Purpose. Conventional two-dimensional vessel wall imaging has been used to depict the middle cerebral artery (MCA) wall in patients with recent small subcortical infarctions (RSSIs). However, its clinical use has been limited by restricted spatial coverage, low signal-to-noise ratio (SNR), and long scan time. We used a novel three-dimensional high-resolution MR imaging (3D HR-MRI) technique to investigate the presence, locations, and contrast-enhanced patterns of MCA plaques and their relationship with RSSI. Methods. Nineteen consecutive patients with RSSI but no luminal stenosis on MR angiography were prospectively enrolled. 3D HR-MRI was performed using a T1w-SPACE sequence at 3.0 T. The presence, locations, and contrast-enhanced patterns of the MCA plaques on the ipsilateral and contralateral sides to the RSSI were analyzed. Results. Eighteen patients successfully completed the study. MCA atherosclerotic plaques occurred more frequently on the ipsilateral than the contralateral side to the RSSI . The occurrence of superiorly located plaques was significantly higher on the ipsilateral than the contralateral side of the MCA . Conclusions. Superiorly located plaques are closely associated with RSSI. 3D high-resolution vessel wall imaging may be a potential tool for etiologic assessment of ischemic stroke. Small subcortical infarction (SSI) is a unique entity with a distinct pathogenesis . TraditiUsing two-dimensional high-resolution MR imaging (2D HR-MRI), several studies have demonstrated that atherosclerotic plaques can be detected in patients with single subcortical infarctions 42–60%) –8. Xu et0% –8. XuIn this study, we aimed to compare the presence, locations, and contrast-enhanced patterns of plaques on the ipsilateral and contralateral side of the RSSI using contrast-enhanced three-dimensional HR-MRI (3D HR-MRI).From September 2013 to March 2014, 19 consecutive patients were prospectively recruited into the current study. Patients were enrolled in this study if the following criteria were met: (1) a recent single infarction  that is believed to be responsible for the clinical symptoms reported within the past 4 weeks: this infarct should have been identified in the territory of the lenticulostriate arteries as confirmed by diffusion-weighted imaging (DWI); (2) no ipsilateral MCA stenosis based on the MRA; (3) one or more risk factors for atherosclerosis . All eligible patients first underwent comprehensive examinations including MRI of the brain , MRA of the MCA, transcranial Doppler, carotid duplex, electrocardiography, and echocardiography. We excluded patients with the following: (1) one or more stenoses at the ipsilateral intracranial internal carotid artery or relevant extracranial arteries; (2) a definite cardioembolic source; (3) other nonatherosclerotic vasculopathies. Among these patients, those who were unable to undergo a second MRI scan for any reason were also excluded. In this study, the term “recent small subcortical infarction” followed the definition described by Wardlaw et al. . The stuThe examination was performed on a 3.0 T MRI system  using a 32-channel head coil. The examination protocol included 3D time-of-flight MRA, DWI, and pre- and postcontrast 3D T1-weighted HR-MRI.3 and (0.7 mm)3, and the average scan time was 8 min . For contrast-enhanced T1w-SPACE, 0.1 mmol/kg body weight of gadopentetate dimeglumine  was manually injected. All subjects included in this study underwent MRI examination within 4 weeks of symptom onset.The 3D HR-MRI was performed using a variant of a 3D turbo spin echo technique known as T1w-SPACE . The tecAll images were analyzed by two experienced readers. One reader (Qi Yang) is a neuroradiologist with 10-year clinical experience; another reader (Yiu-Cho Chung) is an MR scientist working on plaque imaging for more than 10 years. Both were independently blinded to the patients' clinical information and other images before they reviewed the T1w-SPACE images. The discrepancies between the readers were resolved by consensus reading.Image analysis was performed on a workstation . The infarction at the penetrator territory of the MCA was assessed based on a previously published template . The preAs most lenticulostriate arteries arise from the superior wall of the MCA trunk , the locTwo-dimensional short-axis images of the plaques that were generated from the registered pre- and postcontrast T1w-SPACE images were used to find the plaque enhancement ratio as follows. Region of interest (ROI) of the thickened MCA vessel walls was first defined. The mean signal intensities (SI) of the MCA vessel wall at the ROI on the registered pre- and postcontrast T1w-SPACE were measured. They were then normalized by the SI of the nearby gray matter regions in the pre- and postcontrast images using the method described by Lou et al. . Followiκ). A value of κ > 0.8 would indicate almost perfect agreement. Student's t-test was used for quantitative data comparison, while Fisher exact test was used for qualitative data comparison. Statistical significance was defined as a P value of <0.05. All statistical analyses were performed using SPSS software .Continuous data were expressed as mean ± standard deviation. Categorical data were expressed as counts and percentages. The interobserver agreement for the identification of the plaques was assessed by calculating kappa . In addition, the prevalence of superiorly located plaques was significantly higher in the ipsilateral than the contralateral MCA . Weighted κ value of the interobserver agreement for the identification of the plaques was 0.828 .Of the 18 patients , 3D HR-MRI detected 21 plaques that appeared to cause no stenosis on MRA. On precontrast images, 14 plaques were detected and 12 of them were enhanced in postcontrast images. On postcontrast images, MCA atherosclerotic plaques were found in 19 vessels. Superiorly located plaques were found in 17 (89.5%) of the 19 MCAs with plaques. The percentage of atherosclerotic plaques in all the 36 MCAs detected on postcontrast T1w-SPACE images was higher than that on precontrast T1w-SPACE images (52.8% versus 38.9%) .  FigureP = 0.512 using Student's t-test). The percentages of superiorly located plaques with increased SI at both the ipsilateral and contralateral MCA were 58.8% ± 34.9% and 72.2% ± 43.5%, respectively  [–50% 6, , the abiP = 0.044). The superiorly located plaques commonly involve the orifices of penetrating arteries and may play an important role in the development of RSSIs [3D HR-MR can cover the entire range of bilateral MCAs in less than ten minutes and is more time-efficient compared with conventional 2D techniques. It allows the comparison between the ipsilateral MCAs of symptomatic RSSI with the contralateral MCAs. We found that plaques on both sides of RSSI were mostly superiorly located . As plaques occurred more often on the ipsilateral (72.2%) than on the contralateral sides (33.3%), the percentage of superiorly located plaques on the MCA ipsilateral to the RSSI was higher than that contralateral to the RSSI . Despite the varying voxel size used in different patients, the lowest spatial resolution in terms of voxel size in this study is still comparable to other studies using 2D techniques where the voxel sizes were in the range of 0.33 mm3 to 0.36 mm3. Additionally, our study population included only symptomatic patients with a normal lumen, and the results may not be directly applicable to patients with MCA stenosis caused by atherosclerotic plaques.There were several limitations in our study. First, the sample size was relatively small and data from age-matched healthy control subjects were unavailable for comparison. Second, the plaques that were detected cannot be verified by histopathology. Also, the culprit plaques could not be identified as the branch artery ostia cannot be clearly visualized in the images. Third, the spatial resolution of the images varied among different patients (isotropic voxel size varied from 0.125 to 0.343 mmThe current findings indicate that 3D high-resolution vessel wall imaging improves the visualization of MCA plaques and holds promise as a valuable alternative to current 2D MRI techniques. Our results suggest that atherosclerosis may be more prevalent among patients with RSSI than commonly believed. Superiorly located MCA plaques are associated with ipsilateral infarctions and may have an important role in the pathogenesis of RSSI. Plaques found in these patients with RSSI are mostly enhanced. Further studies are required to explore the relevance of these findings to the subclassification of stroke.


