdt may cause severe choroidal ischaemia, while low - fluence pdt minimizes the risk of bruch 's membrane rupture. intravitreal anti - vascular endothelial growth factor administration is an alternative approach to cscr management which has been recently suggested. it is believed that anti - vascular endothelial growth factor exerts its effects by reducing the permeability of the choriocapillaris vessels and tightening the blood - retina barrier. other therapies that have been proposed include the use of focal argon laser photocoagulation targeted at the leakage sites, micropulse diode laser photocoagulation, transpupillary thermotherapy and cortico - steroid antagonists, such as mifepristone and ketoconazole [1, 11, 12, 13 ]. notwithstanding the various treatment modalities, the discontinuation of any exogenous steroids remains the cornerstone of cscr treatment. in our case, as discontinuation of prednisolone was initially contraindicated, it was first decided to proceed with an intravitreal injection of ranibizumab. contrary to previous reports, however, the result was disappointing as the patient 's bcva continued to decline [10, 14 ]. another possible therapeutic option in our patient was the use of focal argon laser coagulation at sites of leakage. this treatment can be a very useful choice in patients with lesions located away from the fovea. however, in this particular case, it was not attempted because it was thought that laser photocoagulation would entail a high risk of rupture of these very prominent pigment epithelial detachments. in addition, it was felt that photo - coagulation through the presumed fibrin that had accumulated in the subretinal space would be of questionable effectiveness. after performing low - fluence pdt and tapering the steroids, the patient 's condition improved dramatically in od. interestingly, the clinical picture and bcva in os had also markedly improved over a period of a few months without any intervention other than steroid withdrawal. it should be noted that the improvement in od could, in principle, be attributed to the discontinuation of prednisolone, rather than pdt. we believe, however, that pdt was essential in our case because the resolution of cscr in od was immediate, whereas the resolution in os occurred over the course of some weeks. additionally, the bcva achieved immediately after pdt remained practically unchanged during our patient 's 1-year follow - up ; this indicates that the maximal therapeutic benefit for od should be attributed to pdt. it is worth mentioning that the existence of yellow - whitish hyperreflective subretinal material presumed to be fibrin is typically associated with severe forms of cscr and may signify a poor prognosis. luckily, our patient 's final bcva in od (6/10) was only moderately reduced compared to the bcva at presentation (8/10). our report indicates that low - fluence pdt may be helpful when immediate steroid withdrawal is not an option in patients with cscr. the authors have not received grant support or research funding and they do not have any proprietary interests in the materials described in the article.	we present a case of bilateral multifocal central serous chorioretinopathy in a 40-year - old male who suffered from myasthenia gravis and was receiving oral prednisolone. due to the severity of the underlying disease, it was not possible to reduce the corticosteroid dose. after initial unsuccessful treatment with an intravitreal injection of ranibizumab, low - fluence photodynamic therapy was performed, followed by gradual tapering of the corticosteroids. visual acuity improved significantly in both eyes. different therapeutic approaches are discussed.
talipes equinovarus is the most common congenital foot deformity diagnosed in newborns. serial manipulative casting by the use of plaster of paris (pop) has been accepted as the primary treatment modality to correct this multiplanar deformity. application of serial manipulative pop casts was first introduced and popularized by kite in the literature ; however, the ponseti technique has been the most popular and demonstrated as the most effective treatment approach. using the ponseti technique, the rates of satisfactory results have been reported as more than 90% by various authors in the literature. the ponseti technique requires casting weekly, with each cast being applied just after the previous cast removal. pop can be more difficult for parents and clinical staff to remove than fiberglass casts. some physicians dealing primarily with the treatment of clubfoot deformity, ask the parents of the patient to soak off the old cast the night before the application of the new cast upon arrival at the clinic. in this case, there is a time gap between the cast removal and the application of the new one. this period can compromise the duration of effective splintage. this may lengthen the time needed for correction and increase the number of casts needed. moreover, the removal of the casts can be stressful for the child as well as the parents. semirigid synthetic softcast is a lightweight material that allows rapid application and removal without any soaking or the use of an oscillating saw. it can easily be applied and dried within an acceptable period, allowing for molding. in addition, there is no time interval between cast removal and application. on the other hand, there are limited numbers of reports regarding the use of fiberglass cast materials in the treatment of clubfoot with the ponseti technique. the main purpose of the present study was to comparatively analyze the effectiveness, advantages, and the complications of using semirigid synthetic softcast with respect to pop cast during the treatment of clubfoot deformity. after having approval from selcuk university faculty of medicine ethical research institutional review board, the present study retrospectively evaluated the clinical data of 196 patients who underwent serial manipulative casting treatment using ponseti technique for clubfoot between september 2009 and october 2010. clubfoot related to neuromuscular disorders such as myelomeningocele, cerebral palsy or arthrogriposis multiplex congenita, the patients who had a past medical history of any failed treatment for clubfoot, and the ones with any accompanying congenital deformity of the lower extremity were excluded from the study. a total of 95 patients (133 feet) treated by an orthopedic referral center using semirigid synthetic softcast were included in group a, whereas the other 101 patients (116 feet) treated by another orthopedic referral center using pop cast were included in group b. group a consisted of 59 boys (85 feet) and 36 girls (48 feet). group b consisted of 61 boys (70 feet) and 40 girls (46 feet). the mean age at presentation was 3.44 3 (range, 014) days in group a and 3.59 3.2 (range, 016) days in group b. serial manipulations and cast treatment was started as soon as possible after the first clinical visit (fig. the feet were scored according to the pirani scoring system at the initial examination and during the last examination before achilles tenotomy. manipulations and serial weekly cast applications were begun immediately according to the ponseti technique. the cavus, adductus, and varus components of the deformity were corrected by positioning the foot in supination and then abducting the foot while counter pressure was applied with the thumb over the head of talus. above - the - knee casts made from the semirigid synthetic cast material (3 m scotchcast soft cast ; health care, st paul, mn) for group a and from the pop for group b were applied. 2). the photograph of a 2 days old baby with bilateral clubfeet at the initial examination. the photograph of a baby applied bilateral above knee casts made of semirigid synthetic softcast according to the ponseti method. all the medical records for the patients were reviewed, especially the clinical evaluation at the initial and final examinations, including pirani scores. any skin problems due to the cast itself (such as pressure wounds) and cast removal (such as skin scratches) were recorded. all the casts were removed in the clinic just before the next manipulation, so that the reduction loss was minimized. when the foot achieved 70 of abduction relative to thigh, percutaneous achilles tenotomy was indicated if there was < 15 of dorsiflexion at the same time. a total of 113 of the 133 feet (84.9%) in group a and 95 of the 116 feet (81.9%) in group b underwent percutaneous achilles tenotomy, followed by 3 weeks of casting. browne splints, as described in the ponseti method, were applied after the removal of the final cast. a final parent satisfaction score was also obtained. according to this 5-point evaluation system (excellent = 5, very good = 4, good = 3, fair = 2, and poor = 1), cast convenience, cast weight, infant tolerance, durability, material satisfaction, and likelihood of recommending the material scores were noted at the end of the final cast removal. number cruncher statistical system 2007 statistical software (ut) was used for the statistical analyses. the descriptive statistics describing the demographic information were expressed as medians, ranges, and interquartile ranges or in medians and standard deviations. a wilcoxon test was used to compare the initial and the final pirani scores before achilles tenotomy. also, a mann whitney u test was used to compare the variables at the initial and final clinical examinations. the mean pirani sores were significantly improved from the first administration of the patients up to the time before achilles tenotomy in both groups (table 1). however, no significant difference was detected between the groups according to the change in the mean pirani score (p = 0.198). the number of casts applied until the patients reached at the achilles tenotomy stage was 3.6 1 (range, 25) in group a and 3.8 0.8 (range, 25) in group b. there was no significant difference between the groups according to the number of casts applied until achilles tenotomy (p = 0.081). in group a, the mean pirani score at presentation was 5.75 0.51 for the feet required a tenotomy and 4.13 0.96 for those that did not. in group b, the mean pirani score at presentation was 5.56 0.49 for the feet required a tenotomy and 4.04 0.90 for those that did not (table 2). the pirani scores before achilles tenotomy or at the end of serial manipulative casting for the ones who did not require an achilles tenotomy were significantly lower than the initial scores in all patients (p = 0.0001). comparison of pirani scores at the initial administration and before the achilles tenotomy comparison of pirani scores between the patients required achilles tenotomy or did not minor complications were noted in 12 patients from group a. nine patients had minor skin irritations (5 skin scratches in the groin, 3 superficial skin abrasions on the dorsal skin of the foot, and 2 superficial heel ulcerations) that did not require any treatment or pausing of the cast applications. seven patients had minor skin irritations (3 skin scratches in the groin and 4 superficial skin abrasions on the dorsal skin of the foot). skin scratches due to oscillating saw during the cast removal were documented in 7 feet. three of those feet required pausing of the cast applications for a 1-week period and thus, increased the total period of serial manipulative casting treatment. the slippage of the cast and skin lesions during the removal of the cast was significantly more common in group b (p = 0.017). the mean parent satisfaction scores were 4 over 5 for cast convenience, 4.5 for cast weight, 3.9 for infant tolerance, 4.5 for cast durability, 4.4 for material satisfaction, and 4.4 for the likelihood of recommending the cast material in group a. in group b, the mean parent satisfaction scores were 3.6 for cast convenience, 2.9 for cast weight, 3.8 for infant tolerance, 4.2 for cast durability, 3.1 for material satisfaction, and 3.3 for the likelihood of recommending the cast material. the history of the clubfoot treatment was assessed in detail by the iowa clinic in 2000. surgical treatment techniques had major problems, such as stiffness, small feet, and poor functional results. the ponseti technique has been used with satisfactory results for more than 20 years. the major advantage of this method is achieving good deformity correction without any major operations. with this technique, many authors reported good - to - excellent results. one of the main issue regarding the ponseti technique is the need for close follow - ups. the ponseti technique requires traditional pop for casting material because it can easily be molded. the standard removal techniques for pop casts include holding the extremity in water for approximately 30 minutes and then unwrapping or using a cast saw or a cast knife, which can be very irritating for the baby and the parents. semirigid synthetic softcast may also be the choice of material used during serial manipulative cast applications. the main advantages of fiberglass materials are radiolucency, lighter weight, improved strength, faster curing time, lower risk of thermal burn, cleaner application and removal, and improved durability. a semirigid fiberglass material has been marketed as having additional advantages over classic rigid fiberglass materials, such as molding ability, flexibility, comfort, and ease of removal and unwrapping. semirigid fiberglass cast materials can be removed easily with unwinding alone in a few minutes. on the other hand some studies have been designed to compare semirigid synthetic and traditional pop cast materials in the treatment of clubfoot. one of them described better functional results with pop, although the cast convenience and parent satisfaction parameters were better with fiberglass material. dimeglio scoring system was used for this study and the number of the patients was 34. our study revealed different results compared to pittner although we used approximately the same parameters such as complete time, different results could be secondary to the scoring system which was used and low number of the patients. they used below knee soft casts and concluded that using below knee soft cast was comparable to those using above knee pop cast. the similar results compared to our study can be secondary to the same scoring system and similar number of patients. this matched the conclusion drawn by another report showing that fiberglass was statically superior in its durability, performance, and ease of removal ; 94% of parents strongly preferred semirigid fiberglass over pop for their children 's serial casting. other advantages of fiberglass materials include its light weight, lack of soiling, and water resistance. we noted pirani severity scores for each patient during the treatment and follow - up visits. overall, we obtained acceptable correction with serial casting alone in 15% of feet and with percutaneous achilles tenotomy in 85% of feet. parental compliance during the treatment is one of the main factors in the nonsurgical treatment. our results demonstrated that using semirigid synthetic softcast for clubfoot treatment provides higher parent satisfaction with lower rates of cast - related complications. furthermore, the fiberglass cast is durable, and its removal is easier than the pop cast. the main limitation of the current study was the retrospective evaluation of prospectively followed patient groups. the different results of similar studies using the scoring system of dimeglio could not be checked for the efficacy of softcast material. on the other hand, the current study was a comparative analysis between the 2 groups of patients with similar clinical features and treated by 2 orthopedic referral clinics using 2 different types of casting material for the ponseti technique. our cohort was a large series included patients with primary idiopathic clubfoot deformity diagnosed and treated immediately after birth. although we did not apply a priori calculation for the sample size, post - hoc analysis was performed and the statistical power of our study in the aspect of achieving a comparison between the 2 groups according to the 2 types of cast material was 0.97 with an alpha value of 0.05. the effect of semirigid synthetic softcast on long - term results and recurrence rate was not evaluated in this study, and thus we recommend that further studies are required. the results that we acquired during the present study were similar to those of previous reports on the effectiveness of the ponseti method of manipulation and casting. semirigid softcast material can easily be used with the ponseti technique for clubfoot treatment because it provides higher parent satisfaction levels and is easier for the physicians to apply and remove.	abstractrandomized controlled clinical trial.the main purpose of the present study was to comparatively analyze the effectiveness, advantages, and the complications of using semirigid synthetic softcast with respect to plaster of paris (pop) during the treatment of clubfoot deformity.the study group consisted of 196 babies (249 feet). a total of 133 feet treated by an orthopedic referral center using semirigid synthetic softcast were included in group a whereas the other 116 feet treated by another orthopedic clinic using pop cast were included in group b. the pirani scores, number of cast applications, time period until achilles tenotomy, any skin problems due to the cast itself, and/or cast removal were recorded. a final parent satisfaction score was also obtained.the mean pirani sores were significantly improved from the first administration to the time before achilles tenotomy in both groups. there was no significant difference according to the number of casts applied until tenotomy. the slippage of the cast and skin lesions was significantly more common in group b. higher parent satisfaction levels were detected in group a.semirigid softcast has been found as superior to pop in the aspects of parent satisfaction and cast - related complication rates.
