Background. We evaluated whether a pilot program providing point-of-care (POC), but not rapid, CD4 testing (BD FACSCount) immediately after testing HIV-positive improved retention in care. Methods. We conducted a retrospective record review at the Themba Lethu Clinic in Johannesburg, South Africa. We compared all walk-in patients testing HIV-positive during February, July 2010 (pilot POC period) to patients testing positive during January 2008–February 2009 (baseline period). The outcome for those with a ≤250 cells/mm3 when testing HIV-positive was initiating ART <16 weeks after HIV testing. Results. 771 patients had CD4 results from the day of HIV testing . ART initiation within 16 weeks was 49% in the pilot period and 46% in the baseline period. While all 421 patients during the pilot period should have been offered the POC test, patient records indicate that only 73% of them were actually offered it, and among these patients only 63% accepted the offer. Conclusions. Offering CD4 testing using a point-of-care, but not rapid, technology and without other health system changes had minor impacts on the uptake of HIV care and treatment. Point-of-care technologies alone may not be enough to improve linkage to care and treatment after HIV testing. AlthougBecause so many patients do not return for their CD4 count results, one improvement to the existing HCT practices that may reduce losses to HIV care and treatment after diagnosis is the use of point-of-care (POC) CD4 testing immediately after HCT , 6–10. TBetween February and July of 2010, the Themba Lethu Clinic piloted the introduction of POC CD4 testing in its HCT program using the BD FACSCount system. By offering same-day CD4 test results, combined with post-CD4 test counseling, the pilot program aimed to improve linkage to HIV care and treatment after HCT. We used retrospective data extracted from program records to evaluate whether this pilot program was successful in achieving this goal.Themba Lethu HIV Clinic was founded in 2004 as part of the scaleup of HIV treatment . The cli3 were eligible for ART [3.Throughout the study periods and until July 2011, adults with a CD4 cell count ≤ 200 cells/mm for ART . This poUnder standard care at Themba Lethu Clinic, HCT patients receive pre- and posttest counseling that emphasizes the importance of CD4 testing for understanding disease progression and determining eligibility for antiretroviral therapy. Patients who test HIV-positive are encouraged to initiate standard CD4 testing immediately after posttest counseling by having a venipuncture blood draw by a nurse in a dedicated phlebotomy room in the clinic. The blood sample is sent to the National Health Laboratory Service (NHLS), where the test is run using the single platform panLeucogated CD4 platform . The NHL3 were “tracked for ART initiation,” which typically involved 2-3 additional visits to the site for medical assessment, education, and counseling prior to ART initiation [At the next visit to the clinic, the CD4 test result is reviewed with the patient. During the study period, patients who had a CD4 count ≤ 250 cells/mmitiation . PatientAt the start of the pilot period, a CD4 flow cytometry machine, the BD FACSCount, was placed in the phlebotomy room, allowing CD4 counts to be conducted at the clinic rather than sending samples to the laboratory. A site phlebotomist was trained in using the machine. During the pilot period, POC CD4 testing was integrated into the routine cascade of care. During the pilot period, the intention was to offer the POC test to all patients testing HIV positive during HCT. During the pilot, patients received the same pre- and post-HIV-test counseling conveying the importance of CD4 testing as during the baseline period, but counselors also explained that the site could complete the CD4 test and provide results during the same visit. Patients consenting to the POC test had two vials of blood drawn from the same venipuncture; one was sent to the NHLS, while the phlebotomist tested the other one in the clinic using the on-site flow cytometry technology. Patients not opting for the POC test had one vial of blood drawn for NHLS testing.Patients who opted for the POC CD4 test were then asked to wait in the clinic until the test was completed. Although a randomized clinical trial using the same technology reported that patients received test results within 45 minutes , during During the pilot, consistent with the standard of care at the site, all patients were counseled to return to the site after one week. Patients who already received POC CD4 count results and were tracked for ART initiation could schedule this second visit to be the next sequential visit in the treatment initiation process . Patients who had already received a POC CD4 count result, but were tracked for the wellness program, received additional counseling on the importance of returning on schedule for their next pre-ART care visit 3 or 6 months later. At this second visit, the tracking decision for further medical care (ART or pre-ART care) could be confirmed for those patients completing the POC test or determined for those who did not, based on the NHLS CD4 result.We extracted data from clinic records for all walk-in HCT patients testing HIV-positive during a baseline (prepilot) period (January 2008 to February 2009) and during the pilot period (February 2010–July 2010) and for up to 8 months of followup. The data for the baseline period were published previously , 4. DataStatistical analysis was conducted using STATA (version 11). A two-sample test of equality of proportions was used to test for differences between the two study periods. We present crude and adjusted risk differences (RDs) and relative risks (RRs) and corresponding 95% confidence intervals. Adjusted relative risks were estimated using a modified Poisson approach . Models Ethical approval for this retrospective evaluation was obtained from Boston University and the University of the Witwatersrand.3 in the pilot and baseline groups, resp.).3 on the day of HCT and should have been tracked for ART initiation at the site. Only 46% and 49% of these patients initiated ART within 16 weeks during the baseline and pilot periods, respectively. Comparing the two groups, there was little absolute  or relative increase  in the proportion of eligible patients initiating ART within 16 weeks of testing positive. As Relative risks adjusted for sex, employment status, and age are reported in Of those patients with CD4 > 250 who were scheduled to return for a medical visit as part of the wellness program see , only 28The results reported in If only the subset of patients offered the POC test during the pilot period is included in the analysis, 54% of those with a CD4 result ≤ 250 initiated ART within 16 weeks of HIV testing compared to 43% in the baseline period . Although the 54% in the intervention period for those offered the test represents an improvement upon the 49% observed in N = 194) accepted the offer to complete the POC test. It is likely that patients walking in to a clinic for HCT were simply unwilling to wait the extra 2-3 hours for their test results and therefore declined the offer. These