Circulating tumour cells (CTC) in patients with metastatic carcinomas are associated with poor survival and can be used to guide therapy. Classification of CTC however remains subjective, as they are morphologically heterogeneous. We acquired digital images, using the CellSearch™ system, from blood of 185 castration resistant prostate cancer (CRPC) patients and 68 healthy subjects to define CTC by computer algorithms. Patient survival data was used as the training parameter for the computer to define CTC. The computer-generated CTC definition was validated on a separate CRPC dataset comprising 100 patients. The optimal definition of the computer defined CTC (aCTC) was stricter as compared to the manual CellSearch CTC (mCTC) definition and as a consequence aCTC were less frequent. The computer-generated CTC definition resulted in hazard ratios (HRs) of 2.8 for baseline and 3.9 for follow-up samples, which is comparable to the mCTC definition . Validation resulted in HRs at baseline/follow-up of 3.9/5.4 for computer and 4.8/5.8 for manual definitions. In conclusion, we have defined and validated CTC by clinical outcome using a perfectly reproducing automated algorithm. The recorded fluorescence images of CK-PE, DNA-DAPI, CD45-APC and a debris-fluorescein (FITC) channel are segmented on the basis of being positive for CK-PE and DAPI and are then presented to a trained reviewer for identification of CTC that are CK-PE positive, CD45-APC negative, ≥4 µm in diameter, DAPI-positive, and have a cell-like morphology. This manual procedure is laborious, time-consuming and can be highly subjective. For example, others have described an inter-reviewer variation in manual CTC enumeration of 4% to 31% (median 14%) In recent years, several studies have reported that a change in circulating tumour cell (CTC) count could indicate whether a therapy for advanced cancer is effective Here we present the results of a new approach to identify CTC in images captured by the system in samples from castration-resistant prostate cancer (CRPC) patients. We recorded images before treatment (baseline samples) and from a follow-up sample taken 2–6 weeks after start of therapy. Our hypothesis was that using survival data as the only training parameter, an automated algorithm could be optimized to define and automatically count CTC with the same fidelity as the manual CellSearch method (mCTC). This algorithm needs to identify automated CTC (aCTC) candidates, characterize them and compare the candidates to a range of known features. Replacement of manual CTC counting with an automated method would significantly reduce cost and importantly, eliminate inter- and intra-laboratory variation that could be clinically important in cases with low CTC counts. Moreover, a consensus definition for what constitutes a CTC is urgently required Development of image analysis algorithms for automated CTC enumeration was performed on stored images from ten CellSearch systems  from patients participating in the prospective IMMC-38 study (NCT00133900) and healthy individuals participating in the IMMC-06 study (NCT00133913) were available All patients had histologically confirmed prostate adenocarcinoma, castrate levels of testosterone (<50 ng/ml) and progressive disease as defined by three consecutively rising PSA values #,6-diamidino-2-phenylindole (DAPI). This enriched sample is transferred to a magnetic cartridge where all ferrofluid labeled objects are pulled towards an analysis surface. The entire analysis surface is imaged by the CellTracks Analyzer II, a four-color semi-automated fluorescence microscope that captures digital images for four different fluorescent dyes using a 10X/0.45NA objective. In addition to the DAPI, PE and APC images, a fourth fluorescence channel (emission 535±25 nm) is imaged as a control channel for exclusion of auto-fluorescent debris. This channel will be termed “FITC” channel. Per cartridge, a whole scan consists of 144–180 4-layer tiff images that are saved for each patient. After imaging by the Celltracks analyzer, the software selects objects that are DNA and CK positive and presents them to an operator in a thumbnail gallery. The operators are trained to reviews these galleries to select the mCTC among the objects. An mCTC is positive for DNA and CK, is negative for CD45, is larger than 4×4 µm and has morphological features that are consistent with those of a cell.The CellSearch system was used to isolate and image EpCAM+ objects. The CellSearch system consists of a CellTracks Autoprep for sample preparation www.diplib.org). An outline of the method is given below and shown in CDs containing up to 180 archived four channel tiff images for each sample belonging to the respective studies were collected for import to a central hard drive. Objects were detected and classified using an automated algorithm developed in Matlab 2009a  using the DIPimage toolbox  and GraphPad Prism v5.After algorithm optimization, all patient samples were processed by the algorithm and the training and validation patient groups were dichotomized on cut-off values ranging 1–10 of aCTC and mCTC to derive HR and median overall survival (OS) for baseline and first follow-up samples. Furthermore, a linear regression was performed for comparison between the aCTC and mCTC count. Classifiers with reduced features were tested to determine the impact of each feature. Pearson coefficient of determination R2), a DAPI-DNA peak value >170 counts and a CD45-APC peak value <60 counts. For every patient sample, the objects meeting these inclusion criteria were added up to arrive at a final aCTC count per patient. The mCTC count was performed by trained reviewers. Time needed for preparation of images for mCTC assignment was similar to the time needed for complete aCTC enumeration; both took 5 minutes. However, enumeration of these mCTC by a human operator takes an additional 8 minutes per sample .The classifier resulting in the optimal aCTC definition that most strongly associated with high HR for baseline and follow-up samples was chosen. The features that most strongly associated with OS were: a CK-PE standard deviation >50 counts, a size range of 75–500 pixels . In the follow-up samples aCTC counts ranged from 0 to 870  compared to mCTC counts of 0 to 545 . The R2 was 0.67 . After all the objects meeting the criteria of the optimal classifier were summed for each patient sample, the aCTC count was compared with the mCTC count. In the baseline samples the aCTC counts ranged from 0 to 3384  compared to mCTC counts of 0 to 5925 . The R2 between aCTC and mCTC was 0.78. Between aCTC and the objects found with the classifiers without CD45 and DAPI the R2 were 0.95 and 0.82, respectively. Between the aCTC and TMP definition, the R2 was 0.56 .To arrive at a clinically relevant cut-off value for aCTC comparable to the mCTC cut-off of 5 used in routine clinical practice, we used the linear regression slope of 1.33 between aCTC and mCTC baseline measurements. This resulted in a cut-off of 4 aCTC. In the scatter plot of Kaplan-Meier plots were generated for 185 baseline and 185 follow-up samples using the standard cut-off value of 5 for mCTC and the cut-off value for aCTC of 4. 2 between aCTC and mCTC was 0.28 . Exclusion of a single outlier resulted in a R2 of 0.90 . In the follow-up samples the aCTC counts range was 0–2490  and a range of 0–3573  with mCTC. R2 between aCTC and mCTC both was 0.83 .To validate the aCTC count an independent data set was used from 100 metastatic prostate cancer patients treated with abiraterone acetate. The set included 93 baseline samples 96 follow-up samples. In the baseline samples the aCTC counts range was 0–1258  and a range of 0–1108  was found for mCTC. RKaplan-Meier plots were generated for 93 baseline and 96 follow-up samples using the standard CTC cut-off of 5 for mCTC and 4 for aCTC. This is the first report of an algorithm-based automated method for unbiased determination of a clinically significant definition for what constitutes a CTC. We used stored images recorded by the CellSearch system from 185 patients with metastatic CRPC. While the algorithm was developed using patients with metastatic CRPC receiving a cytotoxic agent (training cohort), validation on patients receiving the highly active hormonal agent abiraterone acetate  confirmed reproducibility of the enumeration algorithm.For the purpose of the development of the CTC classifier, patients were divided into two groups based on the median aCTC count in the training set of 185 baseline samples. This division was chosen to minimize statistical error in the HR. The median count for the chosen aCTC classifier on the baseline samples was 5 aCTC and resulted in a HR of 3.1. The current standard CellSearch method presents the reviewer with CK+ DAPI+ objects for classification: a threshold of 5 or more mCTC is used to discriminate between patients with a favourable versus an unfavourable prognosis. In an earlier study we reported that this threshold of 5 mCTC could be mainly attributed to error introduced by human interpretation 2 between TMP and aCTC was 0.56 and TMPs were also present in the control group (see Although one would expect that counting tumour related events that occur at a higher frequency -such as TMPs 2 0.28) due to one outlier. For this outlier, the algorithm counted 1258 aCTC, whereas the operator only counted 67 mCTC. Closer inspection of this sample revealed that this sample had a very high density of cells. This resulted in an overestimation of the number of CTC by the algorithm and an underestimation of the number of CTC by the human operator. Kaplan-Meier plots of baseline and follow-up using aCTC and mCTC from the validation set illustrated in The aCTC definition was validated using an independent data set. This validation set showed that the classifier performs well with equivalent HRs to those obtained with mCTC. Correlation with mCTC was quite low (RThe CellSearch system is the first and currently the only clinically validated method for CTC enumeration. The system was introduced in 2004 and its initial users were well-trained clinical researchers. The need for CTC counts in the clinic to manage patients with metastatic disease is however rising quickly and is accompanied by a need for simplification, higher reproducibility and a reduction of time needed to obtain a result, i.e. cost reduction. The introduction of aCTC addresses these issues as the need for extensively trained reviewers is eliminated, the algorithm is perfectly reproducible and no operator time is needed to review the images.In conclusion, we have identified and validated a definition for CTC using an unbiased, automated algorithm that confirms that CK+DAPI+CD45- cells are the EpCAM positive events most strongly associated with survival. Moreover, automated counting of CTC using our classifier compares favourably to manual counting using the CellSearch system.