diabetic nephropathy (dn) is one of the most common microvascular complications of diabetes mellitus (dm) and a leading cause of end - stage renal disease (esrd). china currently has approximately 100 million adults with type-2 diabetes and 150 million people with prediabetes [2, 3 ]. alarmingly, 120 million chinese adults may suffer from chronic kidney disease manifested predominantly as albuminuria. albuminuria is the major determinant of dn and an important risk factor for cardiovascular disease in patients with diabetes. indeed, hospitalized chinese patients with type-2 diabetes showed 44% of microalbuminuria, 12% of macroalbuminuria, and 17% of cardiovascular disease. therefore, type-2 diabetes and chronic kidney disease have become major public health problems in china. renin - angiotensin system (ras) plays a key role in the pathogenesis of diabetic nephropathy. ras blockage including angiotensin - converting enzyme inhibitors (acei) and angiotensin receptor blockers (arb) is currently used for dn treatment [7, 8 ]. furthermore, dn prevention also relies substantially on the prescription of acei and arb in clinical settings. acei such as benazepril have beneficial effects on dn independent of blood pressure - lowering effect and plasma angiotensin ii levels. similarly, arb such as irbesartan also reveal renoprotective effects in landmark clinical trials [1114 ]. in this regard, researches of dual blockade with combined acei and arb are ongoing to maximize the beneficial effects and minimize the adverse events. the disruption of the ras pathway leads to negative feedback and compensatory kallikrein system activation [1517 ]. studies have found that heart tissue kallikrein activity significantly increases the effect of acei in type-2 diabetes, indicating mechanistic interaction between kks and ras. the aim of the present study was to investigate the effect of combined intervention with acei - benazepril and arb - irbesartan on experimental dn rats. we hypothesized that the combined therapy could protect against hyperglycemia - induced chronic renal impairments through renal tissue kallikrein system activation and inflammatory pathway inhibition. male sprague dawley rats (230 20 g body weight) were purchased from guilin medical laboratory animal center. the animals were housed in a specific pathogen - free (spf) laminar flow cabinet. temperature and humidity were maintained at 25 1c and 50 5.0%, respectively. the rats, which were exposed to light, were regulated in 12 : 12 h light - dark cycle. the diet and sterilized water were used to feed these rats. ethical approval for animal studies was according to the animal experimentation ethics committee of guilin medical university. in this study, we used high fat and high sucrose diets (hfd) and conventional diet (5001 rodent diet ; labdiet, st. hfd contains 10% lard, 20% sucrose, 2% egg yolk powder, 3% cholesterol, and 65% conventional diet. after adaptive feeding for one week, the rats were fed with conventional diet (n = 10) or hfd (n = 50) for 4 weeks. then, the hfd - fed rats received intraperitoneal injection with low dose (35 mg / kg) of streptozotocin (sigma, st. streptozotocin was freshly prepared by dissolving in sterile sodium citrate - trisodium citrate buffer (0.1 mmol / l, ph 4.2). meanwhile, the rats fed with conventional received intraperitoneal injection of the same volume of the buffer. six weeks later, dn rats were defined by the following criteria : (1) blood glucose level 16.7 mmol / l ; (2) 1.5-fold increase in urine volume compared to the baseline ; and (3) urinary albumin excretion rate beyond 30 mg/24 h [20, 21 ]. a total of 40 hfd - fed rats met the criteria and eventually were included in this study. subsequently, the rats were divided into 5 groups and each group had 10 rats : (1) normal control ; (2) dn model control ; (3) dn rats treated with benazepril 10 mg / kg / d ; (4) dn rats treated with irbesartan 30 mg / kg / d ; and (5) dn rats treated with both benazepril 10 mg / kg / d and irbesartan 30 mg / kg / d. blood glucose level was determined via taking blood from tail vein once a week, using the precision xtra blood glucose monitoring system (alameda, ca, usa). at the end of 8-week treatments, 24 h urine from each group was collected using metal metabolism cage to calculate total urine volume and measure albumin excretion rate. fasting serum insulin concentrations were measured using enzyme immunoassay and rat insulin elisa kit (mercodia, uppsala, sweden). homa - ir (homeostasis model assessment of insulin resistance) index was estimated with the formula homa - ir = fasting serum insulin (mu / l) fasting serum glucose (mmol / l)/22.5. kidney index here reflects kidney hypertrophy estimated by the kidney weight / body weight (mg / g). all the kidney tissue samples were fixed in 10% buffered formalin and embedded in paraffin. serial sections (4 m) were cut perpendicular to the longest axis of the kidney. sections were stained by primary antibodies including kallikrein 1, the phagocytic immunomarker cd68 (macrophage), the proinflammatory and profibrotic markers transforming growth factor- (tgf-) 1, and -smooth muscle action (sma) (santa cruz, ca, usa), as described in our prior reports [2224 ]. stained slides were counterstained with either dab (brown) for light microscopy or antifading dapi (blue) for immunofluorescence microscopy, followed by examination with a zeiss a2 imaging microscope (carl zeiss, hamburg, germany). when checking the stained slides, the pathologist was blinded for animal grouping. comparisons among the five groups were analyzed by one - way analysis of variance (anova), followed by tukey post hoc multiple comparison test. before injecting streptozotocin, the average concentrations of blood glucose between normal control and hfd - fed groups were not significantly different. however, the hfd stimulated a significant increase in body weight, fasting serum insulin, and homa - ir (table 1). six weeks after injecting the low dose of streptozotocin, hfd - fed rats (n = 40) showed hyperglycemia with average blood glucose level over 16.7 mmol / l, polyuria with 1.5-fold increased urine volume, and albuminuria with urinary albumin excretion rate higher than 30 mg/24 h. all the biochemical outcomes indicate establishment of dn model by hfd plus low dose streptozotocin. to evaluate renal function changes after ras blockade, 24 h urine protein, blood urea nitrogen, and serum creatinine compared with the normal controls, the dn rats showed kidney dysfunction echoed by elevated serum levels of 24 h urine protein (figure 1(a)) and blood urea nitrogen (figure 1(b)) and serum creatinine (figure 1(c)) (all p < 0.05). the renal dysfunction significantly improved after the treatments with acei, arb, or both (figure 1). similar renoprotective effects were observed between the acei and arb treatments (figure 2(a)). interestingly, obvious blood glucose lowering was achieved by the dual ras blockade versus dn control and monotherapy. pathological changes in the dn rats included kidney hypertrophy reflected by the increased kidney index (figure 2(b)), mild - to - moderate glomerulosclerosis, tubular atrophy with basement membrane thickening, arteriosclerosis, and interstitial fibrosis with chronic inflammatory infiltration (data not shown). consistent with the renal function improvement, pathological correction by the combined therapy was superior to any monotherapy. figure 3 shows the immunoreactivity of kallikrein predominantly localized in renal tubular cells and generally negative in glomeruli. the dn control rats showed very fewer cells reactive for kallikrein (figure 3). tubular cells positive for kallikrein substantially increased in rats which received the acei, arb, or both (table 2). cells immunoreactive for the inflammatory marker cd68 were mainly interstitial large macrophages intermixed with chronic inflammatory infiltrates (figure 4). the cd68-positive cells were rare in normal control rats (figure 4(a)) but frequently seen as diffuse infiltrates in the dn rats (figure 4(b)). treatments with the ras blockers prevented the chronic inflammatory infiltrates reactive for the phagocytic marker cd68. the dual blockade was better than the acei (figure 4(c)) and arb (figure 4(d)) to diminish the cd68-stained cell infiltration (figure 4(e)). in general, the density of cd68-positive cells was in parallel to the severity of structural damage including glomerulosclerosis, arteriosclerosis, and interstitial fibrosis. in general, chronic inflammatory cells immunoreactive for the profibrotic marker tgf-1 were interstitial macrophages frequently intermixed with infiltrating lymphocytes (figure 5), consistent with the cd68 reactivity in figure 4. in contrast, both interstitial fibroblasts and vascular smooth muscle cells were positive for the activated fibroblast marker -sma. -sma positivity localized mainly with arteriosclerosis and interstitial fibrosis whereas immunoreactivity of tgf-1 was accompanied by chronic inflammatory infiltration (figure 5). among the five groups of animals, the dn rats showed a few more cells positive for tgf-1 and -sma than normal controls and rats given ras blockers (figure 5). moreover, combined treatment appeared better than monotherapy in attenuating the immunofluorescence of tgf-1 and -sma. this study shows tubular kallikrein activation and inflammatory amelioration likely underlying the renoprotective effects of ras blockades in diet - induced diabetic nephropathy. dual ras blockade with both acei and arb may delay the development of diet - induced diabetic nephropathy through compensatory kallikrein upregulation and inflammatory downregulation. both kallikrein - kinin system (kks) and renin - angiotensin system (ras) have been implicated in the pathogenesis of diabetic nephropathy (dn) [25, 26 ]. tissue kallikrein 1 is a member of the tissue kallikrein family that is mainly responsible for the generation of kinins, while bradykinin (bk) is the principal kinin responsible for the physiological actions. in the kidney, kallikrein 1 consistent with our findings in this study, a reduction of renal kallikrein excretion has been found in diabetic individuals and patients with diabetic nephropathy. early clinical evidence has indicated a significantly lower urinary kallikrein excretion in type-2 diabetic patients with nephropathy than in diabetic patients without nephropathy and in control subjects. in experimental type-1 diabetes, kallikrein protects against the development of microalbuminuria. in streptozotocin - induced diabetic rats, kidney tissue level and excretion of active kallikrein were reduced after 3 weeks compared with age - matched nondiabetic control rats, and despite increased kidney size, renal plasma flow was reduced in the diabetic rats. increased urinary bk levels found in severely hyperglycemic diabetic rats are related to increased filtration of components of the plasma kks and renal kininogen synthesis in combination with decreased renal kinin - degrading activity. furthermore, activated kks under diabetic condition may be beneficial in preventing podocyte loss in diabetic nephropathy. previous reports have shown that intravenous delivery of the human tissue kallikrein gene reduced blood pressure and plasma insulin levels in fructose - induced hypertensive rats with insulin resistance. furthermore, a recombinant adenoassociated viral vector expressing the human tissue kallikrein cdna as a sole, long - term therapy could correct insulin resistance and prevent renal damage in streptozotocin - hfd - induced type-2 diabetic rats. in this study, the intensity of tubular kallikrein expression was related to the severity of diabetic nephropathy defined by polyuria, proteinuria, kidney hypertrophy, and chronic inflammatory infiltration. all these findings suggest that the kks may serve as a therapeutic target in delaying the progression of diabetic nephropathy. both urinary kallikrein excretion and kallikrein excretion rate (24 h excretion of urinary kallikrein/24 h creatinine clearance) in hypertensive diabetic patients with nephropathy were significantly lower than in normotensive patients with nephropathy. the decrease in urinary kallikrein is parallel to the existence of diabetic nephropathy with arterial hypertension. although the basal plasma renin activity in patients with type-2 diabetes was not significantly different from controls, both the renin activity and kallikrein levels increased after furosemide in controls while in diabetics this response was severely blunted, indicating early derangement of the kks and the ras as renal hemodynamic mechanisms heralding the onset of nephropathy. moreover, the suppressed renal kks is evident not only in the whole kidney but in each nephron which is still functioning. furthermore, prior study has revealed that the reduction of kinin metabolism by acei might be involved in the beneficial effects exerted by these compounds in diabetic kidney functions. although an earlier study questions the postulate of a role of the kks in the initiation of essential hypertension, interaction between the kks and ras may partially contribute to the multifactorial nature of diabetic nephropathy. in addition to kidney haemodynamics modulated by the kks - ras counterbalance, inflammatory response appears to change after the initiation of diabetic nephropathy. a recent experimental study showed the tissue kallikrein mediated proinflammatory pathways and activation of protease - activated receptor-4 in proximal tubular epithelial cells.. levels of kallikrein 1 protein were not measured by western blot. due to lack of blood and urine samples, we could not measure the levels of plasma kallikrein 1, urinary kallikrein excretion, and kallikrein excretion rate (24 h excretion of urinary kallikrein/24 h creatinine clearance). our future work will explore interactions between the kallikrein activation and inflammatory pathway by detailed measurements of kallikrein and cytokine levels in samples of plasma, kidney tissues, and urine. understanding of the ras has shifted from the classical limited - proteolysis linear cascade to a cascade with multiple mediators, multiple receptors, and multifunctional enzymes. the homologue of ace, ace2, is insensitive to ace inhibitors and forms angiotensin (1 - 7) [ang-(1 - 7) ] from angiotensin ii the ace2/ang-(1 - 7)/mas axis has putative role as an ace - ang ii - at1 receptor counterregulatory axis within the ras [42, 43 ]. in the present study, a landmark study has shown that the combination of losartan and lisinopril was associated with reduced ang ii formation and elevated cardiac ace2 activity for increased ang ii metabolism. moreover, the renal cortex ace2 formation was upregulated with increased levels of ang-(1 - 7) in the urine of animals after acei or arb. these findings furthermore, ang-(1 - 7) generated by proximal tubular ace2 inhibits ang ii - stimulated mapk phosphorylation and tgf- expression in proximal tubular cells. taken together, the ace2/ang-(1 - 7)/mas axis exerts protective actions in diabetes, chronic kidney disease, hypertension, and other cardiovascular and metabolic disorders. in the present study, we have no intension to promote the combined treatment of dual ras blockade due to the multifactorial nature of diabetic nephropathy. furthermore, a new clinical trial also provides evidence that combination therapy with acei and arb is associated with an increased risk of adverse events among patients with diabetic nephropathy. taken together, new research questions remain for the role of kallikrein as a therapeutic target in diabetic nephropathy [18, 35, 50, 51 ]. in conclusion, kidney tissue kallikrein activation and inflammatory amelioration may underlie the renoprotective effects by the ras blockade in diet - induced diabetic nephropathy. further investigations are required to dissect the mechanistic interactions between the kallikrein - kinin system and renin - angiotensin system.	