same people might also be least likely to return for their CD4 test results a week later and thus remain in care. Women were more likely to accept the test than men, with little difference in acceptance between those employed or not.Second, among the 308 patients offered the POC test during the pilot period, only 63% (N = 110), 59% initiated ART within 16 weeks of HCT. The 59% observed in this subset of patients is substantially better than the 49% and 46% observed for all patients in the pilot and baseline periods. Because a substantial number of patients refused the offer of the POC test, these results very likely overestimate the impact of this intervention on linkage to HIV care and treatment in the general population. Among the subset of patients who were offered the POC test, accepted the opportunity, and had a baseline CD4 result ≤ 250 , which produces a CD4 count in about 20 minutes, can readily be integrated into HIV counseling and testing programs and can have the potential to increase uptake of HIV care and treatment , 7, 15. Although there have been some successful demonstrations of point of care technologies, however, the benefits and costs of adopting these innovations are far from clear and are likely to vary widely by context and approach. We found that a less-rapid point of care technology, while feasible to implement and still creating the opportunity for same-day results, had little effect on patient behavior after a positive HIV test. The results presented here, based on patients presenting for HIV testing at a large, urban clinic, suggest that CD4 testing technology that is point of care but not rapid and that is not supported by other changes to clinic procedures has at best a minor impact on returning to care and initiating HIV treatment. In the pilot we evaluated that a majority of patients tracked for ART initiation on the day of testing HIV-positive did not initiate ART at the site with 16 weeks of HIV testing (52% across both study groups combined) and there was little improvement from the baseline period to the pilot period. Few patients with higher CD4 cell counts (>250) in each study period returned to the site for a next pre-ART care visit within one month of the recommended schedule; the point of care technology made no difference to this outcome.As noted above, results presented in other studies are also modest. The largest improvement was reported in a randomized clinical trial at a nearby public hospital in Johannesburg, where patients were randomized to one of three groups . In this trial, 65% of patients receiving the POC test after HCT and found to be eligible for ART “reported for ART initiation” at a nearby ART clinic within 3 months of HIV testing. Compared to 31% of similar patients reporting in the standard of care arm, the study found a substantial improvement in this outcome (from 31% to 65%), although reporting for ART initiation is not the same as actually initiating ART. If such results held at scale this could indeed be an important benefit. However, given the very small sample size (only 43 patients receiving the POC CD4 test were ART eligible) and the trial conditions, under which patients consented to participate in the study and were then randomized to a study group, it is unclear to what degree such gains would be realized under routine conditions. It seems likely that under routine conditions, the trial site would experience many of the same operational constraints as did our evaluation site.Other studies evaluating the potential impacts of providing CD4 test results more quickly to patients after HIV testing, whether using rapid POC technologies such as the Pima Analyzer or simply calling back patients with their CD4 results, have found modest improvements in linkage to care in fixed and mobile HCT programs , 15, 17.Two tentative conclusions emerge from this evaluation. First, simple technological fixes might not be sufficient to address problems stemming from human behavior. Enrolling in care or treatment after HIV testing is fundamentally driven by patient-level behaviors and decisions, which are influenced by the information and services provided by the site, but not decided by the site. After an HIV diagnosis, some patients may not be able to focus on the implications of a specific CD4 count result, while other patients might very welcome the additional information. Even leaving HCT with clear knowledge of their eligibility for treatment and every intention to return on time to the site (their long-term preference), some patients may not actually make the decision to return on or near the scheduled day, for a range of emotional, social, and economic reasons (an example of short-term preferences at odds with long-term preferences). Offering better information to patients is important, but it is not a complete solution.Second, new technologies may not be effective without health systems adaptations to accommodate and integrate them. In the case of the Themba Lethu Clinic pilot, the desire to process samples in batches meant that patients who arrived for HCT sporadically over the course of the day had to wait significantly longer than might otherwise have been necessary to receive their results. Performing HIV tests only during specified hours, rather than throughout the day, might have minimized this problem, though possibly at the expense of testing access. Similarly, shortages of supplies and staff meant that many patients were not offered POC testing at all, despite the presence of the POC instrument on site. In both instances, health systems changes were needed to support effective use of a new technology. 3 that prevailed at the time of the study. Under the revised CD4 count eligibility criterion of 350, adopted in August 2011, some 80% of patients during the pilot period would have been eligible for ART at the time of their HIV test. The late disease stage at which most HIV-positive South Africans continue to learn of their infection and come forward for care underscores the importance of getting our linkage-to-care interventions right, with and without the benefits of new technologies. Finally, it is important to note that in our study, a majority of walk-in HCT patients were already eligible for ART at the time of HIV diagnosis even under the restrictive threshold of 200 CD4 cells/mm