HER2/neu, a member of the epidermal growth factor receptor family, has been shown to be over-expressed in some tumors. The purpose of this study was to determine the salivary levels and tissue expression of HER2/neu in patients with head and neck squamous cell carcinoma (HNSCC) and their correlation with clinicopathologic parameters.Enzyme-linked immunosorbent assays (ELISA) were used to evaluate the salivary levels of HER2/neu and immunohistochemistry was used to measure tissue expression of HER2/neu in 28 patients with HNSCC and 25 healthy control subjects.The salivary levels of HER2/neu in patients with HNSCC were not significantly higher compared to healthy control subjects. There was no apparent correlation between salivary HER2/neu levels and clinicopathological features such as age, sex, tumor grade, tumor size and nodal status. All HNSCC specimens were positive (membranous or/and cytoplasmic) for HER2/neu, except one sample. Only one HNSCC specimen showed staining purely in the tumor-cell cytoplasm. All control specimens were also positive for both membranous and cytoplasmic HER2/neu but there was a significant difference between the level of cytoplasmic staining in the HNSCC specimens and in the control specimens (P<0.05).In our study, no overexpression of HER2/neu was observed. Thus, identification of HER2/neu levels plays no role in differentiating between normal and squamous cell carcinoma tissues or detecting the carcinogenesis process. Our findings suggest that the use of HER2/neu as a salivary marker of HNSCC is not recommended, because no significant preoperative elevation and no association with clinicopathological features were found.  Squamous cell carcinoma is the most common head and neck cancer , it is fThe HER2/neu (ErbB) protein or epidermal growth factor receptors (EGFR) are a family of four structurally related receptor tyrosine kinases . The c-eThe use of targeted agents against molecular markers belonging to the EGFR family has recently become integrated into the treatment protocols of many malignancies, such as breast cancer -14. DespThe aim of this study was primarily to examine the expression of HER2/neu in normal human oral epithelium and patients with HNSCC, to validate the controversial results of various studies and to determine whether or not HER2/neu could be considered as a useful marker for head and neck cancer. Thus, we investigated salivary levels of HER2/neu in healthy subjects and patients with HNSCC and also compared the tissue expression of the protein between the two groups.Study sample: In this study, 28 patients with HNSCC  who had registered at Khalili and Chamran Hospital  and 25 healthy people  were evaluated. The case and control groups were matched by age and sex. Patients and control subjects who showed signs of significant morbidity, active medical problems, and any systemic or inflammatory disease were excluded from the study. Patients with a histopathological diagnosis of HNSCC as well as a large enough tissue sample with H & E stained slides that could be evaluated, and 25 control subjects with normal oral epithelial tissue were enrolled in the research. Clinical data, such as age, gender, location of the tumor, TNM, and tobacco consumption, were obtained from medical records. The Ethical Committee of Shiraz University of Medical Sciences approved the study. All subjects were informed about the research and agreed to participate in the study by signing an informed consent form. Saliva collection and analysis: Salivary samples were taken from subjects before any surgical procedures or chemotherapy protocols commenced. Prior to the collection of unstimulated whole saliva, subjects were asked to refrain from eating, drinking, smoking or performing oral hygiene procedures for 30 minutes. The lip area was cleaned and each subject rinsed their mouth once with plain water. Typically, patients donated 5 to 10 ml of saliva. Samples were then centrifuged at 2,600×g for 15 minutes at 4ºC. The resulting supernatant was then stored at -80ºC until use . SalivarImmunohistochemical analysis: Sections of tumor tissue (4 µm thick) were mounted on positively charged microscope slides. After dewaxing in xylene, sections were dehydrated in ethanol and rinsed in distilled water. Antigen retrieval was performed using DAKO Target Retrieval Solution . The endogenous peroxidase was quenched with 3% H2O2. Peroxidase-labeled polymer conjugated to goat anti-mouse HER2 was then used to detect the antigen-antibody reaction . The HER2 antibodies  were incubated with the tissue sections for 1 hour at room temperature. Sections were then visualized with 3,3-diaminobenzidine as a chromogen for 5 minutes and counterstained with Harris’s hematoxylin. Slides were washed in tap water, dehydrated, and mounted with glass coverslips. Positive controls were sections of breast cancer tissue that had previously been found to be positive for HER2/neu and negative controls consisted of duplicated sections of the same specimens in which the primary antibody had been excluded and replaced with PBS.In order to quantify the expression of HER2/neu, representative tumor sections were identified on a light microscope. Ten random fields were chosen for each section and the total numbers of positive cells for all 10 examined fields were counted and the percentage of staining was calculated . MembranStatistical analysis: The Mann-Whitney test, Student’s independent t-test, Chi-Squared test and Fisher’s exact test were used for statistical analysis. The level of significance was set at 0.05.The clinical data for the patients with HNSCC included in this study of HER2/neu expression are presented in .All samples of normal oral epithelium were positive for membranous and cytoplasmic HER2/neu. Cytoplasmic staining was limited to the basal and parabasal layers in normal epithelium .All HNSCC specimens were positive for membranous or/and cytoplasmic HER2/neu, except one sample ,3.Only one HNSCC specimen contained only cytoplasmic staining. The mean percentage of membranous staining in HNSCC samples was 63.0 ± 30.8% and in normal epithelium it was 66.4 ± 16.4%. There was no significant difference in the percentage of membranous staining between the patient and control groups. The distribution of membranous immune-staining according to each of the grades is presented in . P<0.001).The distribution of cytoplasmic immune-staining grades is presented in . There was no correlation between the percentage of HER-2 staining and any clinicopathologic factors.There was a high tendency for cytoplasmic staining around keratin pearls in well-differentiated HNSCC samples.There was a significant difference between the grade of cytoplasmic immune-staining in the case and control groups (P= 0.90).The mean salivary level of HER2/neu in patients with HNSCC was 3.12 ± 4.58 ng/ml and for the control group was 13.2± 6.75 ng/ml. There was no significant difference in salivary concentrations of HER2 between the case and the control groups. There was also no significant correlation of salivary levels with the percentage of overall immune-staining in samples of tumor tissue from patients with HNSCC, and there was no significant correlation between salivary HER-2 levels and the clinic-pathological data of patients. However, salivary HER-2 levels had a reverse relationship with the percentage of cytoplasmic staining in tumor samples from patients with HNSCC, but there was no significant correlation, tissue samples, and cell lines .The amplification of the HER2 gene has been demonstrated in many carcinomas of glandular origin, and immunohistochemically-identified expression of the gene has been proven to be closely associated with its amplification level . The ampReports on the role of the HER2/neu proto-oncogene product in HNSCC are less conclusive than those detailing the role of EGFR(HER1), as over-expression of HER2/neu has been shown to be present in anywhere from only a few to all HNSCC specimens investigated and correlations with clinical parameters are controversial -26. In oOn the other hand, some studies have reported over-expression of HER2/neu as a potential useful marker in distinguishing non-cancerous from cancerous tissues ,30. FongIn various studies purely membranous  or cytopIn our study we evaluated cytoplasmic and membranous staining separately to make it clear and found a significant difference between cytoplasmic immune-reactivity in the case and control groups. In the literature, the percentage of HER-2/neu positivity in patients with HNSCC is extremely variable. It is possible that these discrepancies in results may be attributed to an initial lack of standardization of the assay methods . AnotherThere was no significant correlation between HER2/neu expression and age, gender, tumor size, lymph node and distant metastasis, tumoral stage and histologic differentiation in our study. In 1997 Xia et al. found a strong correlation between cerbb2 over-expression and overall survival of patients with OSCC . In a reOne of the limitations of our study was the lack of stage IV tumors in the patients with HNSCC, thus we could not fully judge the relationship between level of tumor stage and HER2/neu expression. In our study, there was prominent cytoplasmic staining around keratin pearls in well-differentiated samples but there was no significant correlation between HER2/neu cytoplasmic staining and tumor grade. In recent years, increasing interest has developed in the use of saliva as an adjunct test medium to help in conventional medical assessment of serious systemic diseases . Due to In our study, there was no significant difference in salivary levels of HER2/neu between the HNSCC and control groups. The salivary level of HER2/neu in patients with HNSCC was lower than in the control group but the difference was not significant. Also, there was an inverse relationship between salivary levels of HER2/neu and the percentage of cytoplasmic staining. One of the reasons for the presence of lower HER2/neu salivary levels in the case group could be the high level of cytoplasmic staining of this marker in HNSCC specimens, which could cause decreased membranous transportation of this marker to saliva. The inverse relationship between salivary levels of HER2/neu and cytoplasmic staining is proof for this claim.In this study there was no significant correlation between the salivary levels of HER2/neu and the clinicopathological data of the patients. Briefly, we found that there was no increase in the presence of HER2/neu protein in saliva in patients with more progressive or aggressive lesions. In a similar study in 2010, Vanessa et al. also found no significant association between the salivary levels of the proteins and patient clinicopathological data, such as age, tumor site, histological grading, T status, nodal involvement of the tumor or stage. Salivary levels of HER-2 also showed no difference between patients pre-surgery and healthy control groups; however, both demonstrated an increase after surgical removal of the tumor .In our cases, no overexpression of HER2/neu was observed. Thus, the protein cannot be used to differentiate between normal and squamous cell carcinoma tissues or identify the occurrence of carcinogenesis.Our findings suggest that the use of HER2 as a salivary marker of HNSCC is not recommended because no significant preoperative elevation of HER2 or association with clinicopathological features was found.