We report the case of a rare solitary small nodular form of malignant hepatic epithelioid hemangioendothelioma in a patient followed by computed tomography and gadoxetic acid (Gd-EOB-DTPA)–enhanced magnetic resonance imaging with histological analysis. This case showed early peripheral septal and nodular enhancement and delayed centripetal enhancing pattern with capsular retraction, mimicking peripheral cholangiocarcinoma, inflammatory pseudotumor, or metastases. The histological and immunohistochemical findings were diagnostic of a malignant hepatic epithelioid hemangioendothelioma. Most hepatic EHEs present as the diffuse multifocal type, which is an advanced stage, and rarely as the nodular type, which represents an early stage4 and more commonly affects adult females with a peak incidence between 30 and 50 years of age.4 Treatment options include liver resection or liver transplantation, and the prognosis is more favorable than that of other hepatic malignancies.4 Metastases have been reported in 27% to 37% of patients at presentation and occur most commonly in the lungs.Epithelioid hemangioendothelioma (EHE) is a rare, low- to intermediate-grade malignant vascular tumor that originates from the soft tissue, visceral organs, bone, lung, brain, and small intestines.Generally, a specific diagnosis for the nodular type is difficult without a biopsy because the radiologic findings are similar to those for some hepatic metastases. The diffuse form of EHE has more specific diagnostic criteria, and peripheral location and capsular retraction are hallmarks of hepatic EHE.We present a case of a rare solitary small nodular form of malignant hepatic EHE.A 22-year-old man without underlying liver disease or clinical symptom visited our hospital for screening. The patient was negative for hepatitis B surface antigen and hepatitis C virus antibody, and the serum α-fetoprotein level was within normal limits. On ultrasonography, a 2.3 cm incidental hepatic lesion was detected that was a well-defined, inhomogeneous hypoechoic hepatic nodule that extended to the hepatic surface . MultideImmunohistochemically, the tumor was positive for CD34 and factor VIII–related antigen, which are endothelial markers . High-ce18F-FDG-PET/CT scan showed no evidence of distant metastasis or other primary tumors within 3 years. Therefore, we concluded that the final diagnosis was a primary malignant hepatic EHE.Postoperative follow-up CT, bone scan, and 6 Hepatic capsular retraction is a rare but specific sign in a variety of tumors, including EHE, liver metastases, cholangiocarcinomas, and fibrolamellar hepatocellular carcinoma.6Although most intrahepatic tumors are commonly associated with mass effects that result in contour bulging, 2.0% to 2.8% of tumors may show capsular retraction.6 In untreated hepatic metastases, capsular retraction can occur in the fibrosis-containing primary tumors such as colon cancer, breast cancer, carcinoid tumor, lung cancer, and gallbladder cancer.Liver metastases are the most common hepatic malignancies, and capsular retraction usually occurs in the subcapsular area, which is associated with systemic chemotherapy.7 However, in our case, biliary dilatation was not identified.Peripheral cholangiocarcinoma may cause capsular retraction because of an abundant fibrous stroma and chronic peripheral biliary obstruction. Therefore, if the hepatic tumor shows capsular retraction, delayed enhancement, and peripheral biliary dilatation, cholangiocarcinoma must be considered.Hepatic EHE is a low- to intermediate-grade malignant vascular tumor, and it is important to differentiate hepatic EHE from other malignant tumors because long-term survival is possible after successful liver resection or liver transplantation.8Epithelioid hemangioendothelioma should not be confused with an infantile EHE, which is histologically benign, common in infants and young children, and resolves spontaneously.4 The adjacent liver parenchyma is normal in the solitary small nodular form, whereas in the diffuse form, there is compensatory hepatic parenchymal enlargement and development of cirrhosis. Histologically, these tumors are composed of central fibrous myxoid stroma and peripheral rich cellularity with active proliferation of epithelioid and dendritic cells.3Most patients are asymptomatic when the tumor is detected and present with multifocal peripheral tumors. The solitary nodular form has been reported in only 13% to 18% of patients.Intravascular growth with neoplastic endothelial cell invasion results in fibrosis, progressive sclerosis, and calcification. The final diagnosis of EHE requires histopathological confirmation with immunohistochemical staining for endothelial markers. The vascular nature of the endothelial origin of the neoplastic cell is confirmed by positive factor VIII–related antigen staining and other endothelial cell markers .9 As a solitary hepatic EHE progresses, the tumor nodules become multifocal and confluent, especially in the subcapsular regions. Hepatic EHE shows intense peripheral enhancement due to active proliferation of epithelioid cells and a delayed or nonenhancing center due to dense fibrous stroma (target appearance). As they progress, fibrous septa within the tumor cause capsular retraction.2 The “capsular retraction” sign is caused by the fibroproliferative reactions of the tumors, which leads to invagination of the nearby liver capsule.11The typical imaging findings are usually based on these histopathologic characteristics. Pathologically, hepatic EHE occurs mainly in the solitary type at the early stage, but with a propensity for invasion of terminal hepatic venules and portal vein branches.3 The MR imaging of hepatic EHE includes heterogeneous intermediate to high SI on T2-weighted images and low SI on T1-weighted images. Heterogeneity on T2-weighted images may correspond to low signal zones of fibrotic, necrotic, or hemorrhagic areas, and higher signal zones of vascular channels and edematous connective tissues. On dynamic contrast-enhanced MR images, peripheral rim enhancement was better delineated. Gadoxetic acid is a hepatocyte-specific MR contrast agent that is increasingly used for liver MR imaging.Unenhanced CT imaging of a hepatic EHE is solid, with heterogeneously lower attenuation than normal liver parenchyma. After contrast enhancement, the tumor displayed no or faint peripheral rim enhancement at the arterial phase and a “halo” sign during the portal venous phase.4 gadolinium-contrast enhanced MR imaging findings of 16 patients with hepatic EHE resulted in high enhancement (37.5%), peripheral and delayed central enhancement (37.5%), concentric layers of variable intensity (19%), and no enhancement (6%). In our case, the lesion was partially surrounded by a nonenhancing hypointense thin rim outside of the thick peripheral rim enhancement.However, these findings are relatively nonspecific. According to Mehrabi et al,12 reported that this avascular outer rim is caused from tumor invasion of hepatic sinusoids, venules, or small portal vein branches.Miller et al4 reported marked capsular retraction in 10.6% of 142 patients with hepatic EHE. We initially thought that our patient had inflammatory pseudotumor. Inflammatory pseudotumors often have low attenuation on unenhanced images, low SI on T1-weighted images, and high SI on T2-weighted images with peripheral enhancement or enhancement of multiple internal septa.13 These imaging features correspond to those of our case. However, capsular retraction adjacent to hepatic nodules is rarely reported in inflammatory pseudotumor.14 When compared with the diffuse type of EHE, which reflects advanced-stage disease, the solitary nodular type, which is considered to be the earlier form of EHE, is often misdiagnosed because of its extreme rarity.4 Therefore, the final diagnosis of hepatic EHE requires histopathologic confirmation with immunologic staining for the endothelial markers.These previously reported characteristic CT and MR findings of hepatic EHE are in agreement with those of our case. Moreover, the hepatic nodule in our case has a capsular retraction with subcapsular location. Hepatic capsular retraction adjacent to a hepatic tumor was first described in the hepatic EHE, which reflects lesion-related fibrosis. Mehrabi et alHowever, based on this case, we concluded that the peripheral location of the tumor, capsular retraction, and peripheral enhancement are hallmark features. Certain clinical features such as the young age of patient and the good clinical condition as well as the positive imaging findings are suggestive of EHE.Therefore, radiologists should be aware of the imaging findings associated with hepatic EHE and be able to suggest immunologic staining for endothelial markers for accurate diagnosis.