Dengue is the most important vector-borne viral infection of man, with approximately 2 billion people living in areas at risk. Infection results in a range of manifestations from asymptomatic infection through to life-threatening shock and haemorrhage. One of the hallmarks of severe dengue is vascular endothelial disruption. There is currently no specific therapy and clinical management is limited to supportive care. Statins are a class of drug initially developed for lipid lowering. There has been considerable recent interest in their effects beyond lipid lowering. These include anti-inflammatory effects at the endothelium. In addition, it is possible that lovastatin may have an anti-viral effect against dengue. Observational data suggest that the use of statins may improve outcomes for such conditions as sepsis and pneumonia. This paper describes the protocol for a randomised controlled trial investigating a short course of lovastatin therapy in adult patients with dengue.A randomised, double-blind, placebo-controlled trial will investigate the effects of lovastatin therapy in the treatment of dengue. The trial will be conducted in two phases with an escalation of dose between phases if an interim safety review is satisfactory. This is an exploratory study focusing on safety and there are no data on which to base a sample size calculation. A target sample size of 300 patients in the second phase, enrolled over two dengue seasons, was chosen based on clinical judgement and feasibility considerations. In a previous randomised trial in dengue, about 10% and 30% of patients experienced at least one serious adverse event or adverse event, respectively. With 300 patients, we will have 80% power to detect an increase of 12% (from 10% to 22%) or 16% (from 30% to 46%) in the frequency of adverse events. Furthermore, this sample size ensures some power to explore the efficacy of statins.The development of a dengue therapeutic that can attenuate disease would be an enormous advance in global health. The favourable effects of statins on the endothelium, their good safety profile and their low cost make lovastatin an attractive therapeutic candidate.International Standard Randomised Controlled Trial Number ISRCTN03147572 Dengue is the most common and important vector-borne viral infection of man, with at least 2 billion people living in areas of risk . ClinicaStatins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. They were first used clinically in the late 1980s and quickly became established as an effective drug for both lipid lowering and mortality reduction in cardiovascular disease . They arin vitro work has demonstrated that lovastatin may have an anti-viral effect in dengue by reducing virion assembly [One of the major features of both dengue and sepsis is widespread vascular endothelial disruption resulting, in part, from exposure to inflammatory mediators -16. Thisassembly ,19.in vitro experiments [in vitro, we believe it is rational to test the drug used in the original work. Furthermore, lovastatin is commonly prescribed and available in Vietnam as a generic and will therefore be immediately available to patients if there is a positive result from the trial.We have chosen lovastatin as the study drug for both scientific and pragmatic reasons. In light of the US Food and Drug Administration’s safety recommendations about the risks of muscle toxicity with simvastatin, there were concerns from the regulatory authorities in Vietnam about the safety of using this agent in dengue. An additional reason for our choice was the dengue anti-viral effect observed with lovastatin in eriments . As thisTypically, severe vascular leakage and shock occur around the fifth day of illness in patients with dengue . It is pAlthough there is extensive experience of using statins as a lipid-lowering agent, and an increase in the amount of observational data from their use in critically ill patients, this will be the first study looking at the use of statins in dengue. We propose to investigate the effect of lovastatin for 5 days in adult dengue patients presenting in the first 72 hours of illness. The rational of a 5-day treatment course is to cover the transient ‘critical’ phase of infection when complications arise, which typically occurs around the fifth day of illness . Our proThis study is a randomised, placebo-controlled, double-blind trial investigating lovastatin therapy in Vietnamese adults with dengue infection. The trial will be conducted in two phases, with an escalation of dose between phases if the results of an interim data review show no safety concerns within the first cohort of patients treated with the lower dose.Patients will be followed for clinical and laboratory endpoints in hospital until study day 6 (or daily as out-patients from discharge to day 6) and reviewed at an outpatient visit on day 28.All patients aged 18 or more presenting to the Hospital of Tropical Diseases, Ho Chi Minh City with a clinical suspicion of dengue, less than 72 hours of fever and a positive rapid test for dengue non-structural protein 1 (NS1) will be eligible for recruitment into the study. Exclusion criteria are: signs or symptoms suggestive of another acute infectious disease, alanine transaminase levels greater than 150 U/l, creatine kinase levels greater than 1000 U/l, liver cirrhosis, myopathy, current or use within past week of statins, pregnancy and lactation. In addition, patients taking medications contraindicated for use with statins, for example, isoniazid for treatment of tuberculosis, will be excluded.The primary endpoint of this study is an evaluation of the safety and tolerability of lovastatin therapy in adult patients with dengue. Comparing the rates of adverse events between randomised treatment arms will assess this.The secondary endpoints of this study are fever clearance time (see definition), plasma viraemia (area under the log-transformed viraemia curve from enrolment until study day 6), platelet nadir between day 3 and 8 of illness, maximum haematocrit between day 3 and 8 of illness, percentage increase in haematocrit between day 3 and 8 of illness from baseline, maximum alanine transaminase (ALT) and creatine kinase (CK) recorded between day 3 and 8 of illness, lowest oxygen saturation recorded between day 3 and 8 of illness, number of patients in each group requiring colloid, and disease progression as defined by one or more of the following: (a) admission to the ICU, (b) diagnosis of shock (see definition), (c) diagnosis of severe bleeding (see definition), (d) development of encephalitis, (e) death. In addition, quality of life scores obtained using a visual analogue scale will be compared between treatment groups. The times from enrolment to the first sample with viraemia less than 1000 copies/ml and the first negative NS1 antigenaemia sample will be compared between treatment groups.1. Fever clearance time is the time from enrolment to the first time the temperature falls to <37.5°C and remains below this level for 48 hours.2. Shock: cardiovascular decompensation requiring fluid resuscitation and considered to be due to plasma leakage.3. Severe bleeding is clinically severe if it results in haemodynamic instability or requires fluid resuscitation or a blood transfusion. Any bleeding resulting in death and any intracranial bleed are considered severe.4. Baseline haematocrit: the haematocrit value obtained at the day 28 follow-up visit, or  the expected age- and sex-matched population value.Randomization to either treatment arm will be in a 1:1 ratio. Randomization will be stratified according to the ward of recruitment. A randomization list will be prepared and maintained confidentially from study staff by the clinical trials pharmacist. Block randomization using variable block sizes will be used.A chronological log of all enrolled patients will be maintained. Each enrolled patient will be assigned the next available sequential study code. The assigned number will correspond to a coded, sealed, pre-packaged bottle containing