Cerebral vessels in the premature newborn brain are well supplied with adrenergic nerves, stemming from the superior cervical ganglia (SCG), but their role in regulation of blood flow remains uncertain. To test this function twelve premature or two-week-old lambs were instrumented with laser Doppler flow probes in the parietal cortices to measure changes in blood flow during changes in systemic blood pressure and electrical stimulation of the SCG. In lambs delivered prematurely at ∼129 days gestation cerebral perfusion and driving pressure demonstrated a direct linear relationship throughout the physiologic range, indicating lack of autoregulation. In contrast, in lambs two-weeks of age, surgical removal of one SCG resulted in ipsilateral loss of autoregulation during pronounced hypertension. Electrical stimulation of one SCG elicited unilateral increases in cerebral resistance to blood flow in both pre-term and two-week-old lambs, indicating functioning neural pathways in the instrumented, anesthetized lambs. We conclude cerebral autoregulation is non-functional in preterm lambs following cesarean delivery. Adrenergic control of cerebral vascular resistance becomes effective in newborn lambs within two-weeks after birth but SCG-dependent autoregulation is essential only during pronounced hypertension, well above the normal range of blood pressure. This phenomenon is called cerebral autoregulation and has been demonstrated in adults, newborns, and even in fetal sheep The extra-parenchymal arteries supplied by the Circle of Willis, including the pial arterioles, are richly innervated by sympathetic nerve fibers Preterm infants are particularly susceptible to dysregulation of CBF and suffer from intracerebral hemorrhage n = 6) that were delivered by C-section on gestational day 127 to 132 (term 147) or on two-week-old lambs (n = 6) following natural delivery. Anesthesia was induced in the ewes (for the preterm lamb group) and two-week-old lambs with an intra-jugular injection of thiopental sodium (10 mg/kg), followed by intubation and mechanical ventilation with 2% isoflurane throughout the surgical instrumentation. The umbilical cord of the preterm lamb was kept intact during the surgery with only the head and thorax exteriorized through a midline incision in the maternal abdomen.Animal protocols were pre-approved by the Institutional Animal Care and Use Committee of Loma Linda University. All sheep were mixed Western breed. Experiments were conducted on either preterm lambs  and pancuronium (0.1 mg kg−1 hr−1).Both preterm and two-week-old lambs were instrumented with bilateral laser Doppler flow probes , inserted through 1.5 mm burr holes drilled 5 mm lateral to the sagittal suture and 15 mm posterior to the coronal suture 2O, positive end-expiratory pressure of 5 cm H2O, inspiratory:expiratory ratio time of 1:3, and respiratory rate of 60 bpm, with subsequent adjustments made to target an arterial PO2 of ∼200 Torr and PCO2 of 35 to 45 Torr. The target arterial PO2 was set higher than the physiologic range to overcome ventilation-perfusion mismatch in the lambs and thereby to ensure cerebral autoregulation was not influenced by hypoxia. Trometamol; tris-hydroxymethyl aminomethane  was administered to treat metabolic acidosis as needed. Rectal temperature was maintained at 39±1 °C by use of a radiant warmer .Both preterm and two-week-old lambs were mechanically ventilated  with initial settings at a peak inspiratory pressure of 25 cm HLambs were placed in the supine position and both SCG were exposed via an incision from the angle of the mandible to the transverse process of the second cervical vertebrae. One SCG was randomly chosen for insertion of two platinum wire stimulating electrodes connected to a Grass S-48 model stimulator .Baseline measurements were made after a four-hour recovery period following placement of the LDF probes. The lambs where then given an intravenous bolus of norepinephrine (5 μg/kg) and angiotensin II (10 μg/kg) to cause hypertension. Systemic hypertension was obtained by pharmacological means because it enabled the study of higher arterial blood pressures than could be obtained by occlusion of the descending aorta, and with the rationale that a favorable comparison with mechanical means would strengthen interpretation of the results. Each bolus was followed by a 5- to 15- minute recovery period until blood pressure returned to baseline. Following pharmacologically induced hypertension lambs were subjected to 100% occlusion of the brachiocephalic trunk for 30 seconds to reduce cerebral perfusion pressure. This was followed by 3 to 5 minutes of recovery to return to a stable baseline. Next, a graded 30-second occlusion was administered to generate a fall in arterial pressure equivalent to 50% of the decrease caused by the 100% occlusion, followed by another recovery period. Finally, a third occlusion was administered to achieve a 25% decrease in arterial pressure compared to the 100% occlusion. The lambs were then subjected to a similar series of 100%, 50%, and 25% occlusions of the descending aorta to generate an increase in pressure perfusing the cerebral circulation. Next, to assess the effects of electrical stimulation of the SCG, electrical impulses were administered directly to the SCG, at an interval of 1 millisecond with a frequency of 8 Hz and intensity of 6 to 8 volts. A total of four stimulations (two at 6 volts and two at 8 volts) were performed on each lamb. Each stimulation lasted for 30 seconds and was followed by a 3- to 5-min recovery period. To confirm that CBF responses were through the SCG itself in three lambs the surrounding tissue was stimulated and found to be without effect.Lastly, the SCG that had been instrumented with electrodes was surgically removed and occlusions of the brachiocephalic trunk and descending aorta were repeated, as were the infusions of angiotensin II and norepinephrine. After completion of the experiment, the lambs were euthanized with Euthasol® .Arterial blood pressure, heart rate, and CBF were recorded digitally at 400 Hz using an A/D converter  and software . Because LDF provides only relative changes in flow, CBF and cerebral vascular resistance were expressed as a percent of baseline values. Cerebral vascular resistance was calculated as mean arterial blood pressure divided by CBF and also normalized to baseline values. The effect of electrical stimulation was measured by averaging CBF signals into 5-second blocks, beginning 30 seconds prior to the beginning of the electrical stimulation and continuing until 30 seconds after the end of the stimulation.Cerebral blood flow and arterial blood pressure during the last 15 seconds of each graded occlusion of the brachiocephalic trunk and aorta, and at the peak response to norepinephrine and angiotensin II injection, were normalized to the 30 seconds of data recorded just prior to each intervention. Autoregulation of CBF was then evaluated by comparison of CBF with mean arterial blood pressure in 2.5 mmHg intervals as previously described n  =  6 for an α of 0.05 and a β of 0.20. All data are presented as mean ± SE.A one-way ANOVA was used to assess the significance of the effects of electrical stimulation of the SCG on mean arterial blood pressure. The effects of stimulation on ipsilateral vs. contralateral (with respect to the side of SCG stimulation) CBF and cerebral vascular resistance time series data . However, within each group baseline mean arterial blood pressures prior to each intervention were comparable before and after removal of the SCG (58±9 mmHg vs. 56±9 for preterm lambs and 74±8 vs. 75±10 for two-week-old lambs). The effect of the blood pressure interventions on upper body arterial blood pressure and CBF are given in The relationship between mean arterial blood pressure and CBF across all ranges of arterial blood pressure studied before and after removal of the SCG is shown in The present experiments demonstrate that the sympathetic innervation of the cerebral vasculature serving the cortex is functionally intact and, if stimulated, modulates CBF in both preterm lambs following cesarean delivery and two-week-old lambs born naturally at term. The work also demonstrates a lack of cerebral autoregulation in anesthetized and mechanically ventilated preterm lambs following cesarean delivery, in contrast to similarly instrumented and anesthetized two-week-old lambs in which effective autoregulation was observed. Finally, the experiments indicate that the SCG does contribute to autoregulation in the two-week-old lambs, but that it is only essential during episodes of supraphysiological hypertension.The development of functional adrenergic innervation of vasculature occurs in stages, with postsynaptic components of the system maturing before presynaptic components are in place in vivo results in decreased CBF in newborn lambs in vivo comparisons of presynaptic function in newborns and adults of the same species have not been performed. Although we did not observe a significant decrease in CBF in the two-week-old lambs, our findings are consistent with these previous newborn reports in that we also observed a significant increase in ipsilateral cerebrovascular resistance to flow in response to unilateral SCG stimulation . In this study the probe was positioned in the parietal cerebral cortex, approximately 0.5 cm below the surface of the brain, a location in the forebrain where blood flow has been shown to be under the influence of sympathetic input Laser Doppler flowmetry is limited in that it can only detect changes in flow in a relatively small volume of tissue (∼2 mm2 to the lambs in both groups may have attenuated cerebral autoregulation. However, this possibility appears unlikely as studies of the administration of high inspired O2 in humans find no change 2. Other limitations include failure to monitor and maintain glucose and renin levels, and an uneven ratio of right-to-left SCG chosen for stimulation and removal, which may have biased the results if the effects of the two ganglia are asymmetrical. Also, the experimental preparation required instrumentation that may have injured cerebral blood vessels, altering blood flow responses. And finally the sheep is a species whose young are quite mature at birth and vascular control is likely to develop more slowly in infants. Furthermore, the anatomy of the vasculature supplying the sheep brain differs from that of the human as blood passes through a carotid rete before reaching the Circle of Willis in the sheep. The effect of these species differences on autoregulation and the role of the SCG are not known.Both the SCG stimulations and interventions in arterial blood pressure in our study were of relatively short duration in order to ensure that the lambs remained in stable condition throughout the experiment. However, this limited time prevented assessment of long-term decreases in CBF, which may be limited by a time-dependent ‘escape’ mechanism in some animal models in utero at an early stage of development, it may be lost with premature delivery and following treatments common to the clinical care of premature infants, such as intubation and mechanical ventilation. The present findings indicate that sympathetic control of cerebral vascular resistance during marked hypertension may be limited immediately following preterm birth. Furthermore, excessive sympathetic activation may potently decrease CBF after birth, contributing to inadequate flow observed in some pathologies.Dysregulation of CBF, resulting in intraventricular hemorrhage and periventricular leukomalacia, is a common problem in the care of premature infants. The present study demonstrates that, although cerebral autoregulation is intact in the fetal sheep