Primary central nervous system (CNS) lymphoma, most frequently a diffuse large B-cell lymphoma, is a rare aggressive lymphoma confined to the CNS, thus requiring differentiation from other brain malignancies such as glioblastoma. Although stereotactic biopsy can confirm the diagnosis, this is invasive, not always feasible and can be inconclusive after steroid use. Hence, cranial magnetic resonance imaging (MRI) with contrast and cerebrospinal fluid analysis are frequently used to make a prompt diagnosis. We report a case of a woman with two brain masses who presented unique diagnostic challenge. Primary central nervous system (CNS) lymphoma, most frequently a diffuse large B-cell lymphoma, is a rare aggressive lymphoma confined to the CNS, thus requiring differentiation from other brain malignancies such as glioblastoma. Although stereotactic biopsy can confirm the diagnosis, this is invasive, not always feasible and can be inconclusive after steroid use.Cranial magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) analysis are commonly used diagnostic tests for primary CNS lymphoma .A 74-year-old woman presented to our hospital with a 2-day history of left hemiparesis and dysarthria. She had left upper motor neuron facial palsy and mild left-sided hemiparesis. MRI of brain with and without gadolinium-based contrast revealed 2.5 cm enhancing lesion within the right posterior frontal lobe and 1.2 cm enhancing lesion within the right occipital lobe. These two lesions were abutting the right lateral ventricle, had little surrounding edema and mild diffusion restriction , 2. The National Comprehensive Cancer Network guidelines on primary CNS lymphoma recommend CSF analysis  for diagnosis in patients with characteristic MRI findings. Brain biopsy is recommended if CSF analysis and ophthalmology evaluation with ocular biopsy of suspicious lesion are not diagnostic . This apCharacteristic MRI findings of primary CNS lymphoma include homogenous and strong enhancement, periventricular location, single or multiple lesions, without necrosis and with relatively less edema , 3. ReceAlthough traditional CSF studies  have low sensitivity and specificity for CNS lymphoma, CSF flow cytometry and immunoglobulin heavy chain rearrangement have moderate specificity. CSF cytology and less commonly available, CSF antithrombin III and micro RNA analysis have high specificity . CSF ana


Pressure garment therapy (PGT) is well accepted and commonly used by clinicians in the treatment of burns scars and grafts. The medium to high pressures (24–40 mmHg) in these garments can support scar minimisation, and evidence is well documented for this particular application. However, PGT specifically for burn donor sites, of which a sequela is also scarring, is not well documented. This study protocol investigates the impact of a low pressure (4–6 mmHg) interim garment on donor site healing and scarring. With a primary purpose of holding donor dressings in place, the application of the interim pressure garment (IPG) appears to have been twofold. IPGs for donor sites have involved inconsistent application with a focus on securing wound dressing rather than scar management. However, anecdotal and observational evidence suggests that IPGs also make a difference to some patient’s scar outcomes for donor sites. This study protocol outlines a randomised controlled trial designed to test the effectiveness of this treatment on reducing scarring to burn donor sites.This study is a single-centre, single (assessor)-blinded, randomised control trial in patients with burns donor sites to their thighs. Patients will be randomly allocated to a control group (with no compression to donor sites) or to an experimental group (with compression to donor sites) as the comparative treatment. Groups will be compared at baseline regarding the important prognostic indicators: donor site location, depth, size, age, and time since graft (5 days). The IPG treatment will be administered post-operatively (on day 5). Follow-up assessments and garment replacement will be undertaken fortnightly for a period of 2 months.This study focuses on a unique area of burns scar management using a low-pressure tubular support garment for the reduction of donor site scars. Such therapy specifically for donor scar management is poorly represented in the literature. This study was designed to test a potentially cost-effective scar prevention for patients with donor sites to the thigh. No known studies of this nature have been carried out to date, and there is a need for rigorous clinical evidence for low-pressure support garments for donor site scar minimisation.ACTRN12610000127000. Registered 8 Mar 2010.Australian New Zealand Clinical Trials Registry identifier  The donor skin is then placed over the original debrided burn wound area to assist in healing. There are sufficient residual epidermal cells in the remaining dermis to allow re-epithelisation to occur over an approximately 14-day period [Thickened erythematous scars can reduce physical function and create a distorted physical appearance for a burn-injured patient –3. When Despite the donor site depth being extremely thin, patients often describe this new wound to be like ‘bad gravel rash’ and report worse pain in this wound than at the original burn or graft site. Pain associated with burn surgery has been shown to delay patients’ return to work and daily functional activity, with donor sites consistently identified as a potential source of concern for burn patients –3, 6–8. A review of the literature published over the past decade demonstrates the advances made in minimising scar formation –15. CompPressure garments appear to enhance the scar maturation process following epithelisation and, if worn for a minimum of 20–23 h/day, also appear to inhibit the development of hypertrophic or abnormal scar tissue. Although the exact mechanism of scar reduction remains largely unknown , 10. TheThe pressure garment is positioned over any dressings that may be needed in the early stage of recovery and is worn until the burn scar has matured or become pale in colour, resembling normal skin. This process of scar maturation is reported to take 18–24 months to complete, but it can take longer in some cases , 19, 20.interim pressure garment for the purpose of this study) can be applied post-operatively around days 3–5 before donning the firmer compression garment. These interim garments are made from a tubular elastic material (similar to Lycra® bicycle pants)  Fig.  and applThrough the course of clinical practice, it has been observed that IPG appears to reduce donor site erythema and reported pain in some patients. Figure The levels of evidence outlining risks and benefits of pressure garment therapy have varied across the literature over the past 45 years. More recently, pressure garments 25–40 mmHg) have been shown to be effective for use in hypertrophic burns scars; however a paucity of evidence exists, along with inconsistent findings regarding the minimisation of erythema for such scars. These recommendations are reported as levels I and II evidence , 17, 21.–40 mmHg Reduction of time to epithelisationReduction of erythemaReduction of scar thickness and/or heightImprovement of overall scar appearanceReduction of self-reported pain levelsThis research study aims to establish evidence for tissue support (4–6 mmHg) in healing burn donor sites and to test the use of interim garments made from tubular support bandages for donor site scar management. Specific aims are to investigate whether low-pressure IPG support to donor sites compared with no IPG results inThe methodology has been documented in a protocol and registered on a publicly available site . The methods are summarised here according to the revised Consolidated Standards for Reporting Trials (CONSORT) statement .The clinical trial has been designed as a single-centre, prospective, single-blind randomised controlled trial.Randomisation will be achieved through computerised tabulation with concealed allocation to each participant via a sealed envelope method. Participants will be randomly allocated into one of two groups: a control group that will have no compression to donor sites (the standard existing treatment) or an experimental group that will be provided with compression to donor sites (the comparative treatment).All new patients admitted to the Professor Stuart Pegg Adult Burns Centre who require SSG surgery for burn management over less than 15 % of their total body surface area (TBSA) will be screened for eligibility to participate in the study by the principal investigator.If eligibility is established, patients will be approached by the principal investigator and a lay explanation of the study procedures and requirements will be provided. Participants will be included once informed consent has been obtained and documented. Child assent will be documented where appropriate.The treatment will be IPG objectively measured in millimetres of mercury using the PicoPress® pressure sensor  , and it The principal investigator will record demographic information, including age, skin type (as assessed using Fitzpatrick skin typing) , cause oIn this study, the commercially available, evidence-based measurement tools described below will be used. These outcome measures will be done every 2 weeks for a period of 2 months.The DermaScan C  is a high-frequency, high-resolution ultrasound scanner which objectively captures and reproduces skin thickness. It can clearly discriminate between hypertrophic scar, normal scar and normal skin and is evidenced as having high inter-rater/intra-rater reliability , 32.The DSM II ColorMeter (Cortex Technology) is a self-contained, battery-operated device which provides objective measurements of erythema and melanin based on the light absorption characteristics of human skin , 34.The PicoPress® pressure sensor has been clinically tested for validity and reproducibility in objectively measuring the pressure in millimetres of mercury of compression garments. It has been shown to be an adequate and reliable instrument for use , 35.The Patient Observer Scar Assessment Scale has been shown to consistently demonstrate valid and reliable measures of scar quality . It is aFor participants in the IPG group, the principal researcher will measure the amount of pressure in each garment and issue new interim garments if needed, thereby ensuring that pressure remains constant. This concludes the assessment and treatment regime. The participant will undergo the same regime at each subsequent appointment for the period of 8 weeks. At this point, they will be released from the study.Measurement of the donor site with the garment removed for a specific length of time (5 minutes)Temperature-controlled roomPosition of donor anatomy dermatome location L3–L5 anterolateral thighA template on the donor site to identify the area to be measuredTime of day when measuredConsiderations to standardize each measurement will include the following:Previous studies using the DermaScan C have shown an effect size of 2.35 (1.69–3.01) between skin-thickness measurements of donor sites and scar sites . For a mOn the basis of data obtained from January 2013 to May 2015 , the ratio is expected to be 3:1. This ratio should accurately reflect the distribution of burns within the general community.p < 0.05. If raw data are not normally distributed, an equivalent non-parametric analysis will be used [Groups will be compared at baseline regarding the important prognostic indications: donor site location, depth, size, age, skin type and time since graft. Data will be entered into IBM SPSS 22.0 for Windows software files  and tested for normality of distribution. Descriptive statistics for all variables will be computed. If parametric statistics are able to be used, a two-way analysis of variance for group × time interaction will be performed to compare skin thickness between normal, donor and scar sites with a planned least significant difference post hoc analysis. Significance will be set at  be used .This study has received ethical approval from the RBWH HREC  and the UQ MREC . The trial has been registered on a publicly available site . The study will be monitored by the data and safety monitoring board, and any adverse or unforeseen events will be reported (as soon as or before they arise) to the appropriate committee for review (RBWH HREC/UQ MREC).Donor sites, regardless of their size, are consistently recognised as a potential source of concern. Interventions that assist to accelerate healing and reduce scar formation would considerably improve post-operative management of donor sites . This stIf IPG is shown to improve donor site scar maturation and healing, it is anticipated that this outcome may assist in improving patient comfort, reduce the incidence of abnormal scarring and reduce the associated psychological stress of this additional wound . FurtherWritten informed consent was obtained from the patient for publication of the images in this article. A copy of the written consent form is held by the authors and is available for review by the Editor-in-Chief of this journal.Eight participants have been recruited to date for a pilot study. A total of 44 participants (22 for each group) is required.