six doses of either lovastatin or visually matched placebo. Blinding will be maintained amongst the attending physicians and nurses by ensuring that the study drug and the placebo have an identical appearance. In addition the administration schedule will be identical.Patients presenting to the out-patients department or in-patient wards with a clinical suspicion of dengue and less than 72 hours of fever will be identified to study staff. Study staff will approach these patients, check eligibility criteria and confirm dengue by NS1 rapid test. Eligible and consenting patients will have screening blood tests sent and provided these are satisfactory will be allotted the next consecutive study number and enrolled to the study.This trial will be conducted in two phases with an escalation of dose if the results of an interim data review show no safety concerns within the first cohort treated with the lower dose. The dose will begin at 40 mg per day in cohort 1 and may continue at 80 mg per day in cohort 2. A DSMB (data and safety monitoring board) review will take place when day 6 study data are available from the 30th patient enrolled in cohort 1. If this review is satisfactory, the dose will be increased to 80 mg per day and recruitment into cohort 2 will commence. Further DSMB reviews will take place when day 6 study data are available from the 30th and 100th patients enrolled in cohort 2.Patients will be assigned to one of two treatment arms:Active medicinal product: 40 mg (stage 1) or 80 mg (stage 2) lovastatin once daily for 5 days.Placebo: visually matched placebo once daily for 5 days.The first dose will be given as soon as practically possible after enrolment.An unblinded study pharmacist will prepare study drug bottles centrally and will distribute the bottles as required. Drugs will be stored in accordance with the manufacturers’ recommendations in a secure area. Lovastatin and the placebo must be maintained below 25°C. All movements of study medication will be recorded. Both individual subject and overall drug accountability records will be kept up to date by the study staff.Patients will be followed by a study physician daily until discharge, and all signs and symptoms recorded in the case report form. An ultrasound scan will be performed on day 6 of illness to detect signs of plasma leakage. Clinical management decisions will remain in the hands of the attending ward doctors. In the event that shock or any other serious complication develops, the patient will be transferred to the appropriate ICU. Details of all adverse events will be recorded on specific forms, together with an assessment as to whether the event is likely to be related to any treatment received, and all serious events will be reported promptly to the DSMB.Quality of life will be measured by questionnaire and visual analogue scale daily.Patients who are fit to discharge on or after study day 3 may be followed as an outpatient until study day 6. All patients will be asked for attend a follow-up visit for review after 4 weeks.Haematocrit, platelet and total cholesterol measurements will be carried out daily or more frequently if clinically indicated. These tests will be repeated at the follow-up visit.Renal and liver function tests, electrolytes and coagulation profiles, will be carried out at enrolment, 48 hours later, day 5 or 6 of illness and at the follow-up visit. If the ALT measured 48 hours after enrolment is greater than 250 U/l, the study drug will be discontinued. It should, however, be noted that hepatic dysfunction might be secondary to dengue infection and could be positively affected by statin therapy.Conventional serological and virological tests will be used to confirm dengue infection and identify the infecting serotype. Plasma samples collected at daily intervals until discharge (and daily until day 6 if discharged before day 6) will be assessed for viraemia levels, NS1 levels, and concentrations of various pro- and anti-inflammatory cytokines .MICB and PLCE1[DNA will be extracted from residual blood samples and genotyped for genetic variants known to be associated with severe dengue, for example, and PLCE1.This is an exploratory study focusing primarily on safety and there are no preliminary data regarding the effects of statins in dengue on which to base a sample size calculation. A target sample size of 300 patients in cohort 2, enrolled over two dengue seasons, was chosen based on clinical judgement and feasibility considerations. In a previous randomised trial in dengue, about 10% and 30% of patients experienced at least one serious adverse event or adverse event, respectively . With 30The primary analysis population will include all patients randomised to placebo from cohort 1 and all patients (regardless of treatment assignment) from cohort 2 according to the intention-to-treat principle. Owing to their low number, patients randomised to low-dose statins from cohort 1 will only be descriptively analysed.The proportion of patients with any adverse events, any serious adverse events, or specific adverse events will be summarised and compared between the treatment arms based on Fisher’s exact test.Pre-defined secondary endpoints will be compared between the two treatment arms based on linear regression for continuous endpoints, logistic regression for binary endpoints, and Cox regression for time-to-event endpoints. For laboratory markers, comparisons will be adjusted for the pre-dose value of the respective marker and the day of illness at enrolment; plasma viraemia and NS1-endpoints will additionally be adjusted for dengue serotype.MICB and PLCE1 genotype using descriptive statistical methods.The clinical, virological and immunological findings will also be correlated with A detailed statistical analysis plan will be finalised prior to unblinding the study data base.This protocol and both the patient information sheet and the consent form have been reviewed and approved by the Institutional Review Board of the Hospital for Tropical Diseases in Ho Chi Minh City, the Oxford Tropical Research Ethics Committee and the Ethics Committee of the London School of Hygiene and Tropical Medicine.All patients entering the study must give informed consent.Each participant has the right to withdraw from the study at any time. The reason for withdrawal will be recorded in the case report form.Patients who enter the trial will be given a unique identification number. This number will be used on both laboratory specimens and case report forms. The study wards and the research unit have the facilities to store study information securely.An independent DSMB will be set up consisting of a biostatistician and senior clinical researchers with expertise in dengue and clinical trials. The DSMB will review the protocol and agree to a data review schedule and reporting requirements before the study commences. All data reviewed by the DSMB will be in the strictest confidence. A DSMB charter will outline its responsibilities and operation.The DSMB will perform a safety review after day 6 data are available for the first 30 patients enrolled (Cohort 1: 40 mg lovastatin daily). This review will be based on a report created by the