Many older people in long-term care do not receive evidence-based diagnosis or management for heart failure; it is not known whether this can be achieved for this population. We initiated an onsite heart failure service, compared with ‘usual care’ with the aim of establishing the feasibility of accurate diagnosis and appropriate management.A pilot randomised controlled trial which randomised residents from 33 care facilities in North-East England with left ventricular systolic dysfunction (LVSD) to usual care or an onsite heart failure service. The primary outcome was the optimum prescription of angiotensin-converting enzyme inhibitors and beta-adrenergic antagonists at 6 months.Of 399 echocardiographically-screened residents aged 65–100 years, 30 subjects with LVSD were eligible; 28 (93%) consented and were randomised . Groups were similar at baseline; six month follow-up was completed for 25 patients (89%); 3 (11%) patients died. Results for the primary outcome were not statistically significant but there was a consistent pattern of increased drug use and titration to optimum dose in the intervention group . Hospitalisation rates, quality of life and mortality at 6 months were similar between groups.This study demonstrated the feasibility of an on-site heart failure service for older long-term care populations. Optimisation of medication appeared possible without adversely affecting quality of life; this questions clinicians’ concerns about adverse effects in this group. This has international implications for managing such patients. These methods should be replicated in a large-scale study to quantify the scale of benefit.http://www.controlled-trials.com/ISRCTN19781227ISRCTN19781227 Evidence-based management of heart failure (HF) reduces mortality and morbidity and improves quality of life. The benefits of drug management for HF have been extensively researched and are included internationally in guidance for the management of HF in older people, though these do not specifically refer to those in long-term care-6. Both Variations in HF management in the long-term care population may be due in part to the difficulty accessing specialist care. DifficuA pilot randomised controlled trial using a PROBE design , compared two models of care: routine GP-led care or an onsite HF team.Residents from 33 of 35 long-term care facilities  in Teesside, North East England, aged ≥65 years without terminal disease and who were permanently resident were eligible to participate  of all eligible residents (including non-participants) in order to assess the representativeness of participants and thus the potential for selection bias. Study data were collected over a 14 month period from April 2009.Participants underwent a diagnostic assessment within each facility, including MMSE, demograParticipants identified using echocardiography as having HF due to left ventricular systolic dysfunction (LVSD) were screened for trial eligibility. Any resident not already prescribed both an ACEi and β-blocker at optimal dose for LVSD was eligible. Residents with an existing diagnosis of LVSD were eligible; those with heart failure with preserved ejection fraction (HFpEF) or with terminal disease were ineligible. Written consent was sought from residents directly or through a consultee when residents lacked cognitive capacity.Residents randomised to the HF service received an assessment visit by a consultant cardiologist who initiated a plan of treatment, followed by visits at one to two weekly intervals within the home by heart failure specialist nurses (HFSNs). The HFSNs enacted the plan, including blood tests, assessment of symptoms and signs, educational advice, and medication titration. The team cardiologist was contacted in the event of any change in a resident’s health status related to heart failure. GPs were notified of other anomalies on the same day as they were detected. Residents were treated according to NICE guidance using drThe team notified primary care providers of any alterations to HF management. Drug prescriptions were managed using usual practice within the care home and the associated general practice. In addition, each patient’s GP was notified by letter each time they were reviewed by the HF team, detailing symptoms and signs as well as medication changes. Once HF management was optimised, participants were discharged from the HF service and a final letter was sent from the team consultant cardiologist, in consultation with a GP with a specialist interest in HF, to the relevant GP notifying current management and advice about ongoing care. For those residents randomised to routine care, echocardiographic test results were communicated to the resident and a letter sent to their GP from the team consultant cardiologist outlining a personalised HF management plan.The original  sample size was based on a difference in proportions receiving ACEi and β-blockers (40% and 70%) and required a total enrolment of 100 residents with LVSD . Allowing for non-participation or loss to follow-up, the study aimed to identify 125 eligible residents. Estimates from previous literature suggested a prevalence of heart failure of 25%, thus the prevalence study required 500 residents in order to identify sufficient eligible residents for the trial. However, previous literature did not differentiate between LVSD and HFpEF – using agreed criteria, screening identified an 8.5% prevalence of LVSD heart failure (and 14.3% HFPEF), rendering the original sample size unfeasible. In response the trial was reconstituted as a pilot study retaining the original trial design and per protocol analyses but aiming to recruit 25–30 participants. Although underpowered to address the primary endpoint, this level of recruitment would show a difference of optimally treated patients (prescribed both ACEi and β-blockers) of 50% .Consenting residents were randomly assigned to either usual care or the HF service. Randomisation used stratified blocks according to NYHA classification . All participants’ notes were reviewed at 6 and 12 months by a blinded assessor. Residents in the intervention arm were discharged back to usual care by 6 months, or at the point of optimal titration if before 6 months. The primary trial outcome was the proportion of residents receiving the guideline-recommended dose of prescribed ACEi and β-blockers at 6 months after enrolment. Titration to a percentage of the recommended  optimum provided a consistent and measurable benchmark for comparing groups, where it was not assumed that 100% was achievable or necessarily desirable for the individual resident. Recognising that the optimal dose would vary for individual patients according to response and tolerability, we conducted secondary analysis on residents receiving ‘any dose’..The optimum dose was defined as ramipril 10mg and bisoprolol 10mg or equivalent; use of an angiotensin receptor blocker (ARB) was counted as equivalent to an ACEi. Since the prescription of spironolactone involved some judgement on the part of the treating physician this was not included in the primary outcome but rates were recorded for both groups, with a target dose of 50mg spironolactone. Secondary analyses included: use of individual drug classes at any dose; the proportion of patients dying or being hospitalized for HF; acceptability to residents of the two forms of care; changes in functional capacity and quality of life (using EQ-5D and EQ-VAS).Data analysis was conducted by intention to treat and using SPSS version 19. Differences in proportions were analysed using Fisher’s exact test, cross tabulations of more than two categories using Pearson chi-square, and continuous variables using unpaired Student’s t-test.This study was approved by Durham University and Leeds (West) national research ethics committees. The investigation conforms with the principles outlined in the Declaration of Helsinki.Of 399 residents screened in the HFinCH prevalence study, 34 (8.5%) were diagnosed with LVSD (19 (56%) mild, 9 (27%) moderate, and 6 (18%) severe). Of these, four were ineligible , thus 30 were eligible for the trial and 28 (93%) agreed to participate. Of the two patients who declined: one was due to relocate and one did not wish to participate.The mean age of trial subjects was 83.6 (range 70–98), all were white British and 12 (43%) were male. Resident data was collected from 24 GPs based at 11 different medical centres. Residents were predominantly categorised as requiring residential care  rather than nursing care; 17 care homes were involved in the trial. Twelve residents (43%) were unable to give consent due to cognitive impairment and a consultee declaration was obtained.Participants were randomised to either the HF service , or to routine care : the two groups were similar statistically at baseline in demographic characteristics, HF severity, comorbidities, signs and symptoms, and prescribed drug use (Table Six month follow-up was completed by 25 residents (86%); the remaining 3 14%) died during follow-up . See Figure 4% died dAt six months, 3 21%) of the intervention group achieved optimal doses of combined ACEi and β-blockers