Background: We studied the role of cerebrospinal fluid (CSF) tap test at idiopathic normal pressure hydrocephalus (INPH) in improving cerebral blood flow velocity indices by transcranial Doppler (TCD) sonography.Methods: Twelve patients with assumed INPH were included in the study. The CSF tap test and INPH grading score was carried out according to the standard protocol. TCD was performed before and after the tap test for assessing blood flow in middle cerebral and anterior cerebral arteries.Results: Five INPH patients (41.7%) had clinical improvement as defined by at least one point reduction in INPH grading scale. The baseline TCD parameters of the middle cerebral artery were significantly higher compared with the control, and those parameters were decreased after tap test in those who improved.Conclusion: Our study showed that improvement in INPH grading score after CSF tap test might correlate with changing in TCD parameter in MCA and TCD parameter might be useful for shunt response in these patients. The improvement of any mentioned clinical impairments after lumbar cerebrospinal fluid (CSF) withdrawal or tap test offers a reliable means for selection of patients likely to respond to shunting in INPH.4 and then improved by Wikkelso et al.5 Wikkeslo et al. believed that improvement in cerebral blood flow (CBF) might have some role beside other factors in CSF tap test response.The CSF tap test fist has been introduced first by Hakim and Adams5Despite more than 60 years of research, our understanding of INPH pathophysiology remains sparse.6Transcranial Doppler (TCD) sonography becomes a noninvasive technique for measurement of cerebral blood flow velocity (CBFV). Besides the mean flow velocity, dynamic changes in the pulsatility index (PI) and the resistance index (RI), as calculated from TCD data, allow for an assessment of the forces acting on the terminal vasculature of the brain.Hence, it is a useful and non-invasive tool to assess the cerebral hemodynamic. The aim of this study was to assess the TCD CBF parameters before and after the CSF tap test and compare it with the INPH grading scale.This was an uncontrolled before and after study. Between January 2008 and February 2009, 12 demented patients with clinical criteria of INPH and enlarged ventricular systems (Evan’s ratio >0.3) were enrolled.2Evan’s index is defined by the maximum width of the frontal horn divided by the maximum inner width of skull.A control group consisting of 12 patients with cerebral atrophy on CT and dementia of different etiologies was included to establish baseline TCD data.CSF tap test was performed at the Neurology Department, Shafa Hospital, Kerman University of Medical Sciences, Kerman, Iran.TCD was performed with trans-temporal window using a 2 MHz Doppler probe  on both sides if possible. TCD was done by a neurologist blinded to the study protocol and parameters, including peak systolic velocity (PSV); end diastolic velocity (EDV); mean cerebral velocity (MSV); PI; and RI were recorded. CBFV in the middle cerebral artery was measured using TCD before and next day after CSF tap test.Clinical state of study subjects before and after the CSF tap test were assessed by the INPH grading scale, which evaluates the three main parts of the NPH syndrome: gait, cognitive function, and sphincter disturbances . It rangThe change of gait was evaluated 1 or 2 days after the tap while change of cognition and urination was evaluated at 1 week.7,8 on all study subjects on 2 consecutive days and at the same hour each day. On the morning of the 1st day, the patients underwent evaluation of gait and cognitive functions. On the 2nd day, these evaluations were repeated, 2 and 8 h after a lumbar puncture (LP) with removal of at least 50 ml of CSF.The CSF tap test was carried out according to Wikkelso et al. protocolWe had analyzed normality of our data and according to the Shapiro–Wilk test, although it had small sample size, but its distribution was normal. Hence, we could use mean and standard deviation for our analysis.The groups were compared using the Student’s t-test or Mann–Whitney U test for the continuous variables. Measures of TCD parameters before and after the tap test were compared using paired t-test. P-values of 0.05 or less were considered as statistically significant.INPH patient’s mean age was 67 years and 58.3% of them were male.We compared the MCA’s mean flow velocity of our INPH patient with the control group at the baseline. The mean flow velocity was nearly 43 cm/s in each group.Following CSF tap test, five INPH patients (41.7%) had clinical improvement as defined by at least one point reduction in INPH grading scale.Interestingly, we observed that in those who improved after CSF tap test, the baseline TCD parameters of the middle cerebral artery  were significantly higher compared to the control, and those parameters were decreased after tap test. Those changes were not statistically significant for anterior cerebral artery (ACA) as has presented in As shown in Our study showed that improvement in INPH grading score after CSF tap test might correlate with changing in TCD parameter in MCA and TCD parameter might be useful for shunt response in these patients.9 Mathew, and his colleagues recommended that assessing CBF changes could assist in both diagnosis and selection of patients for CSF shunting. They observed that regional CBF and velocity reduced in the territory of the ACA in INPH compared to dementia due to brain atrophy. In their study, CBF increased after lowering the CSF pressure by LP in patients with INPH. Patients with more increases in rCBR after lowering CSF pressure showed the most consistent clinical improvement after CSF shunting.10The hemodynamic abnormality in INPH patients is not a new findings. Greitz and his colleagues showed than CBF changes after CSF shunting in INPH patients.10After them, many studies also pointed out the correlation between changing in intracranial pressure (ICP) and CBFV. Increased CBF after shunting has been proposed as responsible for the clinical improvement in patients with INPH.11 This prospective study also shows that MCA blood flow decreases in INPH patients who improve after tap test. A small number of cases prevent a strong clinical pronouncement, but our results are sufficient in showing correlation between clinical improvement and higher MCA blood velocity in INPH patients.It is also previously showed that even moderate ICP changes could limit the modulation of CBF, and that revealed a direct interaction between the CSF space and the cerebrovascular compartment.12-14On the other hand, there are many studies that did not find any significant changes in cerebral hemodynamic after shunting in the INPH patients.15 Traczewski et al. also suggested that restoration of cerebral perfusion after CSF withdrawal was associated with a high likelihood of shunt success.3Our findings are more comparable to a study by Bakker and his colleagues. They showed similar results before and after shunting these patients and concluded that higher cerebral blood velocity before surgery in the INPH patients was related to clinical improvement.The pathophysiological mechanism underlying clinical improvement after CSF tapping in INPH has not been defined clearly yet. It may include restoration of regional blood flow, or metabolic improvement.14Chang has shown that microcirculation and pressure exerted on the nerve fibers in the frontal lobe is closely correlated with dementia in INPH.In our study it seems that, the MCA hemodynamic and INPH grading score are correlated. We have showed that, in INPH patients if MCA flow parameter does not change, his grading scale will also not too.1As said before many of our patients did not respond to CSF tap. In this group of patients, the TCD parameters also did not significantly change. Based on the Kondziella’s hypothesis, there is a certain “point of no return” in pathogenesis of INPH from CSF dynamics. This is probably the reason why despite restored CSF circulation by CSF tap or shunting many patients with chronic hydrocephalus still suffer from severe neurological deficits and did not improve.Our study suggests changing in CBF in patients with INPH after CSF tap may have a role in clinical improvement.Our study had many limitations:First, there are no accurate tests or diagnostic criteria to diagnose the INPH. The available diagnostic tools have limited sensitivity, and specificity and diagnosis is still largely based on measuring CSF dynamics.8Recently, the reliability of CSF tap test has been evaluated by Wikkelso et al. They reported that the positive predictive value the tests was >90% and the negative predictive value <20%. Overall accuracy of CSF tap test was 53%. They also concluded that the CSF tap test did not correlate with outcome in INPH after 12 months. According to them, CSF tap test can be used for selecting patients for shunt surgery but not for excluding patients from treatment.Second, nearly 10% of the patients had closed trans-temporal window that did not let us study intracranial artery by TCD.Finally, larger studies that include more INPH patients should be recommended on this topic.