purpose. the objective of this study is to investigate the effect of dual blockage of renin - angiotensin system (ras) on renal kallikrein expression and inflammatory response in diabetic nephropathy (dn). methods. rats were randomly divided into 5 groups with 10 rats in each group : normal control ; dn model induced by high fat and high sucrose diets ; and dn treated with either benazepril 10 mg / kg / d, irbesartan 30 mg / kg / d, or both. after 8-week treatment, we examined changes in the kidney histopathology, function and immunohistochemical stain of kallikrein, macrophage marker cd68, and profibrotic markers transforming growth factor- (tgf-) and -smooth muscle action (sma). results. dn rats showed enlarged kidneys with glomerulosclerosis, interstitial chronic inflammation and fibrosis, and proteinuria. all the pathological damage and functional impairments were improved after the ras blockades (all p < 0.05). compared with monotherapy, combined treatment further alleviated the kidney impairments in parallel to increased tubular immunoreactivity for kallikrein and decreased immunopositive cells for cd68, tgf-, and -sma. conclusion. the renoprotective effects of the dual ras blockade in diabetic nephropathy may be attributed to improved tubular kallikrein expression and interstitial inflammatory response.
a multiplex pcr solution specifies a forward and reverse primer for each single nucleotide polymorphism (snp) and assigns each primer pair to one of a finite set of tubes. in partitioning snp primers into individual tubes, care must be taken to ensure that all primers within a tube are mutually compatible, i.e. that they do not form primer - dimers through cross - hybridization, which would otherwise reduce target product yield. the multiplex pcr problem is equivalent to partitioning a graph g(v, e) into a set of disjoint cliques, where nodes represent snps, edges connect two snps whose associated primers are tube - compatible and resulting cliques constitute valid multiplex pcr tubes. the problem of partitioning a graph into k k disjoint cliques is np - complete (1). the muplex system is unique in that it provides multiple design alternatives that reveal inherent tradeoffs with respect to multiple competing objectives, such as average tube size, tube size uniformity and overall snp coverage. multiplex pcr is a core enabling technology for high - throughput snp genotyping, serving as a foundation for applications in forensic analysis, including human identification and paternity testing (2), the diagnosis of infectious diseases (3,4), whole - genome sequencing (5), and pharmacogenomic studies aimed at understanding the connection between individual genetic traits, drug response and disease susceptibility (6). for example, in the hme assay (7), genomic sequences containing the snps of interest are first amplified by pcr. after shrimp alkaline phosphatase digestion of excess dntps, a primer extension reaction is carried out to interrogate the snps. the primer extension products (often oligonucleotides 18 to 25 bases long) are then detected by matrix - assisted laser desorption ionization time - of - flight (maldi - tof) mass spectrometry. given the large molecular weight window (45009000 da) and the high resolution of the mass spectrometry, 20 or more snps can be easily and simultaneously genotyped. thus, the throughput - limiting step is often the pcr plex level. in a 384-well format with 20-plex pcr, the per - snp cost can be reduced to just a few cents while a single maldi - tof mass spectrometry can be used to genotype 76 800 snps by a single operator in 1 day. given a set of dna sequences and a snp location at each, the system aims at designing (i) a set of pair forward and reverse primers for each sequence ; (ii) a placement of these primers into maximal size tubes such that the coverage (number of sequences) included in the pcr assay is maximized. the user provides a set of snps and associated flanking sequences in the standard fasta format. these sequences may be entered manually or uploaded from a file. to improve primer specificity, users may instruct the muplex server to filter resulting primer candidates by aligning them against the human genome using blat (8). in addition to the snp sequences, the user specifies primer selection criteria, including length, gc content, positional constraints, and melting temperature tm constraints for individual primer oligos as well as interaction parameters (maximum local alignment score, 3 tail g), and a maximum tm range for all primer pairs within a single multiplex assay. muplex then solves the dual problem of selecting primer pairs for amplifying the flanking sequence of each snp and partitions these primer pairs into multiplex compatible sets each corresponding to a single multiplex pcr tube reaction. as noted above, muplex generates multiple solutions alternative each corresponding to a set of multiplex pcr tubes. each solution is evaluated with respect to the following objectives : total number of tubes required.minimum, average and maximum tube size (multiplexing level).number of unique tube sizes.total snp coverage measured both in terms of the percentage of snps (associated primer pairs) assigned to maximum - sized tubes, as well as the percentage of snps assigned to tubes of any size. total snp coverage measured both in terms of the percentage of snps (associated primer pairs) assigned to maximum - sized tubes, as well as the percentage of snps assigned to tubes of any size. for example, some solutions may achieve higher overall multiplexing levels but at the expense of lower coverage, i.e. by excluding some snps from the solution. in addition, muplex tries to minimize the number of unique tube sizes in order to facilitate automation in a high - throughput genomics environment. the email contains a solution summary allowing quick comparison of each alternative, and details for each solution including the selected primers, their individual properties and assigned tube. muplex employs a number of heuristic algorithms and allows new algorithms to be added over time. solution time depends on the number of solution alternatives requested, the number of snps and the target multiplexing level. for typical problems involving < 100 snps, given a set of dna sequences and a snp location at each, the system aims at designing (i) a set of pair forward and reverse primers for each sequence ; (ii) a placement of these primers into maximal size tubes such that the coverage (number of sequences) included in the pcr assay is maximized. the user provides a set of snps and associated flanking sequences in the standard fasta format. these sequences may be entered manually or uploaded from a file. to improve primer specificity, users may instruct the muplex server to filter resulting primer candidates by aligning them against the human genome using blat (8). in addition to the snp sequences, the user specifies primer selection criteria, including length, gc content, positional constraints, and melting temperature tm constraints for individual primer oligos as well as interaction parameters (maximum local alignment score, 3 tail g), and a maximum tm range for all primer pairs within a single multiplex assay. muplex then solves the dual problem of selecting primer pairs for amplifying the flanking sequence of each snp and partitions these primer pairs into multiplex compatible sets each corresponding to a single multiplex pcr tube reaction. as noted above, muplex generates multiple solutions alternative each corresponding to a set of multiplex pcr tubes. each solution is evaluated with respect to the following objectives : total number of tubes required.minimum, average and maximum tube size (multiplexing level).number of unique tube sizes.total snp coverage measured both in terms of the percentage of snps (associated primer pairs) assigned to maximum - sized tubes, as well as the percentage of snps assigned to tubes of any size. total snp coverage measured both in terms of the percentage of snps (associated primer pairs) assigned to maximum - sized tubes, as well as the percentage of snps assigned to tubes of any size. for example, some solutions may achieve higher overall multiplexing levels but at the expense of lower coverage, i.e. by excluding some snps from the solution. in addition, muplex tries to minimize the number of unique tube sizes in order to facilitate automation in a high - throughput genomics environment. the email contains a solution summary allowing quick comparison of each alternative, and details for each solution including the selected primers, their individual properties and assigned tube. muplex employs a number of heuristic algorithms and allows new algorithms to be added over time. solution time depends on the number of solution alternatives requested, the number of snps and the target multiplexing level. for typical problems involving < 100 snps, muplex is written entirely in java (j2sdk1.4.2_05) and employs the apache jakarta tomcat server () connected to a backend mysql database (). individual solvers operate asynchronously on a network of workstations running a customized distribution of the linux operating system based on fedora core 3 (). these solvers new problems are assigned to the first available solver. as depicted in figure 2, agents encapsulating specific algorithms either create new solutions from scratch, improve or modify existing solutions, or remove unpromising solutions from further consideration. for example, one creator algorithm is based on a best - fit methodology that iteratively assigns snps to the largest open compatible tube. when the tube size reaches the target multiplexing level specified by the user, it is closed, and no further additions or modifications to that tube are made. one improver algorithm eliminates partial tubes in order to reduce the number of unique tube sizes but while incurring reduced coverage, while another attempts at reformulating partial tubes in an effort to identify additional full tubes. efficiency is enhanced during the optimization process by periodically culling unpromising solutions from the population of candidates. the architecture is scaleable in the sense that new algorithms can be readily plugged - in over time, and it is robust in that it does not depend on a single algorithm to generate every viable alternative, and because system load is balanced across a distributed collection of solvers. within a given solution, there is no guarantee that a snp will be assigned, and the results depend on the random order in which snps and primers are processed. resulting coverage critically depends on the number of snps and the target level of multiplexing desired (j. rachlin, c. m. ding, c. cantor and s. kasif, manuscript submitted). the muplex server allows scientists to design multiplex pcr assays while explicitly considering intrinsic design tradeoffs. the consideration of competing alternatives has played a key role in the development of optimization and decision - support technologies in complex domains such as manufacturing and transportation logistics (9,10). here, we have demonstrated the viability of such approaches to the optimization of multiplex pcr assays. future efforts will focus on the development of new algorithms and on allowing users to impose dynamic feedback constraints in an effort to further guide the design optimization process towards solutions that more closely meet the scientist 's particular design objectives. we also plan to develop a distributed version that will run on our 128-processor linux cluster. the muplex homepage. users specify primer selection criteria and provide a collection of snps in the fasta format. the system emails to the user one or more solution alternatives revealing key design tradeoffs. once a problem is submitted and validated, it is assigned to one of the several solvers distributed across the network. each solver instantiates one or more agents (algorithms) that either create new solutions from scratch, attempt to improve an existing solution candidate or eliminate unpromising solutions from further consideration. the collaboration of algorithms in this manner enables the system to produce multiple multiplex pcr solutions that reveal intrinsic design tradeoffs.	we have developed a web - enabled system called muplex that aids researchers in the design of multiplex pcr assays. multiplex pcr is a key technology for an endless list of applications, including detecting infectious microorganisms, whole - genome sequencing and closure, forensic analysis and for enabling flexible yet low - cost genotyping. however, the design of a multiplex pcr assays is computationally challenging because it involves tradeoffs among competing objectives, and extensive computational analysis is required in order to screen out primer - pair cross interactions. with muplex, users specify a set of dna sequences along with primer selection criteria, interaction parameters and the target multiplexing level. muplex designs a set of multiplex pcr assays designed to cover as many of the input sequences as possible. muplex provides multiple solution alternatives that reveal tradeoffs among competing objectives. muplex is uniquely designed for large - scale multiplex pcr assay design in an automated high - throughput environment, where high coverage of potentially thousands of single nucleotide polymorphisms is required. the server is available at.