compared to 0 (0%) in the control group  for the intervention group and 28.7% (SD 29.4%) for the usual care group (p=0.057).Taking each drug in turn, more intervention patients received an ACEi at optimum dose , while numbers receiving a β-blocker at optimum dose were similar . No participants in either group received the optimum dose of spironolactone but two patients  in the intervention group received spironolactone at a lower dose. Comparing the intervention and usual care groups at six months, there were non-statistically significant increases in ACEi use at any dose  and β-blocker use at any dose .The mean time from initial specialist assessment to optimum titration in the intervention group was 4 months (range 1– 6 months) with a mean of 5 titration steps (range 1–15) to optimum or maximum tolerated dose. It was not possible to assess the mean time in usual care from available records.Following the end of the trial, general practice records were assessed at 12 months to establish treatment fidelity and changes in management. Twelve month follow-up was completed by 21 residents (75%); 6 (21%) died during follow-up  and 1 (4%) was lost to follow-up. At 12 months there were no significant differences between HF treatment for each group, and no differences in hospitalisation rates. Comparing 6 to 12 month prescribing trends, there was a substantial reduction in both numbers and doses of β-blockers and ACEi among those discharged from the intervention group . Prescribing trends in the usual care group remained largely stable although at 12 months all β blocker prescriptions were for bisoprolol and 90% of ACEi prescriptions were for ramipril, with the remainder being for lisinopril and perindopril. In the intervention group 3 of 16 patients (21%) were titrated to optimal dose by the HFSN before being discharged from the HF service but this had fallen to 1 of 16 (6%) by 12 months.There was no significant patient morbidity . At 6 mThe diagnostic assessments were acceptable to 395 (99%) of the cohort and all but two eligible participants consented to the trial; receipt of the HF intervention was endorsed by all consenting participants and care home staff as an experience they would be willing to repeat. Five GPs of residents receiving both intervention and control were interviewed as part of a qualitative evaluation : all were supportive of the service.This study challenges the status quo for HF management for older people in care. Findings of this pilot trial demonstrate the feasibility and applicability of evidence-based management in a group of people previously denied optimum care. International research over many decades has demonstrated the sub-optimal diagnosis and management of HF in older residents in care. Reasons for this include clinical reluctance to offer treatments that may have side-effects and which may ultimately reduce length and quality of life. In this study, the intervention group attained a higher level of evidence-based treatment of optimal doses of ACEi and β-blockers than the control group without deterioration in quality of life although results were non-significant due to the sample size. Around half of participants had a pre-existing diagnosis of LVSD and were receiving sub-optimal doses of ACEi and β-blockers but in this trial this was improved. It is possible that the magnitude of change over 6 months might have been even higher if treated cases of LVSD had been excluded in the trial, although drug therapy was already limited in this group.The low numbers of hospitalisations in either group was surprising but reassuring in that the intervention group did not have increased admission rates. This counters often quoted fears of harm through higher (appropriate) doses and types of treatment. An important consideration for this trial was the length of time it would take to reach an appropriate level of treatment (in dose and type) for each patient. We found this could be achieved in the intervention group within 4 months, also suggesting the notional 6 month cut-off for assessment of the primary outcome was a reasonable comparison.It remains uncertain whether treatment levels can be maintained in patients who had received the intervention following discharge back to usual care. Unless there is an ongoing and integrated approach to drugs management, improvements by one care provider may quickly be cancelled by changes made by another. For those discharged from the intervention, rates of prescribing of ACEi and β-blockers had dropped at 12 months following the trial. We were unable to ascertain reasons for the reduction. It would be helpful to understand why GPs altered personalised management plans in some patients when there were no indications that side effects or other drug-associated problems increased. In the qualitative arm of this trial , GPs expressed concern about comorbidities, polypharmacy and side-effects. Further understanding of these issues might allow us to draw definitive conclusions about models of care for heart failure management in the longer term. The introduction of a stand-alone intervention raises important questions about sustainability and capacity building in general practice. Additional steps might have been taken to improve GP knowledge about heart failure, however the purpose of this study was to assess baseline practices from which to inform the development of a subsequent structured interventional study. A subsequent (powered) study should evaluate new and current care pathways in primary care to inform effective heart failure diagnosis and management, measuring a range of end points  as well as a cost-effectiveness analysis.While the findings regarding short-term prescribing differences between GPs and the heart failure team might not be surprising, this is the first study to empirically demonstrate that additional support is required to raise standards of primary care for this group.This trial comprised participants who had been screened for entry into the study and in whom HF status was ascertained. Rates of willingness to participate and retention during follow-up were very high but the trial may have recruited a selected population with less complex needs than the non-screened population. Whilst pilot findings should be interpreted with caution, baseline characteristics were collected for both screened and non-screened subjects. Similarities in these data suggest that selection bias was not a threat to this study.This study excluded patients with HFpEF as it currently lacks a clearly evidence-based rationale for treatment. FurtherOur findings demonstrate the acceptability and feasibility of a tailored heart failure service for older people in care. This study also challenges an implicit orthodoxy that older people in care homes are unwilling or unable to engage in clinical research involving comprehensive diagnosis and management. Optimisation of medication appears possible and larger scale studies are required to quantify the scale of benefit.The authors declare that they have no competing interests.HH: I declare that co-designed the study, led data collection, participated in data analysis, interpretation and writing this report. HC: I declare that I participated in data collection, data analysis, interpretation and writing this report. JMM: I declare that I co-devised the research question, co-designed the study, participated in data collection, led data analysis, interpretation and writing this report. JJM: I declare that I participated in data collection, data analysis, interpretation and writing this report. AF: I declare that I participated in data collection, data analysis, interpretation and writing this report. MdeB: I declare that I participated in data validation, interpretation and writing this report. TH: I declare that I participated in data collection. AB: I declare that I participated in data collection. DW: I declare that I verified data analyses. APSH: I declare that I co-devised the research question, co-designed the study, participated in data interpretation and writing this report. All authors have seen and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2318/12/70/prepub


Genome-wide association studies of two main forms of inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), have identified 99 susceptibility loci, but these explain only ∼23% of the genetic risk. Part of the ‘hidden heritability’ could be in transmissible genetic effects in which mRNA expression in the offspring depends on the parental origin of the allele (genomic imprinting), since children whose mothers have CD are more often affected than children with affected fathers. We analyzed parent-of-origin (POO) effects in Dutch and Indian cohorts of IBD patients.NOD2 were tested in 111 CD trios and a significant finding was re-evaluated in 598 German trios. The UC-associated gene, BTNL2, reportedly imprinted, was tested in 70 Dutch UC trios. Finally, we used 62 independent Indian UC trios to test POO effects of five established Indian UC risk loci.We selected 28 genetic loci associated with both CD and UC, and tested them for POO effects in 181 Dutch IBD case-parent trios. Three susceptibility variants in NOD2  for CD; these results could not be replicated in an independent cohort . A POO effect in IBD was observed for IL12B  and PRDM1 . In the Indian trios the IL10 locus showed a POO effect .We identified POO effects for Little is known about the effect of genomic imprinting in complex diseases such as IBD. We present limited evidence for POO effects for the tested IBD loci. POO effects explain part of the hidden heritability for complex genetic diseases but need to be investigated further. Crohn’s disease (CD) and ulcerative colitis (UC) are the two main forms of chronic relapsing inflammatory bowel diseases (IBD). With a cumulative prevalence of up to 800 per 100,000 in Europe and 570 in North America Prior to the introduction of genome-wide association studies (GWAS), only three loci had been consistently associated with either form of IBD. Over the past six years, multiple GWAS and meta-analyses have yielded a lengthening list of variants associated with CD (71 confirmed independent genetic risk loci) and UC (47 loci) In addition, parent-of-origin effects (POO) may comprise a piece of the missing heritability puzzle in IBD, as suggested by Akolkar et al. In this study, we tested for these two types of POO effects in IBD by using a likelihood ratio