Enhanced expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) is associated with the pathogenic processes of various tumor types. COX-2 and iNOS expression in the immunomodulatory dendritic cells is mediated by the granulocyte macrophage-colony stimulating factor (GM-CSF), which is also expressed by cervical cancer cells; however, whether and how GM-CSF regulates COX-2 and iNOS expression in clinical cervical cancer cells remain unknown. In this study, we found that the COX-2 and iNOS expression was upregulated in the cervical cancer tissues and positively correlated with cancer metastasis and stage. About one-half of the cervical cancer tissues showed strong/moderate GM-CSF expression, while the normal cervical tissues showed >80% positive rate; no GM-CSFR protein was detectable on the cervical cancer cells. The GM-CSF expression was negatively correlated with the COX-2 and iNOS expression in the cervical cancer tissues and the functional negative regulatory effect of GM-CSF on COX-2/iNOS expression was demonstrated in various cervical cancer cell lines. Therefore, in cervical cancer cells, GM-CSF might contribute an antitumor response by inhibiting iNOS and COX-2 expression in a GM-CSFR independent manner. Cervical carcinoma is one of the most common gynecologic cancers diagnosed and represents a substantial threat to the wellbeing and survival of the world's adult female population. While human papillomavirus (HPV) infection (both chronic and persistent) of the cervical epithelium cells is recognized as the major risk factor of cervical cancer, it is not sufficient for cancer occurrence, since most women with HPV infection do not develop cervical cancer , 2.β [The tumorigenic process of cervical cancer involves an array of immunomodulatory cytokines as well, including interleukins (IL) 6, 10, and 12 and transforming growth factor- (TGF-)β , 4, all β , 6, an iβ . In the β . Neverthβ , as wellβ , 10. HowHuman carcinomas are also associated with upregulation of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which are otherwise generally not expressed in normal (noncancerous) tissues, with the exception of placenta, kidney, and brain , 12. HumThe above evidence indicates that cervical cancer cells express GM-CSF and synthases COX-2/iNOS. Intriguingly, it has been reported that GM-CSF can upregulate COX-2 and iNOS expression in skin dendritic cells . However1, Ib2, IIa, or IIb cervical carcinoma stages). The study was approved by the Ethics Committee of Southwest Hospital for Clinical Investigation .Eighty-seven specimens of clinical stage Ib-IIb squamous cell carcinoma and 24 specimens of cervical intraepithelial neoplasia (CIN) were obtained from adult female patients  who had undergone radical hysterectomy with lymphadenectomy. Sixteen normal cervical tissues were obtained from women who had undergone hysterectomy for nonneoplastic indications and had normal findings from the last cervical smear. All specimens were obtained with written informed consent from each study participant. The tissue samples were fixed in 4% buffered paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin for histological analysis. The CIN and primary cervical cancer specimens were graded according to the WHO criteria  and the International Federation of Gynecology and Obstetrics (FIGO) criteria (Ibμm thickness) and mounted on glass slides for IHC analysis according to the published staining methods [α or β , rabbit anti-iNOS , and rabbit anti-COX-2 , followed by incubation  with the appropriate secondary antibodies. Staining with diaminobenzidine (DAB) tetrahydrochloride was performed to localize antigens, and the sections were further counterstained with Mayer's hematoxylin. After dehydration, a protective coverslip was manually mounted.The paraffin-embedded tissue samples were sectioned , SiHa, and Ca Ski (with both of the latter being HPV-16-infected) human cervical cancer cell lines were purchased from the China Center for Type Culture Collection . All cell lines were grown in RPMI 1640 medium  supplemented with l0% heat-inactivated fetal calf serum (HyClone) and 1% penicillin and streptomycin  at 37°C in a 5% CO5 cells/mL) and transfected using the Lipofectamine 2000 Reagent to introduce 5 nM of GM-CSF siRNA  or scrambled control siRNA . For both procedures, at 48 h postinoculation, the cells were harvested for Western blot analysis to verify the expression of hGM-CSF by using an anti-human GM-CSF antibody (R&D Systems) as the primary antibody.To establish overexpression of GM-CSF in the cervical cancer cell lines, the hGM-CSF-expressing plasmid pcDNA3.1(−)/GM-CSF (hereafter referred to as pGM-CSF) and the vector pcDNA3.1(−) were purchased from Invitrogen  and lipid-mediated DNA transfection  was performed. To silence the expression of hGM-CSF, the cancer cell lines were seeded in 6-well tissue culture plates , vector control (VT), and blank control (NT). A p value less than 0.05 was considered statistically significant.All statistical analyses were performed by using the SPSS statistical software for Windows . The chi-square test or Fisher's exact test was used for association analysis between immunostaining results and clinical characteristics. General linear regression model was used to determine possible correlations between GM-CSF negative/weak or moderate/strong expression and COX-2/iNOS protein expression in cervical cancer tissues. Unpaired Student's As shown in Although moderate/strong expression of GM-CSF was detected in 44 out of the 87 (50.6%) cervical cancer tissues examined, this rate was markedly lower than that in the normal cervical tissues (81.3%) . In addip values >0.05); however, both COX-2 and iNOS expressions were positively correlated with lymph node metastasis and FIGO stage (p < 0.05). No significant associations were found to exist between GM-CSF expression and any of the patients' clinical parameters, for either the squamous cell carcinoma or CIN patient groups (p > 0.05).Associations analysis showed that neither COX-2 nor iNOS expression was associated with patient age, tumor size, or HPV infection  and iNOS   .The Ca Ski cervical cancer cells were found to express a low level of endogenous GM-CSF, but the SiHa and Hela cells did not express any detectable endogenous GM-CSF . When tr GM-CSF gene led to undetectable GM-CSF protein in the Ca Ski cells, which otherwise expressed a low level of endogenous GM-CSF protein. As expected, transfection of the vector control did not influence the GM-CSF expression, as compared with that detected in the untreated Ca Ski cells ( GM-CSF, neither on GM-CSF expression nor iNOS and COX-2 expression (Figures Transfection of the cervical cancer cells lines with a specific siRNA sequence targeting theki cells . The SiH Figures .Cervical cancer cells express synthases COX-2/iNOS and GM-CSF , 13–15, κB pathway [Several previous lines of evidence obtained from studies involving patients with cervical cancer have implied the clinical significance and biologic roles of both iNOS and COX-2 , 22–25.  pathway . Similar pathway  showed aα, IL-6, or IL-1β results in upregulation of iNOS [κB [κB pathway [The relationship between GM-CSF and iNOS and COX-2 expression in immune cells is well documented. For instance, incubating peripheral blood mononuclear cells from a healthy donor with GM-CSF alone or in combination with the cytokines VEGF, TNF- of iNOS . In addiiNOS [κB , while i pathway . However pathway , to clea in vitro study may not fully reflect the in vivo effects of HPV infection on the expression of the related factors, which may explain why we did not observe any correlation between HPV infection and the expression of GM-CSF, iNOS, or COX-2 in our clinical cervical cancer tissue samples. Moreover, our finding of no HPV correlation is supported by observations from Sarian et al. [The occurrence of cervical carcinoma is closely related with HPV infection, and HPV may play an important role in development of the disease. It has been reported that HPV18 infection is associated with poor prognosis for patient survival and accelerated tumor growth . Specifin et al. , who demThis study shows that tumor-derived GM-CSF might elicit an antitumor response in cervical cancer by inhibiting the iNOS and COX-2 expression in cervical cancer cells in a GM-CSFR-independent manner. These results suggest that GM-CSF might be a helpful adjuvant that will aid in the design of a novel cervical cancer vaccine. Further studies are necessary to explore the detailed mechanism by which GM-CSF regulates iNOS and COX-2 expression in cervical cancer cells to support the potential applications of GM-CSF in clinical cervical cancer patients.