a two - way process of exchanging or shaping ideas, feelings, and information between dentist and patient has created keen interest in dental society. the best way to achieve effectively exchanging and shaping ideas depend on the credibility of the dentist that is how he defines empathy practically. empathy is to see with the eyes of other, to hear with the ears of another, and to feel with the heart of another (alfred adler). empathy was derived from two greek terms, em and pathos, meaning feeling into and has its origin from the german word einfulung. the basic difference between empathy and sympathy is that empathy means intellectual understanding, while sympathy means sharing sentiments. the american dental education association always emphasized on including empathy as a part of the dental curriculum as it plays an important role in healthy dentist and patient relationship. sherman and cramer reported a similar decline in empathy levels among the dental students during the 2 year of dental training, but in contrary some studies reported vice - versa. in view of such varying empathy findings from different countries, it is the need of the hour to understand empathy levels among dental students in the indian context. the present study is the first of its kind in india and few among the world to explore the empathy level among dental students. to measure the self - reported empathy levels among dental undergraduate and postgraduate students of the dental program (bds, mds)to review the trend of changes in empathy level with experience, age, and gender among dental undergraduate and postgraduate students. to measure the self - reported empathy levels among dental undergraduate and postgraduate students of the dental program (bds, mds) to review the trend of changes in empathy level with experience, age, and gender among dental undergraduate and postgraduate students to measure the self - reported empathy levels among dental undergraduate and postgraduate students of the dental program (bds, mds)to review the trend of changes in empathy level with experience, age, and gender among dental undergraduate and postgraduate students. to measure the self - reported empathy levels among dental undergraduate and postgraduate students of the dental program (bds, mds) to review the trend of changes in empathy level with experience, age, and gender among dental undergraduate and postgraduate students a cross - sectional institutional and self - reporting questionnaire based study was conducted among students pursuing their graduation and postgraduation in two private dental institutions located in the north west part of the country, rajasthan, india. the curriculum in india pertaining dental education offers 5 years course during graduation and 3 years of course during postgraduation. the present study was cleared by the ethical committee of the sdcri and maharaja ganga singh dental college. this research has been conducted in full accordance with the world medical association declaration of helsinki. data were obtained from the 1 to final (4) year students, interns, and postgraduate students enrolled in bachelor of dental surgery and master of dental surgery program, respectively, in these two institutions from january to april 2015. the students were briefly explained about the nature of the study, and their written consent was taken. the inclusion criterion for the present study was that students must have completed 6 months following admission. those students who were either unable to provide the required information or incomplete questionnaire form were excluded. the initial sample consisted of 1041 students but after applying the inclusion and exclusion criteria, the final sample comprised 978 students. health profession students (jspe - hps) version questionnaire (already validated) was administered to assess the empathy level. the questionnaire consists of twenty components using 7-point likert scale (for every single component) and score ranges from 20 to 140 with upper values representing greater empathy. along with jspe - hps questionnaire, there are four subcategories measuring different dimensions of empathy such as perspective taking, compassionate care, standing in patient 's shoes, and personal distress. confirmatory factor analysis was used to recognize the dimensionality of the jspe - hps version using (a) kaiser 's criteria, (b) an eigenvalue (> 1.25 was used), and (c) only retaining items if coefficients were 0.30. after evaluation of results, 3-factor pathway was selected, i.e., perspective taking, compassionate care, as well as standing in patient 's shoes. the above outcome resulted in approximately 37% of the overall described variance. among the components, (armonk, ny : ibm corp) for generation of descriptive, as well as inferential statistics. the statistical significant difference among groups the female : male ratio in the present study was 2.7:1 [table 1 ]. there was a statistically significant difference reported when empathy was tested in relation to gender (between males and females), as well as for age (p < 0.05). the lowest and highest mean empathy score was found in postgraduate (mean = 108.77, standard deviation [sd ] = 9.12) and 1 year (mean = 117.23, sd = 14.19) dental students, respectively. empathy score when tested according to experience using anova and post hoc test was found to be statistically substantial (p < 0.05). different variables retorts on the jefferson scale of physician empathy health profession student 's ten components were computed on subscale-1 and components value arraying between 0.697 and 0.518 describing 22.85% of the variability. the most important component was health - care providers understanding of the emotional status of the patients, as well as that of their families '. five components were computed on subscale-2 and components value arraying between 0.207 and 0.023 describing 10.18% of the variability. five components were computed on subscale-3 and components value arraying between 0.572 and 0.327 describing 9.34% of the variability. two components were common among subscale 2 and subscale 3, i.e., component 14 (i believe that emotion has no place in the treatment of medical illness) and component 11 (patients ' illnesses can be cured only by targeted treatment ; therefore, health - care providers ' emotional ties with their patients do not have a significant influence in treatment outcomes). the required cronbach 's alpha value for satisfactory internal consistency should be above 0.70 and in the present study cronbach 's value for subscale 1, 2, and 3 were 0.79, 0.76, and 0.48, respectively [table 2 ]. component analyses and adjusted score correlations of the jefferson scale of physician empathy health profession student 's few components brought out eminent mean values : patients feel better when their health - care providers understand their feelings i believe that empathy is an important factor in patients ' treatment brought out a mean value of 5.27 (sd = 1.31). the aim of the contemporary study was to define the properties of jse - hps, as well as to measure the empathy among students of dental colleges in sri ganganagar, rajasthan, india. the present study reported that postgraduate students had statistically lower empathy score as compared to all other years of students. this difference may be due to the fact that during the initiation of the dental course, students believe in idealism, i.e., sharing the feeling of the patients but with the progression of the dental course these feelings vanish, i.e., idealism gives way to the realization (means to get through the various level of dental degree meritoriously, requisite is to acquire enormous dimensions of facts). hence, significant perspectives such as sharing the thoughts and feelings of the patients are put aside and emphasis swings on facts - based examinations. the mean empathy score of the present study ranges from 103 to 117 which is similar with the other studies among dental and medical students, while in contrary, other studies reported 7890 mean empathy score. while analyzing the empathy among male and female in the present study, females showed higher empathy level than males which is similar with the other studies, but this was against the studies done by rose. and babar. this might be due to the fact that women 's brains probably show more empathy than men 's brains. when females were probed to recognize other human 's feeling, female 's brain action showed that they themselves feel the same sentiments as well while in contrary male 's brain action showed noetic assessment a more objective position. in the present study, it was found that empathy score differs by age while the same was not reported by babar. subscale-1, 2, and 3 are parallel with the studies described previously. as empathy is an essential component of maintaining healthy rapport between the dentist and patient, thus amending empathy is the vital chore of health training. recently, bonvicini. conducted a study regarding communication skills exercise to find whether it will result in rise of the empathy level or not. the conclusion of the study was that communication skill exercise led to increase in empathy among the students. the present study showed that empathy levels were dropping with the progression of dental course. therefore, the need is to introduce exercise regarding empathy among dental students at the initiation of and during their dental course. however, there is an argument regarding the introduction of training in empathy as newton. the author proposes to introduce administrative modifications which motivate students scholastically and sensitively during their dental course. this will result in interprofessional education, growth of team effort principle, as well as alterations in judgment method. hence, results of the present study can be implemented among other dental institutions also. the limitation of the present study is that valuation of empathy was constructed on subjective evaluation of a validated questionnaire ; therefore, observational approaches such as the history - taking rating scale could be used with jse - hps to measure empathy level in dental students. cross - sectional studies make it impossible to understand the process of changes in empathy level through the years of dental course. dental health educators should consider this fall in empathy seriously as early as possible during the dental course. empathy - related teaching exercises must be implemented among the dental students to endorse the growth of empathy so that fall in empathy should be prevented. more longitudinal research is required to investigate the outcome of communication exercise in cultivating empathy among dental students. the authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article. the authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.	background : empathy plays an important role in healthy dentist and patient relationship. hence, the aim of the study is to (a) to measure the self - reported empathy levels among dental undergraduate and postgraduate students. (b) to review the trend of changes in empathy level with experience, age, and gender among dental undergraduate and postgraduate students.materials and methods : this cross - sectional, questionnaire - based study was carried out in two private dental institutions situated in sri ganganagar, india, with a sample size of 978. data were obtained from the 1st to final year (bds), interns, and postgraduate students from january to march 2015. an empathy level of students was assessed by the jefferson scale of physician empathy health profession students version questionnaire. an exploratory factor analysis using kaiser 's criteria was undertaken to appraise the construct validity and dimensionality. based on the results of the factor analysis, three factors were selected ; labeled as perspective taking, compassionate care, and standing in patient 's shoes.results:the majority of the students was female in a equivalent ratio of 1338:618. there were significant differences in empathy scores by gender and age (p < 0.01). the lowest and highest mean empathy scores were found in postgraduate (mean = 108.77, standard deviation [sd ] = 9.12) and 1st year (mean = 117.23, sd = 14.19) dental students, respectively.conclusion:dental educators should consider the likely decline in empathy among students as early as possible and adopt communication teaching strategies to promote the development of empathy and reduce the risk of further decline.