test developed by Weinberg This study was approved by the institutional review boards  of each of the hospitals and written informed consent was obtained from all subjects personally.NOD2 L1007fs variant from the Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, Germany. These data were available from a previous study, no phenotypic data is available All patients were diagnosed according to standard clinical criteria by endoscopy, radiology and histopathology All Dutch and Indian subjects were genotyped using the Illumina Immunochip (iCHIP) , which is a custom-made genotyping array that contains ∼200,000 single nucleotide polymorphisms (SNPs) focusing on immune-mediated diseases 2>0.5) could be found. Two variants  were also not captured by iCHIP, but we identified perfect proxies using SNAP software: rs12261843 was represented by rs12254167  and rs181359 was represented by rs2266961  2>0.5 flanking the most significantly associated SNP, then extended to the nearest recombination hot-spot, and from there for an extra 100 kb. Comparison of IBD-associated genes with the known imprinted genes resulted in the inclusion of one extra gene, BTNL2; since this is a UC-specific locus it was only analyzed in the Dutch and Indian UC cohorts. SNP rs9268853 is the reported UC risk SNP in the Caucasians and was tested in the Dutch trios, rs3763313 was tested for POO effect in the Indian trios since this is the reported risk SNP in the Indian population. Lastly, we included NOD2 since it is the most strongly associated gene and is most replicated in association studies of CD in populations of western European descent. Three common disease-susceptibility variants  were therefore tested for POO effects in the Dutch CD trios, and subsequently the L1007fs variant was tested in the German replication cohort For this study we used several strategies to select SNPs. First, to gain power we pooled the Dutch UC and CD trios and tested for POO effects in 28 established IBD loci that are both associated to CD and UC et al.A power analysis was performed with Quanto software DNA of 249 complete IBD trios was available for our study, of which four CD (4/115) and two Dutch UC trios (2/72) did not pass the quality control. Therefore 243 IBD trios  were available for the discovery phase of the study. Our findings were then replicated in an independent replication cohort consisting of 598 German CD trios.IL12B gene , with OR >1 indicating that the risk allele is more often transmitted from the mother to the child. In addition, the β term was nominally significant  with OR <1, indicating that offspring have less chance of getting the disease if their mothers carry two risk alleles. The PRDM1 gene showed a nominally significant maternal effect if the mother carried two risk alleles , with OR >1 indicating that the offspring have more chance of getting the disease. The other tests did not result in significant POO effects. However, none of these associations were significant after the Bonferroni correction  were tested for POO effect in the 111 CD trios NOD2 genotyping data was available. Unfortunately, our results could not be replicated in this cohort  (Three established CD variants in  = 0.97) .BTNL2 locus (rs9268853) in 72 Dutch UC trios  where the OR <1 indicates that the risk allele is more often transmitted from the father. This association does not, however, pass the multiple testing correction. This SNP was also tested in the population of western European descent and we could not detect any significant imprinting effect. The NOD2 variant that showed association in the Indian population could not be tested for POO effects since only homozygous wild-type fathers were available, hence all the trios were uninformative  showed inheritance of the disease risk from the mother, while the analysis of independent maternal effect showed protection for disease if the mother carries two risk alleles (β-term). This suggests that the maternal risk allele is expressed and causes a higher disease risk, but simultaneously and independently, if the mother carries two risk alleles the child has a lower risk for IBD due to in utero effects on the fetus. IL12B resides on the established and consistently replicated IBD locus on chromosome 5q33 and it encodes a sub-unit of IL23, which is involved in Th17/IL23R signaling. This pathway has been implicated in several chronic, immune-related diseases such as psoriasis, rheumatoid arthritis, and ankylosing spondylitis Our results suggest a possible contradictory effect of the two types of POO effects we studied for the PRDM1 gene showed a nominally significant maternal effect in the POO analysis in the population of western European descent when mothers carried two risk alleles; the OR of 5.6 supports results from others that IBD is more often transmitted from the mother than the father. The environment in which the fetus develops causes changes that increase the risk for CD. PRDM1 has been associated to several immune-related diseases, but also to various types of lymphomas PRDM1, an aberrant immune response could increase CD risk.The IL10 locus. In contrast to the findings in the population of western European descent, the risk allele was more often transmitted from the father to the child. No epidemiological studies of the Indian population are available to validate the paternal transmission. We could not replicate the POO effects from the Dutch population in the Indian trios nor vice versa. This might indicate that POO effects are population specific. Later in the discussion we will discuss this in further detail.In the Indian trio study we found a nominally significant genomic imprinting effect for the PRDM1 and IL12B loci.Weinberg’s method to detect POO effect is a robust one. Moreover it takes genomic imprinting and maternal effects into consideration simultaneously. It therefore has less power than the standard parental asymmetry test (PAT) that only tests for genomic imprinting, but PAT is invalid if maternal effects are present We do not know why POO effects occur. Humans are diploid organisms and as such, can survive the, on average, 500 recessive mutations that are present in every human being, since most deleterious effects are rescued by the other allele In our study we had sufficient power (>80%) to detect POO effects with an OR of three or higher for each SNP in our study. By adding more tests the significance level must be adjusted accordingly and the power to detect differences is lower. Therefore we chose to only test the 28 overlapping IBD risk loci in a pooled cohort of Dutch CD and UC trios instead of all 99 risk loci. Consequently, bigger cohorts are needed to test the remaining IBD loci for POO effects.None of our findings in one population could be replicated in another population. At least three reasons could explain this fact. First, it might be that the initial findings are false positive findings: the cohorts have a limited size and thus more variation around the mean, resulting in a higher chance of false positive findings. Second, it is unknown for how long genomic imprinting effects are stable in humans. It has been shown from mouse studies that genomic imprinting is stable for at least 3 generations IL12B, NOD2 and PRDM1 might be involved in these phenomena in Dutch IBD trios. It has already been shown that POO effects exist in type 1- and type 2 diabetes, which like IBD are complex genetic disorders and show a substantial overlap of disease susceptibility loci with IBD NOD2 we sought to replicate our findings, but could not confirm the POO effect for NOD2 in an independent German replication cohort. In the Indian population we did identify POO effects in the IL10 gene. We could neither replicate our findings from the Dutch trios in the Indian or in the German population nor our findings from the Indian population in the Dutch cohort. This suggests that POO effects are either false-positive findings or prone to be population specific. We anticipate that future investigations, using larger, multi-ethnical cohorts will help to shed light on these complex and currently little known relationships. Parent-of-origin effects can take various forms and are not restricted to imprinting, but may involve a variety of mechanisms including gender effects, epistasis, epigenetic effects, and environmental influences during pre- or postnatal development. Better understanding of such effects will probably require detailed studies of model organisms in which breeding and environment can be carefully controlled. Given that alleles identified through GWAS account for a relatively small fraction of heritability, parent of origin effects may underlie some of the missing heritability problem. With appropriate family-based study designs data analysis methods and international collaborative efforts it will be possible to screen for parent of origin effects across the entire genome. In addition, epigenetic profiling on a genome scale will likely lead to the identification of novel epigenetic marks in a variety of disorders that may provide a bridge among the parental genome, parental environment, and offspring phenotype. We anticipate that the investigations of alternative models of inheritance, appropriate study design and application of novel technologies will enable a more complete picture of heritability in human traits, leading to new insights in the field of genetics of complex diseases.In conclusion, we aimed to identify genomic imprinting effects and maternal effects acting on the risk alleles of IBD and we showed, for the first time, that Figure S1Power analysis of a. the Dutch trio analysis (181 trios) and b. the Indian trios (62 trios). S1a. Power calculation of the Dutch trio analysis: The power is shown on the x-axis, the different odds ratios (OR) are shown on the y-axis. The different lines represent SNPs with different minor allele frequencies. In red, the regular 80% power cut-off is shown. With an OR of 3, we have sufficient power to detect parent-of-origin effects in SNPs with a MAF of 2.5%. S1b. Power calculation of the Indian trio analysis: The power is shown on the x-axis, the different odds ratios (OR) are shown on the y-axis. The different lines represent SNPs with different minor allele frequencies. In red, the 80% power cut-off is shown. With an OR of 4, we have sufficient power to detect parent-of-origin effects in SNPs with a MAF of 4.0%.(TIF)Click here for additional data file.