Quantitative Bioluminescence Resonance Energy Transfer (qBRET) measurements are the gold standards to prove the direct physical interaction between the protomers of presumed GPCR dimers. For the correct interpretation of these experiments, the expression of the energy donor Renilla luciferase labeled receptor has to be maintained constant, which is hard to achieve in expression systems. To analyze the effects of non-constant donor expression on qBRET curves, we performed Monte Carlo simulations. Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state. We suggest here a new approach to the analysis of qBRET data, when the BRET ratio is plotted as a function of the acceptor labeled receptor expression at various donor receptor expression levels. With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous. The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system. We used this new method to investigate the dimerization of various GPCRs, and our data have confirmed the homodimerization of V G Protein Coupled Receptors (GPCRs) were thought to be monomeric entities for a long time, but results of the last two decades indicate that they can form dimers or higher ordered oligomers A wide range of approaches has been used to prove the direct physical interactions between the protomers of a presumed dimer. While some elegant new methods, such as analysis of receptor mobility In qBRET experiments the protomers of the presumed dimer are labeled with the energy donor Renilla luciferase (Rluc) and an energy acceptor fluorescent protein, respectively. Although the energy donor in BRET is the oxidation of coelenterazine h, the substrate of Rluc, for simplicity we will refer to Rluc as the energy donor in this article. The measured energy transfer is highly sensitive to the distance between donor and acceptor, so BRET ratio  can be excellently used to monitor protein-protein interactions, such as dimerization. BRET is ideal to measure changes of protein-protein interactions  and Stratagene . The cDNA of the human arginine vasopressin receptor 2 was purchased from S&T cDNA Resource Center . Cell culture dishes and plates for BRET measurements were purchased from Greiner Bio-One GmbH . Cell culture media, Lipofectamine 2000 and coelenterazine h were purchased from Invitrogen . Rapamycin was obtained from Merck . HEK293 cells were from American Type Culture Collection .2 vasopressin receptor , first the receptor sequence was amplified from cDNA clone, purchased from S&T cDNA Resource Center . Then the receptor sequence was subcloned using EcoRI and AgeI restriction enzymes into pEYFP-N1 vector containing super Renilla luciferase 1a angiotensin receptor , human β2 adrenergic receptor  and rat CB1 cannabinoid receptor  were previously described 2 angiotensin receptor  was created by exchanging the sequence of eYFP in AT2R-YFP To create RLuc and Venus tagged human V2 at 37°C. Cells were cultured in plastic dishes and were trypsinized prior to transfection. For transient transfection, cells were plated on poly-lysine-pretreated white 96-well plates at 0.75×105 cells/well densities with 0.5 µg/well total plasmid and 0.5 µl/well Lipofectamine 2000.The experiments were performed on Human Embryonic Kidney 293 (HEK293) cells. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% bovine serum, 100 µg/ml streptomycin and 100 IU/l penicillin in 5% COassociation probability depending in the type molecules. Dimers could dissociate in each time step with a probability pdissociation, also depending on the protomers of the dimer. Simulation was performed for 1000 time steps, and BRET values were calculated. Simulated BRET values were calculated as total neighboring donor-acceptor pairs/number of donor molecules. Each simulation was repeated five times. Simulations were written in Python 2.7. Source code is available online.Simulations were performed on a 2 dimensional membrane lattice containing 100×100 hexagons with periodic boundary conditions. Monomers of donors and acceptors were randomly placed on the empty hexagons of lattice. Total number of molecules was varied from 200 to 2000. The molecules were moved in each simulation step. For each monomer molecule a random neighboring hexagon was selected to move to. If the selected hexagon was occupied with another molecule, move was rejected, and not repeated. For moving dimers, both protomers had to arrive onto a free hexagon, and falling apart of dimers was not allowed during a move step. Generally, dimers were allowed to move to the same direction and/or rotate around each other. Besides moving, monomers could form dimers and dimers could fall apart into monomers in each simulation step. For each monomer molecule one neighbor was randomly selected (if there was any), and the two molecules could form a dimer with a p2, 0.7 mM MgSO4, 10 mM glucose and 10 mM Sodium-Hepes, pH 7.4. Measurements were performed in a Varioskan Flash Multimode Reader . Venus fluorescence  was measured at the beginning of each experiment. Coelenterazine h was added at 5 µM final concentration, and total luminescence, luminescence at 485 nm and luminescence at 530 nm were recorded. Intensity ratio was defined as Venus fluorescence/total luminescence. Intensity ratio does not show directly the acceptor/donor ratio, but is proportional with it. From the signals of cells expressing the same amount of RLuc and Venus labeled construct, we approximated that the 1.0 intensity ratio represents an acceptor/donor ratio 1∶1. BRET ratio was calculated as Emission530/Emission485. The BRET ratio of only donor expressing cells was subtracted from the BRET ratio of measured points. In the inducible dimerization experiments, 100 nM rapamycin or vehicle DMSO was added to the cell medium 30 minutes prior the measurements.BRET measurements were performed 24–28 hours after transfection. Medium of the cells was changed to a modified Krebs-Ringer buffer containing 120 mM NaCl, 4.7 mM KCl, 1.2 mM CaClFigures, curve fitting and statistical analysis were performed in GraphPad Prism. All measurements were repeated from n = 3 to 8 (different transfections), and pooled data were plotted. For GPCR dimerization experiments slope of the linear regression for different GPCR pairs was determined for low and high donor expressions, and the difference between slopes was determined by analysis of covariance (ANCOVA). *** indicates significant (p<0.001) difference of slope between the low and high amount donor expressing cells.50 value  is used to determine the presence of dimerization: in experiments conducted with one donor and different acceptor labeled receptors, pairs with low BRET50 value thought to form oligomers, while high BRET50 values indicate weak interaction or the absence of interaction between the investigated receptors. To correctly interpret qBRET curves, it is necessary to hold constant the donor labeled receptor expression, independent of the expression of acceptor labeled receptors  with type I angiotensin receptor (AT1R), β2 adrenergic receptor (β2AdR), CB1 cannabinoid receptor (CB1R) and V2R in transiently transfected HEK293 cells. We also used cytoplasmic expressed Venus protein as a negative control. Cells were co-transfected with constant amount of Renilla luciferase labeled V2R (V2R-RLuc) and increasing amount of the partner receptor labeled with Venus fluorescent protein . To maintain transfected plasmid amount constant, empty pcDNA3.1 plasmid was added. The calculated BRET ratios were plotted as a function of intensity ratio , proportional with acceptor labeled receptor expression/donor labeled receptor expression  dependent of the types of the molecules. Dimers had a probability (pdissociation) to fall apart in each simulation step, which is dependent on the type of the dimer. After 1000 time steps, simulated BRET ratios were calculated based on the number of neighboring donor – acceptor pairs (donor acceptor pairs could be formed by dimerization or by randomly moving a donor and an acceptor molecule to neighboring hexagons). To specify the type of interaction between donor and acceptor molecules we used different passociation and pdissociation values (2 = 0.95), while this was not possible for non-specific interaction (R2 = 0.49) (black dashed curve).To analyze the effect of non-constant donor level on qBRET curves, we performed Monte Carlo simulations. To simulate dimerization, we used the model of Lindermann et al. n values . A graphn values , whereasn values . In the n values  shows thn values  the BRET50 values in Type II plots . In this way, we could reach a wide range of different donor and acceptor expression levels . Measure2 = 0.89), but could not make this for non-specific interaction (R2 = 0.23) in the Type II plots (black dashed curve).The experimental results were in good agreement with the modeling data generated by Monte Carlo simulations. Type I plot shows that in the case of non-specific interaction, the BRET ratio is only dependent on the expression of acceptor labeled construct . HoweverBased on our simulation and experimental results, we analyzed the dimerization of different GPCRs by a modified version of classical qBRET experiments. Instead of trying to maintain constant expression of the donor labeled receptor, we varied the transfected amount of donor and acceptor labeled receptor as well, to reach a wide range of different donor – acceptor expressions. Based on our results, to distinguish specific and non-specific interaction, it is more appropriate to plot BRET ratio as a function of measured fluorescence (acceptor expression) see s. In the2R and CaSR calcium sensing receptor  with AT1R, type II angiotensin receptor (AT2R), β2AdR, CaSR, CB1R and V2R. HEK293 cells were transfected with increasing amount of donor (V2R-RLuc or CaSR-RLuc) and acceptor  labeled receptor coding plasmid. Empty pcDNA3.1 plasmid was added to reach constant total transfected DNA amount. Measured points were sorted into low and high luminescence groups based on the measured total luminescence, similarly to our inducible dimerization experiments (2R- β2AdR (left panel) and V2R-V2R (right panel) interaction. In the case of V2R- β2AdR interaction, the Type I plot shows flat linear relationship; where the donor expression does not influence the slope of the linear regression, indicating the non-specific nature of this interaction. In contrast, in the case of V2R-V2R interaction, the curve is steeper, and the increase of donor expression leads to flattening of the curve, showing the homodimerization of V2R. We performed the same analysis for all of the investigated receptor pairs . The analysis of qBRET curves supposes that with the increasing acceptor expression, the donor expression does not change , but not for non-specific interaction (R2<0.5). However, for the reasons discussed above, this analysis needs a larger amount of data points with ideal acceptor-donor distribution  fit suggests specific interaction, while non-specific interaction results in a worse (R2<0.5) fit. To investigate the performance of these two methods, we performed additional Monte Carlo simulations. Simulations were ran with random acceptor and donor amount (in the [100∶1000] range) and with simulation parameters for non-specific interaction. After the simulation a random Gaussian noise term was added to the acceptor, donor and BRET values to further approximate our experimental setup. Simulations were performed for n = 100 (A) or n = 20 (B) data points. When data sample is sufficiently large (A) both methods showed the non-specific nature of interaction. However, with smaller sample size, but still with a wide range of different donor-acceptor amounts , it is possible to get such a distribution of data points, where in Type II plots  a reasonable saturation curve can be fitted (suggesting specific interaction). In this case Type I plot still shows correctly the non-specific nature of this interaction. Based on these data, we think that when the data sample is not very large (<100 points), Type I plots can better differentiate between specific and non-specific interaction.Type I plots outperform Type II plots in detecting non-specific interaction: Based on our simulations and inducible dimerization experiments, we propose two different analysis methods for qBRET experiments. Both methods require a wide range of different levels of donor and acceptor expressions (left panels). In the first method  BRET ratio is plotted as a function of acceptor expression, and the difference between the slope of linear regression for points with low and high donor amount is investigated. In the second method  BRET ratio is plotted as a function of acceptor/donor ratio, and one-site specific binding curve is fitted on the whole data. Good (R(PDF)Click here for additional data file.Figure S2Fluorescence-Luminescence, Type I and Type II plots of GPCR dimerization experiments: HEK293 cells were transfected with various amounts of different donor and acceptor coding plasmids. Measured points were sorted into low/high luminescence groups based on the total measured luminescence . Fluorescence-Luminescence (left), Fluorescence-BRET ratio (middle) and Intensity ratio-BRET ratio (right) plots were created for different donor-acceptor pairs. Summary of this plot can be found in (PDF)Click here for additional data file.