one of the most important objectives of endodontic therapy is to completely eliminate intracanal microorganisms from the root canal system to create favorable environment for healing and to prevent reinfection in order to achieve long - term success. are polymicrobial in nature with predominant anaerobic microorganisms. bacteria and their metabolic products, enzymes, and toxins play a vital role in the initiation, propagation, and persistence of pulpal and periradicular pathosis. although several factors contribute to endodontic failures, the literature reported that persistent intraradicular or secondary infection is the major etiology for failed endodontic therapy. studies have shown that microflora in failed root canal therapy are different from those found in untreated teeth. enterococcus faecalis (e. faecalis) and candida albicans (c. albicans) are considered to be the most resistant species in infected root canals and they are often associated with endodontic treatment failures. e. faecalis is a gram positive, facultative anaerobic cocci commonly found in persistent root canal infection and commonly recovered in over one - third of the canal of endodontically treated teeth with persisting periapical lesions. c. albicans, the most predominant and commonly isolated yeast in failed root canal treatment with periradicular pathosis. it may gain access to the root canal from the oral cavity as a result of poor asepsis during endodontic treatment procedures or because of coronal leakage. it is a dentinophilic aerobic microorganism able to survive in harsh ecologic environment of root canal. due to complex anatomy of root canal system, irrigants help in removal of debris and microorganisms form the canal that can not be reached by endodontic instruments. an ideal root canal irrigant should have good antimicrobial efficacy, nontoxic to periapical tissues, capable of dissolving necrotic tissue, able to lubricate the canal, and help in smear layer removal. sodium hypochlorite (naocl) has been widely used as root canal irrigant due to its potent antimicrobial activity and tissue dissolving ability. but it has several undesirable characteristics such as tissue toxicity, risk of subcutaneous emphysema, allergic potential, and disagreeable smell and taste. chlorhexidine (chx) digluconate has been suggested as root canal irrigant because of its good antimicrobial efficacy and substantivity. it seems to act by adsorbing onto the cell wall of microorganisms resulting in leakage of intracellular components. at low concentration, it has bacteriostatic effect. at higher concentration, qmix 2 in 1, an experimental irrigating solution, contains a mixture of a bisbiguanide antimicrobial agent, a polyamino carboxylic acid calcium chelating agent ; saline ; and a surfactant. it was evaluated as an effective irrigant similar to 17% ethylenediaminetetraacetic acid (edta) in removing canal wall smear layer after the use of 5.25% naocl as the initial rinse. qmix had stronger antibacterial effects against young and old e. faecalisbiofilm in dentin and had stronger antibacterial effect against e. faecalis present in deep dentin. studies are needed to evaluate the antimicrobial efficacy of qmix 2 in 1 in comparison with naocl and chx against e. faecalis and c. albicans. thus, the aim of the present study is to compare the antimicrobial efficacy of qmix 2 in 1 with 5.25% naocl and 2% chx digluconate against e. faecalis and c. albicans. teeth were cleansed using ultrasonic scalers to render them free from calculus and tissue tags, following which they were stored in physiological saline until use. the teeth were decoronated at the cementoenamel junction using diamond disc (isomet 2000 ; buehler ltd, lake bluff, il) and the root lengths was standardized to approximately 14 mm. pulp was extirpated and working length was established 1 mm short of apex where the file exited the apical foramen. gates glidden drills # 3 (mani inc, tachigi - ken, japan) were used to prepare the root canal orifices (3 mm). apical preparation was done using k - file (dentsply maillefer, ballaigues, switzerland) up to the iso size # 40. canals were irrigated copiously with physiologic saline (0.9 w / v nacl) after each instrument. each canal was then rinsed with 1ml of 17% edta (prime dental product) for 1 min followed by 3 ml of 5.25% naocl (prime dental product, mumbai, india) to facilitate the removal of smear layer. the roots were coated with nail varnish and apical foramen sealed with type ii glass ionomer cement (gic ; gc fuji, tokyo, japan). the roots were sterilized in an autoclave for 15 min at 121c and 15 lb of pressure to ensure complete sterilization within the canal space. the paper points were placed into a test tube containing 1 ml reduced transport fluid, vortexed for 10 s, placed onto brain heart infusion (bhi) agar (himedia laboratories, mumbai, india) and sabouraud dextrose agar (himedia laboratories, mumbai, india), incubated at 37c for 48 h, and examined for growth. a suspension of e. faecalis (atcc 29212) and c. albicans (courtesy ibms institute, chennai, india) were adjusted to 0.5 turbidity on the mcfarland scale (1.5 10 bacteria / ml). e. faecalis suspension of 10 l was injected into group i samples containing 40 teeth and 10 l suspension of c. albicans into group ii samples containing 40 teeth using a micropipette in a class ii vertical laminar airflow cabinet to prevent any airborne contamination. then inoculated specimens with e. faecalis were placed in vials filled with bhi agar and inoculated specimens with c. albicans were placed in vials filled with sabouraud dextrose agar, then inoculum was added every day and incubated aerobically at 37c for 3 days. after 3 days, aliquots were taken from each tooth using a syringe and plated on bhi agar to verify the growth of e. faecalis and sabouraud dextrose agar to verify the growth of c. albicans. at the end of 3 days, teeth were removed from the vials and excess fluid in canal was removed with sterile paper points. the specimens from each group were divided into four subgroups (a, b, c, and d) of 10 teeth each depending on the experimental irrigating solution used. test solutions used were : subgroup ia and iia : 3 ml of 5.25% naocl (prime dental product, mumbai, india), subgroup ib and iib : 3 ml of 2% chx (dentochlor, ammdent, pb, india), subgroup ic and iic : 3 ml of qmix 2 in 1 (dentsply tulsa dental specialities, tulsa, ok), subgroup i d and iid : 3 ml of 0.9% saline (positive control). irrigation was accomplished using 30 gauge prorinse needle (dentsply) placed 1 - 2 mm short of the working length. the time of contact of each irrigant was 1 min and a final flush was done with 5ml of distilled water to terminate the action of irrigant. a small amount of saline solution was introduced into the canal, and an endodontic hand file was used in a filing motion to a level 1 mm short of the root apex. sterile 30 gauge needle was used to collect 0.01ml (10 l) of the sample from the canal. using a bacterial inoculation loop ; the plates were incubated at 37c for 24 h and then number of colony forming units (cfus) was counted. mean log cfus were compared among the groups using analysis of variance (anova) followed by post - hoc games - howell test. results were analyzed with the statistical package for social sciences (spss)/predictive analytics software (pasw) version 18 software. qmix 2 in 1 was the most effective and statistically significant when compared with 5.25% naocl and 2% chx (p 0.001). the main goal of chemomechanical preparation of infected root canals is to completely eliminate intracanal bacteria and prevention of recontamination after treatment. this laboratory study was aimed to evaluate the antimicrobial efficacy of qmix 2 in 1, naocl, and chx against e. faecalis and c. albicans. the in vitro model developed by orstavik and haapasalo has been used to assess antimicrobial efficacy of irrigants. e. faecalis and c. albicans are frequently isolated in root canals with pulpal infection and in secondary / persistent endodontic infections. in the present study qmix 2 in 1 has shown good antimicrobial efficacy and statistically superior to all other irrigants (p 0.01). arslan., concluded that propolis, biopure mtad, 5% naocl, and 2% chx showed good antimicrobial activity against e. faecalis and c. albicans. ruff., investigated the efficacy of 6% naocl, 2% chx, 17% edta, and biopure mtad against c. albicans and concluded that 2% chx and 6% naocl were equally effective and statistically superior to 17% edta and biopure mtad. saline has also shown considerable reduction in the cfu counts of e. faecalis and c. albicans. the mechanical shaping and cleaning of the canals and irrigation with saline served to flush out the debris from the canals. kuruvilla and kamath reported a significant reduction in microbial counts with usage of saline in canals without mechanical preparation. within the limitations of this study, qmix 2 in 1 demonstrated significant antimicrobial efficacy against e. faecalis and c. albicans.	aim / objective : the aim of this study is to compare the antimicrobial efficacy of qmix 2 in 1, sodium hypochlorite (naocl), and chlorhexidine (chx) against enterococcus faecalis and candida albicans.materials and methods : eighty freshly extracted, single - rooted human mandibular premolar teeth were instrumented and autoclaved. samples were divided into two groups of 40 teeth each based on the type of microorganism used. group i was inoculated with e. faecalis and group ii with c. albicans and incubated for 3 days. each group was subdivided into four subgroups based on the type of irrigant used. group ia, iia, 5.25% naocl ; group ib, iib, 2% chx ; group ic, iic, qmix 2 in 1 ; and group i d, iid, 0.9% saline (the control group). ten microliters of the sample from each canal was taken and was placed on brain heart infusion agar and sabouraud dextrose agar. the plates were incubated at 37c for 24 h and colony forming units (cfus) that were grown were counted. data was analyzed with analysis of variance (anova) followed by post - hoc games - howell test.results:the greatest antimicrobial effects were observed in samples treated with qmix 2 in 1 (p 0.001) against e. faecalis and c. albicans.conclusion : qmix 2 in 1 demonstrated significant antimicrobial efficacy against e. faecalis and c. albicans.
the incidence of laryngeal cancer has decreased in the usa in recent years as rates of smoking have declined. however, larynx cancer continues to be a serious problem for individuals suffering from this disease, with treatment frequently affecting the patient 's ability to phonate and swallow. this year, 12630 people in the usa are estimated to be diagnosed with laryngeal cancer and 3610 will die from this disease. the veterans affairs laryngeal cancer study and the radiation therapy oncology group trial 91 - 11 are the basis for the organ - preservation treatment approaches currently employed for advanced laryngeal cancer. definitive radiation treatment with chemotherapy is utilized as the initial treatment strategy for many advanced laryngeal cancers except those with cartilage involvement. however, when there is persistence of disease or recurrence of cancer after chemoradiation, salvage total laryngectomy is often necessary to achieve cure. pharyngocutaneous or salivary fistula is a common complication after salvage total laryngectomy and can lead to serious consequences. fistulas can lead to infection and skin breakdown, prolonging the patient 's hospital stay, at times necessitating operative repair. in very serious cases, the persistent bathing of saliva around major vessels can lead to arterial erosion and subsequent carotid blowout. the reported incidence of pharyngocutaneous fistula for primary total laryngectomy varies from 10 to 35% [413 ]. for salvage total laryngectomy, the reported fistula rate is generally higher, varying in the literature from 25 to 50% [1419 ]. many groups have attempted to identify the risk factors for salivary fistula with salvage total laryngectomy, and associations have been reported for poor preoperative nutrition, low hemoglobin, prior tracheostomy, liver disease, and diabetes [4, 12, 16 ]. with some exceptions, most groups have found that a prior history of radiation and/or chemotherapy predisposes patients to a higher risk of salivary fistula after total laryngectomy [911, 2022 ]. in an attempt to reduce the incidence of salivary fistula after salvage total laryngectomy, the use of pedicled or free vascularized tissue transfer to reinforce while there is some data that vascularized flaps may provide benefit, the conclusions have thus far been conflicting [6, 2326 ]. this study aims to identify what factors play a role in development of pharyngocutaneous or salivary fistulas in patients undergoing salvage total laryngectomy. a secondary aim was to separately analyze the predictive factors for development of minor fistulas that are managed conservatively and major fistulas that are severe enough to require surgical intervention. a retrospective chart review was performed for all patients who underwent salvage total laryngectomy for laryngeal squamous cell carcinoma at the university of california, san francisco (ucsf). we included all patients who had laryngeal squamous cell carcinoma treated primarily with radiation therapy or chemoradiation who subsequently were found to have recurrent or persistent disease, requiring a salvage total laryngectomy between january 1, 2002, and january 1, 2012. oncologic resection was completed within the department of otolaryngology - head and neck surgery at ucsf. during this time period, eight different attending surgeons performed these laryngectomies. data collection was performed using all electronic medical record systems in place at ucsf, and patient records were screened for inclusion in this study using procedure codes for total laryngectomy. patients who had salvage total laryngectomy for reasons other than cancer such as chronic aspiration or dysfunctional larynx were excluded. demographic data was then collected including information about sex, race, ethnicity, and tobacco or alcohol use. oncologic data regarding the primary tumor including the american joint committee on cancer (ajcc) stage, tnm stage, and detailed histopathologic data were collected. salvage laryngectomy surgical details regarding extent of pharyngeal resection or concurrent neck dissection were reviewed. the type of neopharyngeal closure was also evaluated with regard to whether a single-, double-, or triple - layer closure was performed. data regarding use of pedicled pectoralis muscle flaps or free tissue transfer was also collected. inlay method refers to using the skin paddle of the pectoralis myocutaneous flap to reconstruct a portion of the neopharyngeal wall. the onlay method refers to placing a pectoralis myofascial flap on top of the neopharyngeal closure without actually augmenting the neopharynx wall. the occurrence of a salivary fistula for each of these patients was determined by reviewing the discharge summary for the hospital stay following the salvage total laryngectomy as well as the clinic note for the first postoperative visit. a fistula was defined as any documented clinical suspicion or clear evidence of salivary leak, on a continuum from erythema of the neck to saliva within the surgical drain to frank wound breakdown and leakage of saliva. major salivary fistulas were defined as those that needed revision surgery for closure of the leak. data analyses were performed with sas, version 9.3 (sas institute, cary, north carolina). normally distributed data were analyzed using independent sample t - testing and nonparametric data using mann - whitney testing. of these, 133 patients were excluded because they did not receive prior radiation or chemoradiation or their laryngectomy was not done for cancer. in total, there were 48 patients who met inclusion criteria for the study. radiation metric data was available for 27 patients ; 40/48 patients received radiation treatment at an outside hospital. total radiation dose varied from 6000 cgy to 7920 cgy (mean 6900 cgy, median 7000 cgy). for 40 patients, it was possible to assess whether there was persistence versus recurrence of tumor. there was persistence of tumor in 13 patients, occurring 1.9 months to 4.8 months after radiation treatment. there was recurrence of tumor in 27 patients, occurring 6.2 months to 24 years after radiation treatment (median 11.4 months). once recurrence of persistence