A causal role of mutations in multiple general transcription factors in neurodevelopmental disorders including autism suggested that alterations in global levels of gene expression regulation might also relate to disease risk in sporadic cases of autism. This premise can be tested by evaluating for changes in the overall distribution of gene expression levels. For instance, in mice, variability in hippocampal-dependent behaviors was associated with variability in the pattern of the overall distribution of gene expression levels, as assessed by variance in the distribution of gene expression levels in the hippocampus. We hypothesized that a similar change in variance might be found in children with autism. Gene expression microarrays covering greater than 47,000 unique RNA transcripts were done on RNA from peripheral blood lymphocytes (PBL) of children with autism (n = 82) and controls (n = 64). Variance in the distribution of gene expression levels from each microarray was compared between groups of children. Also tested was whether a risk factor for autism, increased paternal age, was associated with variance. A decrease in the variance in the distribution of gene expression levels in PBL was associated with the diagnosis of autism and a risk factor for autism, increased paternal age. Traditional approaches to microarray analysis of gene expression suggested a possible mechanism for decreased variance in gene expression. Gene expression pathways involved in transcriptional regulation were down-regulated in the blood of children with autism and children of older fathers. Thus, results from global and gene specific approaches to studying microarray data were complimentary and supported the hypothesis that alterations at the global level of gene expression regulation are related to autism and increased paternal age. Global regulation of transcription, thus, represents a possible point of convergence for multiple etiologies of autism and other neurodevelopmental disorders. Autism is a severe neurodevelopmental disorder with characteristic social and communication deficits and ritualistic or repetitive behaviors that appear by age three. Many etiologies have been suggested and numerous risk factors have been identified Research on gene expression in autism has previously focused on identifying specific or a limited group of genes related to disease Addressing the impact of variability in global levels of gene expression regulation on neurodevelopment in mice, we recently reported that the pattern of the distribution of gene expression levels, as assessed by variance in the distribution, accurately predicted mouse behavior in genetically identical animals. Specifically, we found that increased variance across the total distribution of gene expression levels in the hippocampus predicted increased levels of open-field exploration, a hippocampal dependent behavior. Developmental epigenetic interventions that modified the variance in the gene expression distribution in the hippocampus also modified mouse behavior in tandem In the current study, we used our previously established strategy of studying the overall pattern of the gene expression distribution to test the hypothesis that autism would be associated with alterations in global levels of gene expression regulation. To do this, we compared the pattern of the gene expression distributions, as assessed by variance across the total gene expression distribution, in the blood of children with autism and controls  according to the DSM-IV criteria, and confirmation of the diagnosis by ADOS Expression profiling was performed at Translational Genomics (TGen), a member of the NIMH Neuroscience Microarray Consortium. Total RNA was extracted from peripheral blood lymphocytes (PBL) within 30 minutes of the blood draw using the Qiagen Qiaquick kit . Isolated total RNA was double round amplified, cleaned, and biotin-labeled using Affymetrix's GeneChip Two-Cycle Target Labeling kit  with a T7 promoter and Ambion's MEGAscript T7 High Yield Transcription kit  as per manufacturer's protocol. Amplified and labeled cRNA was quantified on a spectrophotometer and run on a 1% TAE gel to check for an evenly distributed range of transcript sizes. Twenty micrograms of cRNA was fragmented to approximately 35–200 bp by alkaline treatment  and run on a 1% TAE gell to verify fragmentation. Separate hybridization cocktails were made using 15 micrograms of fragmented cRNA from each sample as per Affymetrix's protocol.Two hundred microliters (containing 10 micrograms of fragmented cRNA) of each cocktail was separately hybridized to an Affymetrix Human Genome U133 Plus 2.0 Array for 16 h at 45 degree Celsius in the Hybridization Oven 640. The Affymetrix Human Genome Arrays measure the expression of over 47,000 transcripts and variants, including 38,500 characterized human genes. Arrays were washed on Affymetrix's GeneChip Fluidics Station 450 using a primary streptavidin phycoerythrin (SAPE) stain, subsequent biotinylated antibody stain, and secondary SAPE stain. Arrays were scanned on Affymetrix's GeneChip Scanner 3000 7G with AutoLoader. Scanned images obtained by the Affymetrix GeneChip Operating Software (GCOS) v1.2 were used to extract raw signal intensity values per probe set on the array. A scaling factor of 150 was used to normalize array signal intensity in MAS 5.0.Arrays were scanned over 1 day on 2 different machines. Arrays scanned on the same machine and in the same day were considered to be from the same scan batch. Rescanning of a limited number of samples indicated that there were no significant differences between machines, nonetheless, for all comparisons groups were balanced for the scan batch. Gene expression levels were not adjusted for possible batch effects as algorithms that attempt to adjust for batch effects also alter the gene expression distribution. When samples could not be prepped simultaneously they were balanced for group membership (autism vs. control). To statistically control for possible confounds related to scan batches in our analysis of gene expression variance, batch number was entered into an analysis of covariance. For traditional analysis of gene expression, experimental groups were balanced with respect to batch membership.Affymetrix .cel files were imported into Affymetrix Expression Console version 1.1. Data was pre-processed and summarized by Microarray Analysis Suite (MAS) 5.0 and Robust Multiarray Analysis (RMA). For the analysis of gene expression distributions, MAS 5.0 was used because the algorithm does not alter the gene expression distribution, whereas, RMA utilizes quantile normalization of probes prior to summarization and, therefore, has the potential to remove group level differences in gene expression distributions. Because of the numerous advantages in its handling of noise in the measurement of gene expression levels and background subtraction, RMA was used for traditional gene expression analyses looking for specific gene expression differences between groups. Because group level differences in the distribution of gene expression levels between groups were found, gene expression levels were also quantile normalized after the summarization step for traditional gene expression analyses. Quantile normalization adjusts all data sets such that they have identical distribution patterns. Probe sets were then filtered for those that were called present in at least 50 out of the 146 subjects . A p-value of .05 was used as a threshold for significance. A fold-change of 1.1 was used as a cut off for magnitude of change. All microarrays met manufacturers recommended quality control criteria. Present calls ranged from 37.4% to 49%, mean 43.7%, SD 2.7%. Actin 3′to5′ ratios ranged from .726 to 5.15, mean1.37, SD 0.5. There were no significant group level differences in quality control measures.Variance across the distribution of gene expression levels was the primary measure used in the present study. The measure of overall variance compresses data from the expression levels of 54,675 probe sets covering approximately 47,000 unique transcripts including 38,500 human genes on the Affymetrix Human HG-U133 Array into a single variable. We previously found that overall variance in gene expression levels in the hippocampus of mice accurately predicted hippocampal-dependent behaviorDAVID (david.abcc.ncifcrf.gov) Functional Annotation Bioinformatics Microarray AnalysisWe found significant differences in variance in gene expression between children with autism and controls. When the paternal age of the control father was considered it was found that variance in gene expression of children with autism was only significantly different from children of younger fathers. Additionally, we found that variance in gene expression was significantly different between children of older fathers and children of younger fathers. To validate these findings we subdivided our data sets into 6 subgroups to allow for within study “replications”. In all cases subgroups of the same type  were equally balanced for paternal age and scan batch. The validation sets were as follows: Autism1 (n = 41), Autism2 (n = 41), Control young father1 (n = 14), Control young father2 (n = 14), Control old father1 (n = 14), Control old father2 (n = 14), Control 1 (n = 28 - combination of control young father1 and control old father1), and Control 2 (n = 28 - combination of control young father2 and control old father2).http://www.ncbi.nlm.nih.gov/geo) public database. The microarrays used in the transcription inhibitor study were the same used in the current study, Affymetrix Human Genome U133 Plus 2.0 Array. Microarray data was pre-processed and summarized as described above using Expression Console and the MAS 5.0 algorithm.In order to assess for changes in gene expression variance in response to globally targeting transcription, we downloaded a publically available dataset in which a new class of transcription inhibitors, sappyrins, were compared to actinomycin D. The microarray study analyzed gene expression in A549 human lung cancer cells that were treated with transcriptional inhibitorsJMP8 and Statview software  were used for statistical analyses. The statistical analyses used in the study are listed below.To determine if parametric tests were appropriate, the Shapiro-Wilk Test was performed for the primary measures: variance in gene expression and paternal age. When assessed across the entire study or across all control subjects, variance in gene expression was not normally distributed, but was normally distributed within experimental groups: 1) autism, 2) children of older fathers, and 3) children of younger fathers. Paternal age was normally distributed across the entire study population and within relevant experimental groups: 1) autism and 2) controls. Based on these analyses of normality, parametric tests were used when appropriate.2) estimates the proportion of variance in a dependent variable  that is accounted for by an independent variable . Fisher's transformation was used to calculate a p-value from the Pearson's r. For our analyses we considered a p-value of .05 to be statistically significant.Pearson's correlation coefficient (r) between two variables is defined as the covariance of the two variables divided by the product of their standard deviations. The square of the Pearson's correlation coefficient , subject age, and scan batch . All possible interaction terms were also included in the model. For our analyses we considered a p-value of .05 to be statistically significant.http://www.graphpad.com/quickcalcs/contingency1.cfm) was used to assess for a significant overlap between gene lists. A chi-square with Yates correction was used to calculate chi squared and a two-tailed p-value.An internet based 2×2 chi square contingency table , we tested whether subject age had an effect on the relationship between diagnosis and overall variance. An analysis of covariance (ANCOVA) demonstrated that even when controlling for subject age, variance in gene expression continued to be significantly decreased by the same amount in the blood of children with autism compared to controls . When scan batch was included with subject age in the ANCOVA the results also remained significant . Importantly, parameter estimates for the relationship of diagnosis to variance were virtually unchanged in the ANCOVAs indicating that increasing p-values were related to increases in the degrees of freedom in the analysis, and not to a decreased association.2 = .08, p = .03, parameter estimate  = −.054 Std Dev/year of paternal age)  . Using aBecause increased paternal age and autism were both negatively related to overall variance in gene expression, it was predicted that the overall variance in gene expression for children of older fathers would be similar to that in children