Autophagy is demonstrated to be involved in acute ischemic stroke(AIS), which, however, is confined to cells and/or animals levels. The aim of this study was to determine two autophagy biomarkers, Beclin1 and LC3B, in cerebrospinal fluid (CSF) and serum of patients with AIS, and to evaluate a possible correlation between levels of Beclin1 and LC3B and severity of neurological deficit and clinical outcome of stroke patients.Levels of Beclin1 and LC3B were quantified by ELISA in CSF and serum collected from 37 AIS patients and 21 controls. The clinical severity at stroke onset was determined by the National Institute of Health Stroke Scale (NIHSS) and the neurological outcome was determined by the Modified Rankin Scale (mRs) and the improvement in NIHSS between stroke onset and 3 months later. Associations between autophagy biomarkers and infarct volume, NIHSS and mRs were assessed using Pearson analysis.The levels of Beclin1 and LC3B were increased both in CSF and serum of AIS patients relative to controls. In CSF, they were positively correlated with infarct volume and NIHSS scores, and negatively correlated with mRs scores, but no significant association was observed in serum. Moreover, AIS patients with higher levels of Beclin1 and LC3B in CSF had significantly higher improvement in NIHSS.CSF and serum levels of autophagy biomarkers are altered in AIS patients. CSF levels of autophagy biomarkers are associated with infarct volume, clinical severity of and neurological outcome. Acute ischemic stroke (AIS) is a major cause of death and disablement all over the world . DespiteRecent studies have revealed that autophagy may play an important role in ischemic stroke , 10. ManThe present study was designed to investigate the role of autophagy in AIS patients, with Beclin1 and LC3B used as markers of autophagy. Our study showed that levels of Beclin1 and LC3B greatly increased both in CSF and serum of patients with AIS. More interestingly, we found that Beclin1 and LC3B in CSF were correlated with infarct volume, clinical severity and neurological outcome.This study was performed in Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University from Mar. 2013 through Dec. 2014. To accurately evaluate the effect of autophagy on acute ischemia injury, the patients who met the following criteria were included: (1) the first ever stroke, without history of cerebrovascular disease; (2) within 24 h after onset; (3) age ≥18 years; (4) evidence of previous infarct lesion by brain computed tomography (CT) or magnetic resonance (MR); (5) willing to sign a informed consent document. The exclusion criteria were as follows: (1) concomitant systemic disease such as cardiovascular disease; (2) concomitant malignant disease; (3) history of diabetes; (4) history of surgery or trauma recently; (5) with autoimmune disease or treated with hormone or immuneinhibitors; (6) with neurological degenerative disease, such as Alzheimer’s disease, Parkinson’s disease, motor neuron disease, and multiple system atrophy; (7) receiving thrombolysis treatment. Forty-two AIS patients were included in the experimental group, and 5 patients were excluded due to the loss to follow-up when we assessed the mRS and NIHSS scores 3 months later after the stroke.24h − NIHSS3 months [Detailed neurological examination and brain magnetic resonance imaging (MRI) 3.0T with diffusion weighted images (DWI) were performed in all AIS patients. Acute cerebral infarction was defined as an area of high signal intensity on the DWI. The edge of each infarct lesion was draw by the manual approach and the total infarct volume of each patient was calculated automatically by software Volume Viewer 2  as previously described , 36. To 3 months . The MRIThe control subjects were selected from 21 age- and gender-matched patients with no central nervous system (CNS) disease, including psychoneurosis (n = 15), benign paroxysmal positional vertigo (n = 3), trigeminal neuralgia (n = 1), hypokalemic periodic paralysis (n = 1) and progressive muscular dystrophy (n = 1).This study was approved by the Ethical Committee of the Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University and conducted in accordance with the principles of the Declaration of Helsinki. All the participants were informed about the study and agreed to participate by signing an informed consent form.CSF and peripheral blood samples of patients with AIS were collected within 10–24 h of the onset of neurological symptoms. CSF samples were taken during lumbar puncture between the L3/4 or L4/5 intervertebral space using a 25-gauge needle and held immediately on ice. Peripheral blood samples were collected by venepuncture in heparinized tubes and stored on ice. The CSF samples were centrifuged at 800 rpm for 5 min at +4 °C, and the blood samples were centrifuged at 3000 rpm for 15 min at +4 °C. All samples were stored at −80 °C until they were analyzed.Concentrations of two autophagy biomarkers, Beclin1 and LC3B, in CSF and blood were measured by commercial enzyme-linked immuno sorbent assay (ELISA) kits following the manufacturer’s instructions . Briefly, for Beclin1 and LC3B evaluation CSF and serum samples were diluted 5 times. The optical density (OD) was measured by Microplate reader  at a wavelength of 450 nm. A standard curve linear regression equation was calculated according to standards’ concentrations. Then, the concentrations of Beclin1 and LC3B in CSF and serum were calculated by the regression equation.t-test for continuous variables or Chi square test for dischotomous variables. The Mann–Whitney U Test was used to compare the autophagy biomarkers between the AIS patients and controls. Differences in good outcome group and poor outcome group assessed by the improvement in NIHSS were evualted by the Student’s t-test. Pearson’s correlation test was used to assess the linear dependence between autophagy biomarkers and infarct volume, NIHSS and mRS. P <0.05 were considered to be statistically significant.All statistical analyses were performed by SPSS Statistics, version 19.0 software . All graphs were generated by GraphPad Prism version 5.00 . All data were expressed as mean ± standard deviation (SD). Differences in characteristics between the AIS patients and controls were evaluated using the Student’s Patients were enrolled between Mar. 2013 and Dec. 2014. From the 42 AIS patients who were primarily included, 5 patients were excluded due to the loss to follow-up. Thus the final analysis was conducted on 37 AIS patients and 21 control subjects. Their demographic clinical characteristics are presented in Table In the observed population, samples were collected within 10–24 h of stroke onset. Firstly, we analyzed the change of levels of Beclin1 and LC3B both in CSF and serum of AIS patients. Concentrations of Beclin1 and LC3B in CSF and serum of AIS patients were significantly higher than that of control subjects  was selected as the second outcome measures. ΔNIHSS ≥3 was considered to be good outcome and ΔNIHSS <3 was considered to be poor outcome. As a result, 19 cases of AIS patients gained good outcome and 18 cases gained poor outcome. We found that levels of Beclin1 and LC3B in CSF were significantly higher in good outcome patients than in poor outcome patients  and LKB1 kinases, therefore inducing autophagy [In CNS, autophagy can be activated by cerebral ischemia/hypoxia, nutrient deprivation, oxidative stress, energy crisis and neurotoxins –44. The utophagy . Howeverutophagy . ConsistIn this study, levels of Beclin1 and LC3B in CSF were positively correlated with infarct volume and severity of neurological deficit, whereas this relation was not observed in serum. This is likely to be due to: First, the autophagy biomarkers in serum can not accurately reflect their levels in brain, and they may be affected by other organs of bodies when stroke takes place. Second, the sample size in this study was small.More importantly, increased concentrations of Beclin1 and LC3B in CSF were found to be associated with good outcome, suggesting autophagy plays a protective role in AIS. Accumulating evidence suggests that autophagy enhanced functional recovery after stroke. It was repeatedly demonstrated that autophagy played a neuroprotective role in ischemic stroke. Urbanek et al. reported that rapamycin can effectively prevent neuro damage by induction of protective autophagy . AutophaThere were some limitations in the present study. First, it is difficult to determine concentrations of Beclin1 and LC3B in CSF from normal persons as controls. Second, because of ethical concerns, our study was restricted to CSF and serum within 10–24 h of AIS, lacking of kinetic observation of alternations of Beclin1 and LC3B. Despite the limitations of the present study, our findings provide new evidence that autophagy was involved in AIS and was associated with clinical outcome.In summary, we observed that autophagy biomarkers in CSF and serum levels of AIS patients were increased and that levels of Beclin1 and LC3B in the CSF were associated with good clinical outcome, implicating a thorough involvement of autophagy in ischemic injury and suggesting a bright intervention target in AIS treatment.