was diagnosed, salvage surgery was scheduled, with a mean interval from date of diagnosis to surgery of 38 days. reconstruction methods were varied : 9 had single - layered primary closure, 14 had double - layered primary closure, 7 had triple - layered primary closure, 3 had pedicled pectoralis myocutaneous inlay flap without free tissue transfer, 9 had pedicled pectoralis myofascial onlay flap without free tissue transfer, and 4 had free tissue transfer. one patient had a temporary esophagostoma and pharyngostoma created in anticipation of future reconstruction, but, for unclear reasons, this patient never had definitive reconstruction. one patient had a stapler assisted closure of the neopharynx. among patients who had primary closure it could not be determined through chart review how the second or third layers were closed if applicable. twenty - three of the 48 patients had clinical evidence of a fistula for an overall fistula incidence of 47.9%. nine patients had a major fistula requiring operative repair for a major fistula rate of 18.8%. we analyzed various preoperative patient and tumor characteristics to determine whether any were associated with fistulas (table 1). there were no statistically significant associations between overall fistula rate and sex, ajcc tumor stage, or t or n status at the time of initial diagnosis. age was similar between those who developed fistulas compared to those who did not (64 versus 65 years, p = 0.72). there was not a significant difference in fistula rate whether patients received prior radiation at an academic medical center or a community medical center. the use of chemotherapy was not associated with a significant difference in the overall fistula rate. performing a concurrent neck dissection with the total laryngectomy next, comparisons of several closure techniques were made to determine whether differences in fistula rate could be identified (table 2). there was no significant difference in fistula rate for patients who had a complete pharyngectomy versus those that had either a partial or limited pharyngectomy. the rate of fistula was observed to decrease with increasing number of layers of primary closure, from 66.7% for single - layer closure to 28.6% for triple - layer closures. the fistula rate for pectoralis muscle flap onlay (22.2%) was lower than any of the primary closure techniques. we found that those who developed major fistulas were older (71 versus 63, p = 0.03). sex, ajcc tumor stage, or t or n status at initial diagnosis was not associated with major fistulas. no significant difference in major fistula incidence was detected whether patients were radiated at an academic or community hospital, whether they received chemotherapy, or whether a concurrent neck dissection was performed. the various neopharyngeal closure techniques were examined for major fistulas (table 4). comparing patients who had complete pharyngectomy to those who had a partial or limited pharyngectomy, compared to multilayer primary closures, there was a trend toward a higher major fistula rate with single - layer closures (44.4% versus 12.82%, p = 0.09). the rate of major fistula was observed to decrease with increasing number of layers of primary closure, from 44.4% for single - layer closure to 0% for triple - layer closures. no patients (0 of 7) with triple - layer closure had a major fistula, and only 1 of 9 patients (11.1%) with pectoralis muscle flap onlay had a major fistula. major salivary fistulas requiring reoperation occurred in 9 patients (18.8%), which is within the broadly documented range of salivary fistula rates (1050%) reported in the literature [419 ]. it is notable that only a few of these studies delineate how a salivary leak is defined. fistulas can vary widely in their presentation, from a small leak having minimal impact on postoperative course to large volume salivary drainage leading to prolonged hospital stay and potential catastrophic consequences requiring surgery. for example, grau. defined a fistula as those salivary leaks lasting more than 2 weeks. in our study, we counted any clinically evident fistula regardless of severity or length of time that the leak was present. we also sought to categorize fistulas into minor and major fistulas to determine whether more specific risk factors for salivary leaks could be identified. we did additional analysis on those fistulas requiring operative intervention, which we defined as major fistulas. the way in which the salivary fistula rate is calculated is important since it varies the magnitude of the fistula rate and modifies how we find different risk factors. among the preoperative factors studied, our study did not reveal a difference in salivary leak rates with regard to sex, ajcc stage, t or n status at initial diagnosis, or whether radiation treatment was performed at our institution versus a community hospital. other possible risk factors such as the addition of adjuvant chemotherapy, extent of pharyngeal resection, or concurrent neck dissection were not associated with an increased salivary fistula rate in our study. other groups have reported various preoperative factors that are associated with salivary fistula after total laryngectomy. the majority of the literature shows a trend towards higher fistula rates in patients with a history of radiation to the larynx [911, 2022 ]. the magnitude of the radiation dose seems to be important. in a study by vendelbo johansen., the fistula rate for salvage total laryngectomy was 25% if patients received 57 gray (gy) compared with 92% for those receiving 72 gy. other studies have corroborated this finding that higher dose and larger radiation field contribute to fistula formation. in a number of studies, the addition of adjuvant chemotherapy increases by up to twofold the risk of fistula formation when compared to radiation alone [28, 29 ]. one study found a significant increase in fistula rate if salvage total laryngectomy was done within 4 months of radiation. other studies have similarly found a higher wound complication rate for surgeries done soon after radiation [8, 11, 13, 31 ]. besides history of radiation or chemoradiotherapy nonglottic tumors or advanced t3 or t4 tumors tend to have elevated rates of pharyngocutaneous fistula after salvage total laryngectomy. patients with nutritional deficiencies, hypothyroidism, or hypoalbuminemia are at higher risk as well [4, 19, 32 ]. the link between postlaryngectomy fistula formation and previous radiation can be explained through radiation therapy 's cellular mechanism of action. radiation induces cell death through dna damaging mechanisms. though preferentially affecting rapidly dividing cells such as malignant tumors, radiation also damages normal cells such as connective tissue and muscle. on a microscopic level, radiation leads to progressive fibrosis and obliterative endarteritis of the blood vessels, which in turn inhibits future wound healing. chemotherapy has been thought to be an effective radiosensitizer, inducing more cellular damage, more fibrosis, and obliteration of the microcirculation. it has been hypothesized that placing nonradiated vascularized tissue into the compromised recipient wound bed can improve wound healing and reduce the incidence of salivary fistulas in those patients who have undergone prior radiation and/or chemotherapy. we examined whether the neopharyngeal closure technique correlated with the overall (major and minor) fistula rate. we observed a decrease in overall fistulas and major fistulas with increasing the number of primary closure layers. increasing the number of layers of closure may minimize the risk for the suture line dehiscing. to our knowledge, this is the first study to examine the impact of varying the number of layers of primary closure on fistula incidence for salvage total laryngectomy. however, possibly due to small sample size, none of the differences we observed in fistula rates for the various closure techniques reached statistical significance. providing vascularized tissue from outside the previous radiation field as an onlay over some studies have shown that prophylactically placing a pectoralis myofascial flap over the suture line can reduce the incidence of salivary leak while other studies have failed to find this difference [57, 34 ]. similar conflicting results have been found with the utilization of free tissue transfer to augment the neopharyngeal suture line [15, 35 ]. one study showed that placing a pectoralis flap in an inlay fashion reduced the salivary leak rate when compared to an onlay fashion, citing that skin holds sutures better than fascia or muscle [6, 24 ]. all these studies suffer from low statistical power and lack a standardized method of defining salivary leaks. placing vascularized tissue in the wound bed may not reduce the overall incidence of salivary fistula, but it may mitigate the severity of the leak. perhaps prophylactic placement of vascularized tissue converts cases that would have resulted in a severe fistula into a mild fistula that can be managed conservatively. in our study, the use of a pedicled pectoralis muscle onlay flap was noted to reduce the overall and major fistula rates compared to single - layer closures ; however the difference was not significant possibly due to small sample size. nonetheless, it seems likely that certain subgroups of patients more prone to poor wound healing would benefit from vascularized tissue overlying the neopharyngeal suture line. further studies are needed to explore and define appropriate recommendations for closure techniques in salvage total laryngectomy. it is also interesting to speculate whether a multilayer closure for total laryngectomy in patients without a history of prior radiation treatment has an impact on the occurrence of salivary leaks. a future study could help determine whether there is any added benefit to performing more than a single - layer closure for previously untreated patients undergoing total laryngectomy. the utilization of various closure techniques may have been predicated on certain preoperative or intraoperative findings that raised the surgeon 's fear of having a fistula. our study had a small sample size and was thus underpowered to detect statistical differences that may truly exist. we observed a trend with greater number of layers of neopharyngeal closure techniques or onlay pectoralis flaps and decreased rates of pharyngocutaneous fistula, findings that may have been statistically significant with a larger sample size. these limitations are not unique to this study, and the majority of single - institution reports on this topic are afflicted with these same drawbacks. although there is a paucity of data from the literature to guide the surgeon as to how to prevent pharyngocutaneous fistulas after salvage total laryngectomy, we believe that the optimal management of these challenging cases should begin with identifying those patients at highest risk for developing fistulas. the literature suggests that this high risk group includes patients who have been treated previously with chemoradiation or those with poor nutritional status. in these patients, the surgeon should carefully select the method of neopharyngeal reconstruction to decrease the risk of fistula. our study suggests that it may be beneficial to increase the number of pharyngeal closure layers or to use a pedicled pectoralis muscle onlay flap. in this study, salivary fistulas were a common complication after salvage total laryngectomy, occurring with varying severity in 47.9% of cases. reoperation due to salivary fistulas was performed for nearly 1 in 5 salvage total laryngectomies. in contrast to previous studies, we did not find any clinicopathologic variables associated with fistulas, which may be related to the small sample size. the overall fistula and major fistula incidence was decreased with increasing the number of layers of primary closure and with pectoralis muscle onlay flaps.	background. salivary fistula is a common complication after salvage total laryngectomy. previous studies have not considered the number of layers of pharyngeal closure and have not classified fistulas according to severity. our objective was to analyze our institutional experience with salvage total laryngectomy, categorize salivary fistulas based on severity, and study the effect of various pharyngeal closure techniques on fistula incidence. methods. retrospective analysis of 48 patients who underwent salvage total laryngectomy, comparing pharyngeal closure technique and use of a pectoralis major flap with regard to salivary fistula rate. fistulas were categorized into major and minor fistulas based on whether operative intervention was required. results. the major fistula rate was 18.8% (9/48) and the minor fistula rate was 29.2% (14/48). the overall (major plus minor) fistula rate was 47.9%. the overall fistula and major fistula rates decreased with increasing the number of closure layers and with use of a pectoralis major flap ; however, these correlations did not reach statistical significance. other than age, there were no clinicopathologic variables associated with salivary fistulas. conclusion. for salvage total laryngectomies, increasing the number of closure layers or use of a pectoralis major flap may reduce the risk of salivary fistula.
resistance training is a commonly prescribed and broadly researched rehabilitative strategy for older adults to maintain or improve muscle strength and function. resistance training interventions typically emphasize high - load, strengthening exercise ; however, muscle power (force velocity) has emerged as an important muscle performance characteristic in this population [17 ]. resistance training using high movement speeds and high external resistance or high movement speeds and low external resistance [911 ] have demonstrated positive impact on both muscle power and some functional performance tests. a recent meta - analysis revealed that various forms of high - speed resistance training (i.e., power training) were more effective at improving muscle power with only a small impact on function compared to traditional slow - speed strength training. in older adults, muscle power declines at up to twice the rate than muscle strength (3 - 4% versus 1 - 2%), mostly due to declines in velocity compared to force [13, 14 ]. thus, interventions that potentially improve muscle power and the velocity component of power may be critical in this population, especially with regard to function. different functional tasks, however, may require power with a greater velocity component or a greater force component depending on the nature of the specific task (e.g., moving the lower limb quickly to keep from falling versus slowly getting up from a chair) ; thus, different resistance training protocols may be able to deliver different aspects of power to transfer to functional task performance. we believe resistance training programs to improve velocity could have a significant impact on functional tasks related to safety in this population. for example, exercise that improves the ability to rapidly move the lower limb from the accelerator to the brake while driving would have significant public health implications because motor vehicle accidents are one of the leading causes of injury - related deaths in older adults. one recent study has shown that ankle power training increased foot movement speed compared to control using a left and a right foot switch. because of the importance of rapidly braking an automobile, however, we believe it is critical to replicate both the equipment found in an automobile and the mechanics of the braking motion. few studies in older adults have focused on the key components of muscle power (velocity and force) and how these variables are impacted by different resistance training protocols. the purpose of this study was to examine the effect of high - speed power training on muscle power and its components in older men and women and how changes in those parameters with training impacted braking speed using a driving simulator. we hypothesized that high - speed power training would improve power and the velocity component of power at lower external training resistances and transfer successfully to improve braking speed compared to slow - speed strength training. eligible participants had to be between the age of 6590 years, ambulatory with or without an assistive device (cane or 3 post walker only) and community dwelling. exclusion criteria consisted of