DSMB statistician containing unblinded summary tables of baseline demographics, serious adverse effects, adverse effects and disease progressions, as well as viraemia curves. If no safety concerns are identified, the lovastatin dose will be increased to 80 mg daily and recruitment will commence in cohort 2. Enrolment will continue in cohort 1 while awaiting the outcome of the DSMB review. Additional safety reviews will take place after the day 6 data are available for the 30th and 100th patients in cohort 2.Dengue remains a significant global public health challenge with costs to both the infected individual and the struggling health systems of dengue-endemic countries. At present, treatment is limited to supportive care. A therapeutic that can attenuate disease and prevent progression to severe disease would represent a highly significant advance with enormous benefits for both patients and health systems. There is growing observational evidence from the critical care field to suggest that statins may have a beneficial role in a number of conditions, such as sepsis, acute lung injury and pneumonia. Statins have beneficial pleiotropic effects, including stabilizing and anti-inflammatory effects on the endothelium. As endothelial dysfunction is so important in dengue pathogenesis, stabilizing effects at this site may prove to be clinically beneficial. In addition, statins have an excellent safety profile and a low cost. In view of this, we are optimistic about the potential benefit of lovastatin in dengue.We expect that patients will start being recruited to this trial in November 2012.ALT: alanine transaminase; CK: creatine kinase; DSMB: data and safety monitoring board; FDA: US Food and Drug Administration; IFN-γ: interferon-gamma; IL-6: interleukin-6; IL-10: interleukin-10; MICB: MHC class I polypeptide-related sequence B; NS1: non-structural protein 1; PLCE1: phospholipase C epsilon 1; TNF-α: tumour necrosis factor-alpha.The authors declare that they have no competing interests.JW, NVVC, TTH, RP, CS, BW and JF conceived of the study and participated in its design. JW developed the study protocol with NTT, LTHT, NVH, MW, LM and NTPD. All authors read and approved the final manuscript.


Bdnf and Kcnj13. To analyze these data from a systems perspective, we applied weighted gene co-expression network analysis (WGCNA) to identify groups of co-expressed genes (modules) and their central (hub) genes. One such module contained genes upregulated in the epileptogenic region, including multiple epileptogenicity candidate genes, and was found to be involved the protection of glial cells against oxidative stress, implicating glial oxidative stress in epileptogenicity. Another distinct module corresponded to the effects of chronic seizures and represented changes in neuronal synaptic vesicle trafficking. We found that the network structure and connectivity of one hub gene, Sv2a, showed significant changes between normal and epileptogenic tissue, becoming more highly connected in epileptic brain. Since Sv2a is a target of the antiepileptic levetiracetam, this module may be important in controlling seizure activity. Bioinformatic analysis of this module also revealed a potential mechanism for the observed transcriptional changes via generation of longer alternatively polyadenlyated transcripts through the upregulation of the RNA binding protein HuD. In summary, combining conventional statistical methods and network analysis allowed us to interpret the differentially regulated genes from a systems perspective, yielding new insight into several biological pathways underlying homeostatic anti-epileptogenic effects and epileptogenicity.Neither the molecular basis of the pathologic tendency of neuronal circuits to generate spontaneous seizures (epileptogenicity) nor anti-epileptogenic mechanisms that maintain a seizure-free state are well understood. Here, we performed transcriptomic analysis in the intrahippocampal kainate model of temporal lobe epilepsy in rats using both Agilent and Codelink microarray platforms to characterize the epileptic processes. The experimental design allowed subtraction of the confounding effects of the lesion, identification of expression changes associated with epileptogenicity, and genes upregulated by seizures with potential homeostatic anti-epileptogenic effects. Using differential expression analysis, we identified several hundred expression changes in chronic epilepsy, including candidate genes associated with epileptogenicity such as  Epilepsy is a neurological condition with myriad etiologies that is characterized by the recurrence of spontaneous seizures There are several models of MTLE that utilize the injection of a neurotoxin  to induce a brief period of status epilepticus. After a latent period, these animals will go on to develop spontaneous, recurrent seizures The study of epilepsy at the molecular level has been greatly aided by the use of genome-wide expression microarrays, which provide expression information for all genes that are expressed within a biological sample. Several studies have used gene expression data to identify candidate genes involved in epileptogenesis, such as Cystatin C and Homer 1A Here, we used a model of MTLE where kainic acid (KA) is injected into one hippocampus to induce status epilepticus The University of California, Los Angeles, Institutional Animal Care and Use Committee approved all procedures involving animals described in this study. The ARC protocol ID is #2000-153-32. Total of twenty-seven oubred male (250–300 g) Wistar rats were used in these studies. Adult male rats were given atropine (0.04 mg i.m.), anesthetized with isofluorane 2%, and unilaterally injected with kainic acid  in the right posterior CA3 area of hippocampus , microarray (n = 10).Eight – twelve months after kainic acid injection rats selected for electrophysiological experiments were anesthetized with isofluorane, 2%. Pairs of tungsten wires (60 µm in diameter) with 0.5 mm vertical tip separation were placed in the right angular bundle to stimulate perforant path afferents to the hippocampus . Fixed recording microelectrodes also consisted of pairs of tungsten wires with 1.0 mm vertical tip separation. They were implanted bilaterally at symmetrical points in the dentate gyrus (DG) and CA1 region of anterior hippocampus , DG region of the posterior hippocampus ; EC  and piriform cortex .in vivo recordings in freely moving rats, five 4-channel MOSFET input operational amplifiers mounted in the cable connector served to eliminate cable movement artifacts. Physiological data were recorded wide-band 0.1 Hz to 3.0 kHz and sampled at 10 kHz/channel (16 channels) with 12 bit precision using RC-Electronics  software. Location of electrodes was determined on the basis of the shape of responses to perforant path stimulation. Recordings were performed during 1 month 5 days a week and 8–10 hours a day. After completion of electrophysiological experiments rats were given overdose of Nembutal and perfused for following histological verification of the location of the recorded electrodes and for neo-Timm staining for estimation of mossy fiber sprouting During Recorded seizures