Donor cell leukemia (DCL) represents a rare complication of allogeneic transplantation. The precise incidence remains unclear, though it may be higher following umbilical cord blood transplants. Here, we present an unusual case of a patient with B-ALL who presented with a donor derived myeloid sarcoma of the heart following a double cord blood transplant. To our knowledge, it is the first case of sarcomatous or chloromatous presentation of DCL following a UCBT. Donor cell leukemia (DCL) represents a rare complication of allogeneic transplantation. It was first recognized as a distinct entity in 1971 by Fialkow et al. .The first two cases of donor cell leukemia after umbilical cord blood transplant (UCBT) were reported, more recently, in 2005 , 4. AgaiHere, we present an unusual case of a patient with prior history of B acute lymphoblastic leukemia (B-ALL) who presented with a donor derived myeloid sarcoma of the pericardium following a double cord blood transplant. To our knowledge, this is the first case of a sarcomatous or chloromatous presentation of DCL following UCBT.A 41-year-old man was admitted to our hospital 567 days after a double UCBT for B-ALL with t. His treatment history in brief is as follows. After cycle 3A of Hyper-CVAD, he developed symptoms concerning relapse. A bone marrow biopsy confirmed B-ALL. His regimen was changed to Augmented Hyper-CVAD. A repeat bone marrow biopsy was negative for disease.2, and 200 cGy TBI (Cy/Flu/TBI). GVHD prophylaxis included CSA and MMF. After transplant, bone marrow (BM) and peripheral blood (PB) chimerism was 100% donor.Given his relapse during treatment, the patient was referred for bone marrow transplant (BMT) evaluation. He was HLA typed and had neither a sibling nor a suitable volunteer unrelated donor match. Thus, he underwent a double UCBT. He received reduced-intensity conditioning prior to his UCBT with cyclophosphamide 50 mg/kg, fludarabine 200 mg/mHis posttransplant course was complicated by graft-versus-host-disease (GVHD) of skin and the gastrointestinal tract. The GVHD was managed with a brief course of steroids and cyclosporine. Bone marrow and peripheral blood chimerism was 100% donor.2 for 10 days and responded well to the treatment. A bone marrow biopsy performed 8 weeks after the completion of the cycle revealed no evidence of AML. Additional cycles of treatments were held secondary to infectious complications.At approximately day 515 after transplant he was noted to be thrombocytopenic and 8% blasts were reported on peripheral blood. A bone marrow biopsy was performed, which revealed a donor derived acute myeloid leukemia (AML), with 22% blasts showing an immature myeloid phenotype by flow cytometry , negative for CD10 and CD19, both of which had been expressed in the prior B-ALL. His peripheral blood and bone marrow chimerism remained 100% donor. FISH was negative for t. He received 1 cycle of decitabine at 20 mg/mHe was readmitted to the hospital on day 567 after transplant with fever, cough, pleuritic chest pain, and tachycardia. A chest X-ray showed small pleural effusions. He was noted to have EKG changes with T-wave inversions in leads V3–V5 . He had Histopathological evaluation revealed an infiltrate of atypical cells, seen in sheets and clusters throughout the pericardium and focally within adjoining adipose tissue. By immunohistochemistry, the atypical cells showed an immature myeloid/monocytic immunophenotype: CD34 focal+, CD43+, MPO focal+, CD117 focal+, CD68 weak+, CD163 focal+, PAX5−, CD56−, TdT−, and CD79a focal weak+ .Chimerism studies of the biopsied tissue revealed that the lesion was entirely donor derived. A bone marrow biopsy showed normal hematopoiesis with full donor chimerism. Thus cardiac myeloid sarcoma represented an isolated site of disease.Unfortunately, the patient continued to clinically deteriorate. He developed multiorgan failure requiring vasopressor support, hemodialysis, and an intra-aortic balloon pump. He expired on day 579 after transplant.The umbilical cord blood registry was contacted and the donor was reported to be in good health with no evidence of leukemia.Donor cell leukemia (DCL) is a rare complication following allogeneic transplants, with an estimated incidence of 0.1% . Acute mThe pathogenesis of DCL remains unknown at this time. The most basic explanation is that the graft harbors occult malignant cells which progress into clinically overt disease after the transplant. In a systematic screening of unselected cord blood samples, approximately 1% of the sampled products harbored small clones positive for TEL-AML1 and 0.2% harbored AML-ETO . HoweverThe extreme proliferative demands on transplanted cells following transplantation may further drive this process. The rapidity of proliferation may increase the probability of replication errors and leukemogenicity , 8. Stem MLL gene rearrangements [Another theory suggests that the residual effects of conditioning chemotherapy may damage the graft and cause a therapy related neoplasm as is classically associated with topoisomerase II inhibitors and alkylating agents. In a review by Wiseman, 96% of the patient with DCL had been exposed to either of these two classes of chemotherapeutics during conditioning and 47% had cytogenetic abnormalities consistent with t-AML including −7 andngements , 11.Not surprisingly, given the comparatively recent introduction of UCBT, fewer cases of DCL leukemia have been reported in the literature when compared with peripheral blood (PBSCT) or bone marrow transplants (BMT). Despite this young history, some interesting trends have emerged. AML predominates as the most common type of UCBT DCL, whereas AML and ALL are similarly represented in DCL following BMT or PBSCT. There also appears to be a shorter interval to the development of DCL in UCBT, with the median time to development of DLC being 14.5 months for UCBT versus 36 months for BMT or PBSCT. Abnormal karyotypes, particularly monosomy 7, are much more common in UCBT DCL than in BMT or PBSCT .Perhaps, UCB is particularly sensitive to residual leukemogenic effects of conditioning chemotherapy. The benefit of time and further research will likely provide more information about the true incidence and mechanism of DCL in UCBT. Given the predominance of AML type DCL in UCBT, it is possible that in the future we may also see more cases of donor myeloid sarcoma, as was the case with our patient. Improved molecular testing, including VNTR, will allow us to detect this entity with greater precision and avoid mistakenly categorizing this phenomenon as relapsed disease.Donor cell leukemia (DCL) represents a rare complication of allogeneic transplantation.The incidence may be higher following cord blood transplants.Here, we present an unusual case of a patient with B-cell acute lymphoblastic leukemia (B-ALL) who presented with a donor derived myeloid sarcoma of the heart following a double cord umbilical blood transplant (UCBT).Acute myeloid leukemia (AML) is the most common type of UCBT DCL.There is a shorter interval to the development of DCL in UCBT compared to bone marrow or peripheral blood stem cell transplantation and abnormal karyotypes are more common in UCBT DCL.UCB may be uniquely sensitive to leukemogenic effects of conditioning.Improved molecular testing will allow precise detection and avoid mistakenly categorizing DCL as relapsed disease.