history of heart disease, severe visual impairment, presence of neurological disease, pulmonary disease requiring the use of oxygen, uncontrolled hypertension, hip fracture or lower extremity joint replacement in the past 6 months, and current participation in structured exercise. a study physician determined medical eligibility for all participants. eighty - nine individuals were randomized to one of three groups : high - speed power training (hspt : n = 30), slow - speed strength training (ssst : n = 30), and control (con : n = 29) (see consort diagram figure 1 for details). eight participants withdrew during the intervention, 1 in hspt, 3 in ssst, and 4 in con, three of which were study - related (hspt (n = 1) and ssst (n = 2)). using an intention - to - treat design, the data from 72 older men and women (hspt : n = 25 ; str : n = 25 ; con : n = 22) were analyzed in this study. this project was approved by the university of missouri institutional review board and written consent was obtained from all participants. the study compared 12 weeks of explosive high - speed power training with traditional slow - speed strength training. primary outcome measures included muscle performance : leg press and knee extension one - repetition maximum (1rm), muscle power across a range of external resistances (4090% 1rm), and the corresponding velocity at peak power and force / torque at peak power. the primary measures were chosen to determine whether high - speed training would improve critical muscle performance variables (power and speed) necessary for function in older adults. for brevity only leg press measures are presented. a secondary outcome measure included high - speed function : braking speed using an automobile driving simulator. this secondary outcome measure was necessary to demonstrate whether changes in primary outcomes translated to actual functional performance. tertiary outcome measures included ratings of perceived exertion (rpe) and total work performed during the training. these latter measures were included as an exploratory analysis of how individuals responded to the different exercise regimens and to ensure that the volume of exercise in both training groups was comparable, respectively. participants reported to the laboratory for 2 weeks of baseline measurements. on visit 1, body mass was recorded on a platform scale to the nearest 0.1 kg with the subject fully clothed. global cognitive function was assessed by the mini - mental state examination, and the geriatric depression scale was administered to assess depression over the previous week. number of falls in the past year and daily medications were assessed via questionnaire. on visit 2 and 3 the following week, all muscle performance and functional measures were repeated to establish reliability. if baseline 1rm measurements deviated by more than 10% in repeated attempts, a third measure was obtained. at the end of baseline testing, participants were randomized to treatment. following the 12-week resistance training intervention, posttraining muscle performance and functional measures were obtained. volunteers randomized into hspt and ssst exercised 3 times per week for 12 weeks using computer - interfaced keiser a420 pneumatic leg press and seated knee extension resistance training equipment (fresno, ca). for hspt, each training session consisted of 3 sets of 1214 repetitions at 40% 1rm. participants performed an explosive movement at high speed during the concentric phase of each repetition, paused for one - second, and performed the eccentric portion of the contraction over 2 - 3 seconds. volunteers randomized into ssst also exercised 3 times per week for 12 weeks with each training session consisting of 3 sets of 810 repetitions at 80% 1rm. repetition number was higher in the hspt group to more closely equate work performed between groups and to remain consistent with resistance training guidelines for exercise using lower external resistances. the participants performed each movement at a slow velocity (2 - 3 s for concentric phase of the repetition), paused for one second, and performed the eccentric portion of the contraction over 2 - 3 seconds. the control group met three times a week for warm - up and stretching exercises, but performed no resistance training. hspt and ssst performed the same warm - up and stretching exercises as con. leg press and knee extension 1rm were obtained using keiser pneumatic resistance training equipment fitted with a420 electronics. as the exercise arm is moved through its range of motion, a piston is driven into a cylinder where it encounters the mechanical resistance of the air pressure in the system. the a420 equipment captured measures of peak power, peak power velocity, and peak power force / torque during the concentric portion of each contraction by sampling the system pressure at 400 hz and making calculations based on an appropriate algorithm. the seat of the recumbent leg press and seated knee extension machines were positioned to ensure the hip and knee joint were at 90 to 100 degrees of flexion. the 1rm is defined as the maximum load that can be moved throughout the full range of motion once while maintaining proper form. the 1rm was obtained by progressively increasing resistance until the subject was no longer able to push out one repetition successfully. the borg scale was used to assist in evaluating when 1rm (combined with perceived maximal effort) was reached. peak muscle power was obtained at 40%, 50%, 60%, 70%, 80%, and 90% of the 1rm approximately 30 minutes after 1rm testing [10, 11 ]. participants were instructed to exert as fast as possible at each relative percentage of the 1rm. three attempts were made at each resistance and the greatest peak power output obtained at each resistance was used in the analysis. the corresponding peak power velocity and peak power force / torque were obtained for each external resistance from 4090% 1rm. the 1rm was measured biweekly in hspt and ssst only and relative training intensity was adjusted accordingly to ensure adequate overload during training. posttraining muscle performance measures were obtained using loads relative to the initial baseline 1rm to evaluate change in muscle performance variables from baseline to posttraining across a range of external resistances 4090% 1rm typically encountered in daily tasks. we compared these changes relative to the baseline 1rm because external resistances in the environment are typically fixed and do not increase as you get stronger. this more closely reflects how changes in power and speed apply to real world functioning. sample sizes for the evaluations were : hspt (n = 21), str (n = 23), con (n = 17). the simulator consisted of an adjustable car seat, steering column, and depressable accelerator and brake mounted on a steel frame attached to a computer and wide - screen monitor (figures 2(a) and 2(b)). the participant was seated with hands on the steering wheel and right foot depressing the accelerator. slam on the brakes when a visual stimulus changed from green (go) to red (stop). pilot work showed that most subjects tend to lift the foot from the accelerator to the brake utilizing the hip, knee, and ankle during emergency braking situations in our laboratory, therefore, we instructed all participants to utilize this strategy instead of simply pivoting the foot between the pedals. in addition, this strategy closely replicated the mechanical movement of the limb during the leg press training. the computer recorded two events (in ms) : initial reaction time, or the time for the participant to react to the red light and lift the foot from the accelerator, and the braking speed, the time from movement of the foot off the accelerator and onto the brake. rating of perceived exertion (rpe) during the training intervention was performed using the borg scale. the borg scale consists of a set of numbers that correspond to a specific exertion level. the scale ranges from 6 (no exertion at all) to 20 (maximal exertion), and is commonly used in older adults to determine training intensity. participants were asked to rate the exertion they felt immediately following each set of leg press and knee extension exercise. the average of the three rpe measures for each leg press and knee extension exercise was calculated to obtain a measure of exertion per exercise session. the rpe per session was then averaged across the total number of exercise training sessions attended during the intervention (12 weeks 3 visits per week) to provide a measure of average daily rpe. measures of average daily leg press and knee extension rpe were used in separate analyses. work (f d) was calculated as the mechanical resistance encountered (f) multiplied by the distance the piston traveled into the cylinder (d) during each exercise repetition using the keiser pneumatic a420 electronics software. work for each repetition of leg press and knee extension exercise was obtained during each training session and summed for a measure of total work per session. total work was then averaged across the total number of exercise training sessions attended during the intervention (12 weeks 3 visits per week) to provide a measure of average daily work. measures of average daily leg press and knee extension work were used in separate analyses. associations among variables of age, sex, body mass index, cognitive function, depression, medications, and falls were evaluated using pearson 's r. when significant associations were found, those variables were used as covariates in all analysis of variance (anova) models. to evaluate baseline differences in subject characteristics, a one - way anova (continuous variables) or chi - square (categorical variables) was run. to evaluate baseline differences among groups in leg press muscle performance (peak power, peak power velocity, and peak power force) at each condition (4090% 1rm) and braking speed, a univariate anova was run. to evaluate differences among groups in leg press muscle performance, the change scores in peak power, peak power velocity, and peak power force with training were calculated (posttraining value minus baseline value) at each condition (4090% 1rm) and a univariate anova was run covarying for the baseline measure. to evaluate differences among groups in braking speed from baseline to posttraining, the change score was calculated (posttraining value minus baseline value) and a univariate anova was run covarying for the baseline measure. if significant group main effects were found, tukey 's hsd test was performed. to determine differences between groups in average daily rpe and work performed during the 12 week intervention there were no differences among groups in age, sex, body mass index, depression, cognitive function, number of medications or falls in the past year (see table 1). univariate anova showed no group main effects for baseline leg press peak power (all p 0.09), leg press peak power velocity (all p 0.28), or leg press peak power force (all p 0.17) at any condition (4090% 1rm). univariate anova showed no group main effects for baseline reaction time (p = 0.67) or braking speed (p = 0.44). these findings indicate that subject characteristics, muscle performance, and function were similar among all groups at the start of training. the changes in leg press peak power, leg press peak power velocity, and leg press peak power force values at each condition (4090% 1rm) (see table 2) were compared using a univariate anova. there was a significant group main effects for leg press peak power and peak power velocity at each condition (all p 0.007). post hoc tests showed that for both measures (peak power and peak power velocity) hspt was greater than con across all external resistances (4090% 1rm ; all p 0.02), while ssst was only greater than con from 7090% 1rm (all p 0.04) (see figures 3 and 4). there was no significant group main effect for leg press peak power force at any condition (all p 0.10). these findings indicate that hspt exerted a broader training effect than ssst when comparing the change in baseline power and speed across a range of typically encountered external resistances. baseline reaction time and braking speed values were 322.9 ms (95% ci : 293.8352.1 ms) and 221.9 ms (95% ci : 188.5255.4 ms), respectively, for hspt ; 303.1 ms (95% ci : 270.2335.8 ms) and 236.1 ms (95% ci : 206.6265.6 ms), respectively, for ssst ; 311.3 ms (95% ci : 269.4349.8 ms) and 252.0 ms (95% ci : 211.7292.3 ms), respectively, for con. there was no significant group main effect for the change in reaction time from the onset of the visual stimulus to the movement of the foot off the accelerator after training (change = 0.79 ms (95% ci : 3.11.5 ms) for hspt, 0.07 ms (95% ci : 1.92.1 ms) for ssst, and 1.5 ms (95% ci : 4.41.4 ms) for con ; p = 0.72). univariate anova demonstrated a significant group main effect for the change in braking speed with training (p = 0.02). the speed at which the lower limb was moved from the accelerator to the brake improved 15.3% in hspt (change = 3.4 ms (95% ci : 4.8(2.0 ms))) and 2.7% in ssst (change = 0.68 ms (95% ci : 3.01.7 ms)), but worsened by 2.2% in con (change = 0.58 ms (95% ci : 1.72.8 ms)). there was no difference between groups in average daily leg press work performed (hspt : 7235 j (95% ci : 63468123 j) versus ssst : 6876 j (95% ci : 58007953 j) p = 0.59) or knee extension work performed (hspt : 4002 j [95% ci : 35144490 j ] versus ssst : 3839 j (95% ci : 31764501 j) ; p = 0.68) during the 12-week intervention. there was a difference in average daily rpe between hspt and ssst (p < 0.001) during both leg press and knee extension exercise. leg press rpe averaged 12.2 (95% ci : 11.512.9 ; light to somewhat hard) for hspt and 15.1 (95% ci : 14.316.0 ; somewhat hard to hard) for ssst training while knee extension rpe averaged 14.6 (95% ci : 13.915.2 ; somewhat hard to hard) for hspt and 17.0 (95% ci : 16.217.8 ; very hard) for ssst training. these results indicate that despite being exposed to similar workloads during training, hspt perceived the exercise to be easier. the major finding from this study was that high - speed power training significantly improved muscle performance and braking speed in older men and women using a driving simulator. high - speed power training and slow - speed strength training both improved power in older men and women ; however, high - speed power training also improved the velocity component of power compared to slow - speed strength training and this improvement likely contributed to the improved ability to move the foot quickly from the accelerator to the brake. because speed is trainable in older adults, the utilization of high - speed movements during resistance training may result in the transfer to functional tasks that require high - speed movements. the benefits to muscle performance obtained at high - speed and low external resistance also occurred without a compromise in muscle strength and at lower perceived exertion compared to slow - speed strength training. we evaluated muscle performance by comparing peak power, peak power velocity, and peak power force across a range of external resistances typically encountered in daily task performance. because external resistances in the environment are fixed and do not increase as you get stronger, we compared the change in peak power, peak power velocity and peak power force relative to the baseline 1rm, which may more accurately reflect how improvements in muscle performance apply to real - world functioning. we hypothesized that high - speed power training would increase peak power and peak power velocity at the low external training resistances while slow - speed strength training would increase peak power and peak power force at the high external training resistances because of the principle of training specificity. however, hspt improved peak power and peak power velocity across the entire range of external resistances (4090% 1rm), while ssst improvements were limited to external loads closest to the training loads (~7090% 1rm). although we did not hypothesize that slow - speed strength training would improve peak power velocity, it makes sense that following training (and strength gain) it would be easier to move the same absolute load faster. still, this did not occur across all external resistances as it did with high - speed power training. training at 40% 1rm, however, increased peak power and peak power velocity across the entire range of external resistances, demonstrating that