with autism. To confirm this hypothesis, we performed a median split of subjects by paternal age (median  = 31 years) and assessed for differences in overall variance in gene expression between children with autism and controls in groups of children from older and younger fathers. As predicted, we found that the overall variance was the same in the blood of children from older fathers as in the blood of children with autism from fathers of any age . We founTo evaluate for a possible mechanism underlying decreased variance in gene expression in the blood of children with autism or from older fathers, we analyzed for specific gene expression differences between study groups. Gene expression patterns in the blood of children with autism and control children of older fathers were compared to gene expression patterns in the blood of control children of younger fathers. First, it was noted that in the blood of children with autism and children of older fathers, there were many more significantly down-regulated genes than up-regulated genes . In the To determine if these overlapping genes were enriched for particular biological pathways, DAVID was used to perform pathway analysis. As background, we used the 25,146 probe sets that were above our background cut-off requiring that they were called present in at least 50 out of 146 samples. Pathway analysis indicated that multiple pathways associated with transcriptional regulation and supported by multiple independent databases, e.g. GOTERM, BIOCARTA, OMIM disease, etc., were enriched within the shared set of genes that were down-regulated in children with autism and children of older fathers . ImportaTo evaluate the robustness of our findings, experimental groups were sub-divided to perform statistical cross-validation. For instance, the group of 82 children with autism was divided into 2 groups of 41 children that were balanced for paternal age and microarray scan batch (Autism1 and Autism2), the group of children with older fathers (>30 years old) was divided into 2 groups of 14 children (Old father1 and Old father2), and the group of children of younger fathers (< or  = 30 years old) was divided into 2 groups of 14 children (Young father1 and Young father2). With respect to variance in gene expression , cross-vCross-validation of pathway analysis indicated that these findings were highly robust. Significant gene lists from subgroups  indicated that in all cases, significantly down-regulated genes in children with autism or children of older fathers were enriched for genes involved in transcription . It was Pathway analysis showed that regulators of transcription were enriched in the subset of genes that were down-regulated in children with autism and control children of older fathers. This suggested that dysregulation of transcription might be a mechanism underlying the decreased variance in gene expression in these groups of children. To explore this hypothesis, we used a publically available dataset to examine whether inhibition of transcription can lead to decreased variance in gene expression. In the gene expression study, a human lung cancer cell line, A549, was treated with transcription inhibitors for four hours. Treatment with high levels of sapphyrin , a new class of transcription inhibitor, or actinomycin D  led to a decrease in variance in gene expression when compared to control treatment with mannitol . Low levStudying the variance in the distribution of gene expression represents a new approach to gene expression analysis. Because changes in a limited number of genes could not account for a change in the shape of the entire gene expression distribution, changes in variance suggest alterations at global levels of gene expression regulation. This “birds-eye view” of gene expression levels contrasts with gene-centered approaches that seek to identify specific genes or pathways that differ between groups. While gene specific approaches are useful they may miss changes occurring at the global level of gene expression regulation. The two approaches, therefore, represent distinct yet potentially complimentary approaches to understanding the full magnitude of effects on gene expression regulation. Here we used our novel approach of studying the gene expression distribution to associate decreased variance in gene expression levels with the diagnosis of autism and a risk factor for autism, increased paternal age. We complimented this global approach with gene specific analyses that indicated that changes in global levels of gene expression regulation might be related to large-scale changes in transcriptional regulation. Finally, we reinforced the potential role of transcription regulation by demonstrating with a publically available dataset that inhibition of transcription could, in fact, lead to decreased variance in the distribution of gene expression levels.Global levels of gene expression regulation may be particularly important in understanding the pathological basis of diseases such as autism where multiple systems are affected. This is true because alterations at the global level of gene expression regulation could be shared across systems even when the tissue specific genes affected by these global changes were different. For instance, in the current study, even though gene expression patterns in the brain and blood are known to be different, it is possible that in the brains of children with autism, variance in gene expression is decreased as it is in the blood. Thus, it is possible that a common mediator, a change at the global level of gene expression regulation, could offer a unifying explanation for multi-systemic effects of disease. By understanding that global levels of gene expression regulation can be disrupted in disease, it becomes clear that targeting global levels of gene expression regulation might also be therapeutic, an idea strongly supported by successful treatments such as HDAC inhibitors in mental health disorders and cancerThe association of paternal age with overall variance in gene expression in the blood of healthy controls is interesting. In our study, decreased variance in gene expression was a risk factor for the diagnosis of autism. That another well established risk factor for autism, increased paternal age, was associated with a similar decrease in variance raises the interesting possibility that the associated increased risk of autism in children of older fathers could be mediated by changes in global levels of gene expression regulation or by paternally transmitted age-related factors that are linked to changes in the global regulation of gene expression. In other words, an effect on global levels of gene expression regulation transmitted by older fathers might act to increase the likelihood that neurodevelopmental abnormalities will occur and thus increase the rates of autism in children of older fathers. Determining whether this hypothesis is correct would require a large-scale prospective study in which statistical models assessed whether increased rates of autism in children of older fathers were statistically mediated by effects on variance in gene expression.de novo germ line mutations and global changes in DNA methylation The association of variance in gene expression with paternal age is also interesting because it suggests that fathers may transmit factors influencing global levels of gene expression regulation in offspring. Paternal age has previously been associated with another genomic phenotype, increased telomere length, indicating that, in principal, global genomic properties can be sensitive to paternal ageThe parallels of the current study to our work in mice also bear discussion. We found in mice that epigenetic factors acted during early brain development to elicit changes in adult behavior that were accompanied by changes in the variance in gene expressionAlterations in the overall variance in gene expression in peripheral blood lymphocytes are also of interest. Children with autism have immunological abnormalities and it has been previously reported that gene expression differences were found in immune cells of children with autism Finally, traditional approaches to gene expression analysis suggested a possible mechanism for the changes in gene expression variance. We found enrichment for genes involved in transcriptional regulation in genes that were down-regulated with respect to paternal age and autism. Analysis of the effects of transcription inhibition on gene variance was supportive of the hypothesis that down-regulation of transcriptional regulators may account for the decreased variance in gene expression in the current study. Also of interest we found enrichment for genes regulated by zinc, an important component of many transcription factors. Since it has been demonstrated that neuronal plasticity is often transcription dependent Multiple lines of evidence including genetic linkage Figure S1Cross-validation. Figure describes the work flow used for cross-validation. Briefly, experimental groups were divided in half to create 6 subgroups that were balance for scan batch . a) Shows cross-validation of changes in gene expression variance. The means of autism1, autism2, older father1, and older father 2 are lower than the means of younger father 1 and 2, but only the autism subgroups are significantly different. b) Demonstrates a significant overlap between down-regulated genes in autism1 vs. young father1 and autism2 vs. young father2 . c) Demonstrates a significant overlap in down-regulated genes in old father1 vs. young father1 and old father2 vs. young father2 .(TIFF)Click here for additional data file.Table S1Cross-validation of pathway analysis of down-regulated genes. Pathway analysis was done on significant gene lists  from cross-validation datasets . The table illustrates that cross-validation datasets highlighted the same enrichment of biological pathways related to transcription and zinc.(PDF)Click here for additional data file.Table S2Non-overlapping down-regulated genes. Pathway analysis was done on significant gene lists  of genes that were down-regulated in autism or in children of older fathers and not overlapping with the other group.(PDF)Click here for additional data file.Table S3Up-regulated Pathways. Pathway analysis was done on significant gene lists  of genes that were up-regulated in autism or in children of older fathers.(PDF)Click here for additional data file.Table S4Pathway analysis with increased stringency for gene list selection. Pathway analysis was done on significant gene lists  of genes that were down-regulated in autism and in children of older fathers. Table shows that the same pathways were enriched as with lower stringency gene list selection.(PDF)Click here for additional data file.


Endoscopic discectomy method is a novel technique that is increasingly used in spine surgery. Previous studies have reported that some common complications like dural adhesions are lower in this technique compared with the other techniques. Furthermore, treatment outcomes are reportedly higher because of minimal invasion. Thepresent study aims to determine the outcome of percutaneous endoscopic discectomy.A total of 130 patients underwent the lumbar disc prolapse operations during 2008 to 2012 in all of them the entire procedure was performed endoscopically. All procedures were carried out from a posterior approach using a 4-mm Hopkins 0 degrees-telescope placed in the working insert equipped with channels for suction tube, operative instruments and nerve root retractor (ENDOSPINE instrumentation (Karl STORZ GmbH and Co. KG). The pre- and post-operation pain was assessed using a Visual Analog Scale (VAS). Furthermore, the treatment outcome was assessed using modified MacNab criteria before operation, 24 hours, one month, 2 months, 6 months, one year, and two years after operation . Good to excellent outcome was achieved in 89% of patients, which is comparable with the results of classic microdiscectomy. The mean age of patients was 35.6 years old and the mean length of follow-up was 3.4 years. There was significant reduction in the severity of back pain and lower limb symptoms at 6 months and 2 years post-operation. There was significant improvement in all aspects of the Quality of Life scores at 6 months and 2 years post-operation. In 3 patients the dural sac was lacerated but none of the tears was exceed a few mm in length with not association with neural injury.Findings of this study showed that percutaneous endoscopic lumbar discectomy is associated with improvement in back pain and lower limb symptoms. It has the advantage that it can be performed on a day case basis with short length of hospitalization and early return to work thus improving quality of life earlier.Endoscopic discectomy, Spine surgery, Lumbar disc prolapse operation, Outcome