high - speed power training exerts a broader training effect than slow - speed strength training. a key question was whether improvements in muscle performance with high - speed power training would impact the braking speed functional task. interestingly, it is not uncommon to find studies where functional tasks do not improve with resistance training or power training. reviews and meta - analyses [22, 23 ] demonstrate clearly a very small impact of resistance training on function, with improvements observed mostly with gait - related tasks. bean. did not find significant changes in function following equipment - based training or training using weighted - vest exercises, respectively. other power training studies have shown only small changes in sometimes half or fewer of the functional tasks in a battery of tasks [4, 24 ]. it may be that participants in many resistance training studies are healthy with a greater reserve capacity in their functional abilities. these types of participants are likely closer to their functional threshold, where even large increases in strength or power would result in little or no increases in function. or it may be that the transfer of the resistance training task did not closely represent the complex movements required for the functional tasks. the transfer of a resistance training task to a functional task is most likely when the muscle activation patterns required for functioning are those that have been repeatedly practiced through the training task. thus, the optimal transfer of training to function demands specificity between the training task and functional task. because performance of the braking speed task utilized in this study required considerable movement velocity and a similar movement pattern to the leg press exercise, we anticipated that high - speed training would impact this measure of function to a greater degree than slow - speed resistance training. improvement in muscle performance with high - speed power training were closely linked to the improvements in braking speed. when we measured the effect of this relatively simple but explosive movement (moving the foot from the accelerator to the brake) that closely approximated the explosive nature of the exercise (leg press training specifically), we did find positive transfer of training to function. functional tasks across a range of external resistances, high - speed power training demonstrated greater improvements compared to slow - speed strength training. as a result, the similar braking task may have benefitted from this global improvement in power and speed with high - speed training. we believe these findings have significant public health implications for older adults who continue to operate motor vehicles. a previous study by webber and porter found that power training of the ankle plantar flexors and dorsiflexors at 80% 1rm significantly increased speed of movement from one foot switch to a second foot switch compared to control. because older drivers are at greater risk for injury - related deaths while driving, we believed it was critical to explore this question further by using a driving simulator with an accelerator and brake that more closely represented the equipment and mechanics utilized in a braking maneuver. we calculated that at 60 mph (88 feet per second) with an average deceleration rate of 20 feet per second (coefficient of friction of 0.75), the time to brake a vehicle is 4.4 s (88/20) and 194 feet (half the initial velocity the time required to stop (0.5 88 4.4 s)). however, at initial velocity (60 mph), there is a delay in applying the brake due to reaction time to a stimulus and movement time from the accelerator to the brake. in our study, groups had an average of 0.310 s of reaction time plus an additional 0.235 s to move the foot from the accelerator to the brake. reaction time alone (0.310 s 88 ft / s) added an additional 27 ft to the distance (194 + 27 = 221 ft). if we calculate a 15.3% improvement in braking time for hspt (0.198 88 = 17 ft) and a 2.2% worsening for con (0.240 88 = 21 ft), differences in stopping distance between the groups will be 242 ft for con (221 + 21) and 238 feet for hspt (221 + 17). considering that mere inches may be critical to avoid collision - related injury at high speeds, a difference of 4 feet could have significant safety implications. finally, older adults performing high - speed power training perceived the exercise to be easier than those performing slow - speed strength training during the 12-week intervention despite both groups performing the same amount of work during the training. these findings could have implications for the retention of older adults in resistance training programs outside the laboratory. in addition, for older adults who have previously been involved in resistance training programs maintaining continued participation in this type of exercise has proven difficult. one study reported a 50% decline in the number of older adults participating in resistance training exercise during followup from 12 weeks of facility - based resistance training. research suggests that moderate intensity exercise is a stronger predictor of whether adults maintain continued participation in exercise than high - intensity exercise [27, 28 ] ; thus, an exercise protocol such as high - speed power training which is perceived as less strenuous could be a part of the strategy to reverse this trend toward reduced participation in resistance training exercise for older adults. high - speed power training and traditional slow - speed strength training both improved peak muscle power after 12 weeks of training ; however, high - speed power training increased velocity compared to traditional strength training. when examining the power and velocity required for real world functional tasks across a range of external resistances, high - speed power training exerted a broader training effect than slow - speed strength training. these benefits occurred without a compromise in muscle strength and at lower perceived exertion (while performing the same amount of work) than when performing slow - speed strength training. most importantly, high - speed power training improved braking speed using a driving simulator, suggesting that when the explosive nature of the training closely mimicked both the motion and the speed at which the task was performed, there was a positive transfer of training to function.	we examined whether high - speed power training (hspt) improved muscle performance and braking speed using a driving simulator. 72 older adults (22 m, 50 f ; age = 70.6 7.3 yrs) were randomized to hspt at 40% one - repetition maximum (1rm) (hspt : n = 25 ; 3 sets of 1214 repetitions), slow - speed strength training at 80%1rm (ssst : n = 25 ; 3 sets of 810 repetitions), or control (con : n = 22 ; stretching) 3 times / week for 12 weeks. leg press and knee extension peak power, peak power velocity, peak power force / torque, and braking speed were obtained at baseline and 12 weeks. hspt increased peak power and peak power velocity across a range of external resistances (4090% 1rm ; p < 0.05) and improved braking speed (p < 0.05). work was similar between groups, but perceived exertion was lower in hspt (p < 0.05). thus, the less strenuous hspt exerted a broader training effect and improved braking speed compared to ssst.
membranous glomerulonephritis (mgn) is the most important cause of nephrotic syndrome in adults. among patients > 60 years old, secondary mgn is more common, especially due to underlying malignancy. in 2009, the m - type phospholipase a2 receptor (pla2r) was identified as the major podocyte antigen involved in idiopathic adult mgn, while anti - pla2r antibody is not detected in secondary mgn. this patient did not have any underlying disease that could cause secondary mgn, and anti - pla2r antibody was detected in her serum, so idiopathic mgn was diagnosed. this is the oldest reported case of idiopathic mgn, and it is discussed with reference to the literature. in july 2014, a 95-year - old japanese woman was admitted to our hospital with a 3-month history of bilateral edema of the lower extremities. when she developed chronic diarrhea at 90 years of age (in 2009), laboratory tests revealed that serum creatinine (cre) was 1.6 mg / dl, albumin was 3.2 g / dl, and urinary protein excretion was 0.32 g / day. laboratory tests revealed a white blood cell count of 4,600/l, a red blood cell count of 3.7110/l, hemoglobin of 11.7 g / dl, and platelet count of 21.310/l. in addition, total protein was 6.3 g / dl, albumin was 2.4 g / dl, serum urea nitrogen was 26 mg / dl, serum cre was 1.55 mg / dl, c - reactive protein was 1.6 mg / dl, and lactate dehydrogenase was 257 iu / l (normal : 119 - 229 iu / l). immunological tests showed that antinuclear antibody, anti - double - stranded dna antibody, and u1-nuclear ribonucleoprotein antibody were all negative. igg was 1,340 mg / dl, iga was 427 g / dl, and igm was 266 mg / dl. the serum level of c3 was 123 mg / dl (normal : > 86 mg / dl), c4 was 19 mg / dl (normal : > 18 mg / dl), and ch50 was 38 u / ml (normal : > 30 u / ml). the patient was negative for hepatitis b virus antibody, hepatitis c virus antibody, and treponema antigen. urinary protein excretion was 9.1 g / day, and the sediment contained 5 - 10 erythrocytes per high - power field. no findings suggestive of malignancy were revealed by imaging studies such as computed tomography and ultrasonography. serum anti - pla2r autoantibody was positive according to the method of akiyama.. light microscopy of a renal biopsy specimen containing 36 glomeruli revealed global sclerosis in 22 of them (fig. 2a), but there was no definite spike formation, bubbling, or thickening of the glomerular basement membrane (gbm) (fig. 2b). in the tubulointerstitial region, atrophy or fibrosis affected 50 - 60% of the cortical tissue (fig. 2c), while the interlobular arteries showed mild to moderate fibroelastic intimal thickening (fig. analysis of igg subclasses revealed dominant staining for igg4 (igg4 > igg1 > igg3 > igg2 ; fig. electron microscopy showed subepithelial electron - dense deposits (edd) in the gbm, but these subepithelial edd were not associated with spike - like protrusions arising from the basement membrane (fig. anasarca was treated with diuretics (furosemide at 80 mg daily) and dietary restriction (6 g of salt and 700 ml of water daily), achieving weight loss from 56.4 to 51.0 kg. because the patient did not want steroid therapy due to adverse effects such as bone disease, cyclosporin microemulsion (neoral at 50 mg daily) was added, and the level at 2 h after administration was titrated in the range of 600 - 1,000 ng / ml. hemodialysis was started when cre was 6.0 mg / dl and urea nitrogen was 102 mg / dl because of exacerbation of pleural effusion and generalized edema along with an increase in weight to 58 kg at 2 months after diagnosis. light microscopy of a renal biopsy specimen containing 36 glomeruli revealed global sclerosis in 22 of them (fig. 2a), but there was no definite spike formation, bubbling, or thickening of the glomerular basement membrane (gbm) (fig. 2b). in the tubulointerstitial region, atrophy or fibrosis affected 50 - 60% of the cortical tissue (fig. 2c), while the interlobular arteries showed mild to moderate fibroelastic intimal thickening (fig. analysis of igg subclasses revealed dominant staining for igg4 (igg4 > igg1 > igg3 > igg2 ; fig. electron microscopy showed subepithelial electron - dense deposits (edd) in the gbm, but these subepithelial edd were not associated with spike - like protrusions arising from the basement membrane (fig. anasarca was treated with diuretics (furosemide at 80 mg daily) and dietary restriction (6 g of salt and 700 ml of water daily), achieving weight loss from 56.4 to 51.0 kg. because the patient did not want steroid therapy due to adverse effects such as bone disease, cyclosporin microemulsion (neoral at 50 mg daily) was added, and the level at 2 h after administration was titrated in the range of 600 - 1,000 ng / ml. hemodialysis was started when cre was 6.0 mg / dl and urea nitrogen was 102 mg / dl because of exacerbation of pleural effusion and generalized edema along with an increase in weight to 58 kg at 2 months after diagnosis. in 2009, beck. identified the m - type pla2r as the major target podocyte antigen in adult idiopathic mgn and showed that it was expressed by podocytes and colocalized with igg4 in glomerular immune deposits of patients with idiopathic mgn. a pla2r band was detected in the serum of 26 patients (mean age 48.1 14.9 years ; range 21 - 88) of 37 patients (70%) with idiopathic mgn, while it was not found in 8 patients with secondary mgn, 22 patients with other diseases, and 30 healthy controls. qin. evaluated chinese mgn patients with the method of beck., and they detected anti - pla2r autoantibodies in the serum of 49 patients (mean age 49.2 14.9 years ; range 18 - 77) of 60 patients (82%) with idiopathic mgn, in 1 of 20 patients with lupus - associated mgn, in 1 of 16 patients with hepatitis b - associated mgn, and in 3 of 10 patients with tumor - associated mgn.. investigated circulating anti - pla2r antibodies in japanese mgn patients using a highly sensitive western blotting method under nonreducing conditions with human glomerular extract (at dilutions of 1:25, 1:10, and 1) as the primary antibody. anti - pla2r antibodies were detected in 53 of 100 patients (aged 67 9 years at diagnosis) with idiopathic mgn compared to 0 of 31 patients (aged 61 12 years) with secondary mgn. studied 309 patients with idiopathic mgn and reported that 74 were elderly (60 years old), with the oldest subject being 83 years old. the incidence of chronic renal insufficiency was significantly higher in elderly patients than in younger patients (60 years, including 2 patients aged 80 and 85 years. there was a higher incidence of an identifiable cause for the nephropathy among the older patients, who were also more likely to have hypertension and more severe renal impairment than the younger patients. abrass reported on the treatment of elderly patients with mgn, stating that the only consistent recommendation is to avoid high - dose corticosteroids. she also mentioned that drug dosages should be modified and monitored carefully and that elderly patients may be particularly prone to the side effects and infectious complications of immunosuppressants. when we investigated positivity for anti - pla2r antibodies at the diagnosis of idiopathic mgn among patients attending our hospital, we identified 7 patients aged 75.1 10.2 years (range 65 - 95) including the present case. our patient was older than those reported by previous authors, such as beck. (48.1 14.9 years ; range 21 - 88), akiyama. (49.2 14.9 years ; range 18 - 77). if nephrologists performed renal biopsy more frequently in elderly patients over 90 years of age with nephrotic range proteinuria, other such cases might be discovered. in conclusion, we encountered a 95-year - old japanese woman with nephrotic - range proteinuria, and renal biopsy revealed stage 1 mgn with igg4 dominance. this patient did not have any underlying disease that could have caused secondary mgn, and anti - pla2r antibody was positive, so idiopathic mgn was diagnosed. corticosteroid therapy was avoided, but hemodialysis was required for generalized edema, and the patient is currently doing well on hemodialysis. this case reminds us that idiopathic mgn with anti - pla2r antibodies can occur even at the very advanced age of 95 years. the present report adhered to the declaration of helsinki and these patients have given their consent for the case report to be published.	a 95-year - old woman was admitted to our hospital for evaluation of bilateral lower - limb edema persisting for 3 months. serum creatinine was 1.55 mg / dl, and urinary protein excretion was 9.1 g / day. renal biopsy revealed stage 1 membranous glomerulonephritis (mgn) with immunoglobulin g4-dominant staining. this patient did not have any underlying disease such as infection with hepatitis b or c virus or malignancy, and anti - phospholipase a2 receptor (pla2r) antibody was detected in the serum. accordingly, idiopathic mgn was diagnosed. corticosteroid therapy was avoided, but hemodialysis was required to treat generalized edema. the patient is currently doing well. this is the oldest reported case of idiopathic mgn with positivity for anti - pla2r antibody.

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