were classified on the basis of electrophysiological onset pattern, signal averaging and power spectral analysis. For high frequency oscillations (HFOs) analysis raw records were band-pass filtered between 80–500 Hz, using a Butterworth filter with roll off 3 dB, which does not introduce any visible phase lag. Then HFOs were detected by DataPac  software followed by averaging and power spectrogram analysis.Ten rats were taken for microarray studies: five with documented behavioral spontaneous seizures and five rats without spontaneous seizures. Rats were anesthetized with isofluorane, brains were quickly removed and sectioned by vibratome into 400 µm slices. Dentate gyri were removed under dissecting microscope and placed in −80C for further microarray experiments. Horizontal sections were taken approximately 6.2 mm below the bregma. The comparisons were performed between the area of dentate gyri (DG) adjacent to the KA lesion and non-lesioned contralateral side in each animal.RNA extraction was performed using Trizol (Invitrogen). RNA concentration and quality were evaluated using Nanodrop ND-1000 spectrophotometer (NanoDrop) and Agilent 2100 Bioanalyser (Agilent). We used 100 ng of RNA as the initial starting template, which was labeled with Cy-3 or Cy-5 cytidine -5′-triphosphate (CTP) using the low input fluorescent linear amplification kit (Agilent). The labeled cRNA concentration was verified (Nanodrop) and RNA quality was checked on an Agilent Bioanalyzer (Agilent).Two commercial oligonucleotide microarray platforms, the Rat Microarray carrying 20,500 probes representing 15,703 unique GeneBank IDs  and the CodeLink Rat Whole Genome array with 33,664 probes representing 24,329 unique GeneBank IDs  were used to identify gene expression changes. Both platforms contained a total of over 54,164 distinct oligonucletide probes. Due to partial overlap in the gene lists of Agilent and CodeLink microarray platforms, the total list of probes corresponded to 29,080 unique GeneBank sequences that represented 14,439 unique genes with known full-length cDNA sequence (including RIKEN genes) and additional 14,641 expressed sequence tags (ESTs). 9,879 genes were represented by more then one probe, which typically recognized different parts of the transcript. For the Agilent microarrays, the right dentate gyrus from one animal was labeled with Cy-5, and the left dentate gyrus from the same animal was labeled with Cy-3. These were then hybridized to the same slide. The dyes were then swapped, and the same samples were hybridized to another slide. For the Codelink microarrays, all samples were labeled and hybridized in a random order to two chips, each containing twelve arrays. As a result, each individual sample was hybridized to at least three different microarrays (2 Agilent & 1+ Codelink). Microarray hybridization and scanning was performed according to manufacturers protocols. We have deposited the raw data at GEO under accession numbers GSE27015 (Agilent) and GSE27166 (Codelink), and we can confirm all details are MIAME compliant.http://www.r-project.org/). We used available libraries from Bioconductor (http://www.bioconductor.org/) to analyze the one-color and two-color microarray designs. For the two-color Agilent data, we used the linear models package (LIMMA). After the removal of flagged data, we used the default method for background subtraction and used quantile normalization to normalize values between arrays. We used the empirical Bayesian algorithm to determine differential expression and fold changes between different conditions. For the one-color Codelink data, we removed all of the flagged data, as well as all of the control probes. We used the quantile method to normalize the data. A Bayesian ANOVA was used to compare different conditions and determine differential expression.We imported the raw data from the Agilent and Codelink microarrays into R <0.10 and fold change >1.2 for both platforms. Using BLAST, we then mapped probes between the two platforms to determine those that targeted the same exon of the same gene. If a differentially expressed probe targeted the same exon as a non-differentially expressed probe on the other platform, then we required that the two probes be moderately correlated (r>0.5) for that gene to be called differentially expressed. All probes that targeted an exon uniquely and met the differential expression criteria were called differentially expressed.We performed four major analyses to identify differentially expressed genes. 1) Differential expression between the injected and non-injected hippocampus in animals without seizures. 2) Differential expression between the injected and non-injected hippocampus in animals with seizures. 3) Differential expression within the injected hippocampi between animals with seizures and animals without seizures. 4) Differential expression within the non-injected hippocampi between animals with seizures and animals without seizures.http://david.abcc.ncifcrf.gov/) to identify over-represented functional categories within each group of differentially expressed genes The genes that were identified as differentially expressed were used for gene ontology and network analyses. We used DAVID (kME) by calculating the absolute value of the Pearson correlation between the expression of the gene and the ME. The kME of a gene is related to its module centrality and importance to organization of the rest of the module To identify the gene expression patterns in the dataset in an unbiased manner, we performed weighted gene co-expression network analysis (WGCNA) affy package, using optimal parameters kME for those genes within each of the conserved modules. The average kME from our genes of interest was compared to the average kME for a random group of genes that was the same size, and we calculated a Z-score for each of our epilepsy-related modules within all of the consensus modules in the human cortical transcriptome.To determine which cell types that genes in a module were associated with, we used two published resources. The first was a database of gene expression from purified mouse neurons, astrocytes, and oligodendrocytes To validate the expression changes, we used qRT-PCR as previously described In situ hybridization was performed as previously described Gene specific probes were designed to be ∼500 bp and directed against same regions that the microarray probes targeted . In situWe examined an animal model of focal epilepsy in which KA is injected into the hippocampus of one hemisphere. Out of the twelve animals used for detailed electrophysiological and behavioral characterization of seizures, we observed that five (42%) animals had clear behavioral seizures during the monitoring session and seven (68%) did not have clinically observable seizures. Within the seven animals that did not display behavioral seizures during video monitoring, electrographic seizure activity was recorded in two animals. This is consistent with the fact that some animals with KA treatment demonstrate electrographic ictal events without evident behavioral seizures. A majority of the recorded seizures began in the posterior hippocampus of the lesioned hemisphere , which sTo understand the pathological processes initiated by KA that are involved in the ability to generate seizure activity (epileptogenicity), we undertook detailed examinations of multiple animals with and without seizures on both morphological and histological levels. The study design allowed us to compare animals with and without seizures, despite the fact that all animals had experienced an initial neurotoxic lesion. Cell death is thought to be important in the development of epilepsy Ten additional animals were used for analysis of gene expression in chronic epilepsy. The dentate gyri of five animals with documented chronic behavioral seizures were isolated by microdissection, as well as equivalent regions in five animals that did not develop epilepsy Methods; . We tookWe identified differentially expressed genes from both microarray platforms independently (see Methods). For each comparison (see below), we used a false discovery rate (FDR)<0.10 and a fold change >1.2 as criteria to determine differential expression. We were confident in differentially expressed genes at these liberal FDR and fold change thresholds because we will use several comparisons between platforms and conditions to help eliminate false positives. Because different probes that are associated with the same gene may correspond to different exons and measure different splice variants, we carefully compared all differentially expressed probes across the two platforms for their specific location within their target genes (see Methods). To perform this comparison, we used all differentially expressed probes and identified the corresponding probes on the other platform that targeted the same gene. In the simplest case, a probe uniquely targeted a gene and there was no corresponding probe on the other array, and we considered the gene differentially expressed (n = 519). Within the group of genes that were targeted by a probe from each platform, we determined whether or not the two probes targeted the same exon. If a differentially expressed probe did not have a corresponding probe targeting the same exon, then the gene was considered differentially expressed because the probe may represent a differentially regulated splice variant (n = 450). However, if the differentially expressed probe and corresponding probes from the other platform targeted the same exon, we compared the probes using a Pearson correlation, and if the probes were moderately correlated (r>0.5), we considered the gene to be differentially expressed (n = 440). We used this conservative correction based on the correlation to limit false positives caused by noisy or non-specific probes.The experimental design permitted us to separate three main effects on gene expression of major interest: 1) the effect of the KA-induced lesion that is unrelated to epilepsy , 2) the effect of chronic seizures on the brain , and 3) gene expression changes associated with the ability to generate spontaneous seizures . We reasoned that the effect of neuronal injury due to KA injection (L) would be seen in the lesioned hippocampus of both animals with and without seizures . In addiTo identify the specific effect of the lesion, we identified genes that were differentially expressed between the lesioned and non-lesioned hippocampi of animals without seizures (L-O = L). This analysis led to the identification of 24 differentially expressed genes that were specifically associated with the long-term effects of the lesion and independent of any epileptic processes because these animals did not have seizures . InteresGfap and Vim were differentially expressed, and they are known to be present in reactive astrocytes Gene ontology (GO) tools, such as DAVID, annotate the functional aspects of genes based on published literature and can identify any enrichment in these functional categories among a list of genes. We used GO analysis to begin to characterize the biological functions of the 24 genes specifically associated with the response to the lesion, and despite the fact that this list is small and therefore relatively underpowered for such analyses, this group was significantly enriched for genes involved in cell death (p = 7.49e-3) and the acute inflammatory response (p = 1.45e-2) . In addiTo identify the specific effects on gene expression due to chronic seizures alone, we identified differentially expressed genes between the non-lesioned hippocampi of animals with and without seizures (S-O = S). This analysis led to the identification of 385 differentially expressed genes that were solely related to the effects of chronic seizures and unrelated to the effects of the lesion . BecauseTo investigate the functional consequences of the effect of chronic seizures at the molecular level, we used GO analysis on the differentially expressed genes due to seizures in both the lesioned and non-lesioned hippocampi. These groups of genes shared many enriched biological processes, including synaptic transmission (p = 5.91e-6 & p = 3.25e-6), secretion by cell (p = 9.15e-5 &p = 3.78e-4), and regulation of neurotransmitter levels (p = 6.47e-4 & p = 2.14e-4) . Thus, cKcnj13, an inwardly rectifying potassium channel, and Ptgds, which is involved in prostaglandin metabolism  were present in the list and dramatically down-regulated in the epileptogenic region, as well as survival genes, such as bcl-2 related protein Bok. These data suggest that neuronal survival or function may be compromised within the lesioned hippocampus, increasing the epileptogenicity of that region.We hypothesized that candidate genes and pathways associated with epileptogenicity would be specifically differentially expressed within the lesioned hippocampus of animals with seizures. Two of the previous analyses identified differentially expressed genes that corresponded to epileptogenicity. The first group represented the 619 genes differentially expressed between the lesioned and non-lesioned hippocampi in animals with seizures , which atabolism ; Table 3in silico tissue dissection Standard analysis of differential expression allowed us to identify some of the pathological processes and dysregulated genes that are present in this animal model of epilepsy. But, while we identified strong, consistent effects associated with the lesion and seizures, the effects associated with epileptogenicity were subtler. We have recently demonstrated the power of systems biology approaches that utilize network methods to organize gene expression data through the use of weighted gene co-expression network analysis (WGCNA) kME), which is a measure of module centrality. We have previously shown that these central or hub genes are important for module function and organization WGCNA grouped the 11,000 expressed genes into forty co-expression groups or modules  of each of these genes, we were able to identify the central or hub within the module, and we found that the most highly connected gene was Nfe2l2 (kME = 0.97), which remarkably, was previously found to be differentially expressed between the lesioned and non-lesioned hippocampi in animals with seizures. This gene and the other hubs within this module are illustrated in the network plot , which represent another aspect of the anti-oxidant system that is regulated by Nfe2l2By determining the network position (ork plot . Nfe2l2 Nfe2l2 (kME = 0.97), Hmox1 (kME = 0.88), Mag (kME = 0.95), Klk6 (kME = 0.93), Apod (kME = 0.94), and Rdx (kME = 0.96)  . For eackME, with each module representing a cell type. For example, a gene with a greater kME with the neuronal module would be considered a neuronal gene, whereas a gene with a greater kME in the oligodendrocyte module would be considered an oligodendrocyte gene. We calculated the average kME for all genes in the yellow module within all of the cell type specific modules identified in Oldham et al., (2008), and we compared these values to random groups of genes and summarized the average kME values as Z-scores , Ppp2ca (kME = 0.97), Rab5b (kME = 0.97), Ppm1a (kME = 0.92), and Pam (kME = 0.92). We compared the expression of each of these genes between animals with seizures and animals without seizures. Expression in the lesioned hemisphere and non-lesioned hemisphere was compared independently, and we found that each gene was significantly upregulated due to the effect of chronic seizures in both hemispheres , Ywhag (kME = 0.92), Ppm1a (kME = 0.97), and Ppp2ca (kME = 0.92) all showed moderate to high expression within the granule cell layer of the dentate gyrus in both epileptic and non-epileptic animals , there was a highly significant over-representation of HuD targets (p = 2.12e-18), suggesting that HuD could play a role in regulating the transcripts within this module. To confirm that the expression of HuD was increased due to chronic seizures in this animal model, we performed qRT-PCR and found that HuD was significantly upregulated in both hemispheres of animals with seizures when compared to animals without seizures . To determine whether these modules were truly comparable, we compared the top five hundred most highly connected genes in the epileptic seizure-related module and both of the non-epileptic modules, and we found a highly significant overlap in both comparisons (p<1.0e-10). These data both validate the co-expression relationships that we have identified using totally independent data sets and demonstrate that the functions of these modules are comparable. To determine whether connectivity within the seizure-related module was conserved, we compared the connectivity (kME) of each gene in the seizure-related module to the conserved module of both non-epileptic datasets , than in the non-epileptic modules (kME = 0.04 & kME = 0.52). These data suggest that Sv2a has gained a more important role in synaptic function in epilepsy than under normal conditions.Because the blue module was enriched in neuronal genes and synaptic vesicle trafficking is common to all neurons, we hypothesized that this module should be present in other data sets; thus, finding such co-expression relationships independently would provide another level of validation of this module. We examined two different microarray datasets that examined gene expression in non-epileptic samples. The first dataset contained gene expression data from specific neuronal sub-types datasets . We obseSv2a and Arf1 using gene expression from epileptic animals (kME) in the epileptic and non-epileptic networks for each gene that had probes that targeted the same exon. We used this difference to prioritize the genes because genes that had large differences in connectivity between the networks were likely to be very important for the pathological processes that occur in chronic epilepsy  and the epileptogenicity module . These data demonstrate that there are consistently differentially expressed genes due to epileptogenicity, which validates several of the changes observed in this study.Comparison of our data to previous studies was useful in contextualizing our results in terms of previously observed effects in animal models of MTLE. One major review of gene expression studies in epilepsy identified fifty-one consistently differentially expressed genes Fos, which was also differentially expressed in Gorter et al., (2006). Fos is a transcription factor that immediately increases expression in response to seizure activity Fos demonstrated increased severity of KA-induced seizures and increased hippocampal cell death Fos has been shown to upregulate neuronal protective factors such as BdnfFos, however, Bdnf was down-regulated in the epileptogenic region. This decrease in Bdnf expression has been described in other models of chronic epilepsy Tac2 and Cart, were among the down-regulated epileptogenicity candidate genes, and these genes have roles in regulating both neuronal activity and survival The first major goal of this study was to identify gene expression changes associated with the ability of the epileptogenic region to initiate spontaneous seizures or epileptogenicity. Using differential expression analysis, we found that lesion-induced changes were present in animals with and without epilepsy but that these changes were more dramatic in animals with seizures. Although we initially found that the lesions were grossly similar, these data suggest that the lesions were distinct molecularly. After correcting for lesion-induced changes, we identified forty candidate genes associated with epileptogenicity that were enriched for genes involved in neuronal survival. Interestingly, one candidate gene that was significantly upregulated in the epileptogenic region was Fos, was highly connected within this module (kME = 0.85), providing further validation of these genes using another analytic method. The most highly connected gene within this module was the transcription factor Nfe2l2 that is involved in the coordination of multiple different pathways that are protective against oxidative stress We then used WGCNA to examine these important genes and processes associated with epileptogenicity from a systems perspective. The ME for the yellow co-expression module had the greatest correlation with epileptogenicity, although there was one animal without seizures that displayed up-regulation of these genes, which suggests that this animal may have had seizure activity without observable epilepsy. In addition, one of the epileptogenicity candidate genes, Nfe2l2 knockout mice develop widespread astrogliosis and myelin degeneration, suggesting that Nfe2l2 plays a role glial survival Nfe2l2 are more susceptible to KA-induced seizures than wild-type animals Sod2 are more susceptible to KA-induced seizures and a subset of animals develops spontaneous seizures Nfe2l2 within the lesioned hippocampus, may be a key biochemical feature responsible for epileptogenicity within that region.The differential expression and network analyses make specific predictions about the processes and cell types that are altered in the epileptogenic region, which may provide insight into the mechanisms underlying epileptogenicity. It has been shown that there is increased glial differentiation from progenitor cells within the subgranular zone in KA-treated animals with spontaneous seizures Vglut1 can be upregulated in both hemispheres by a unilateral epileptogenic lesion Another goal of this study was to identify homeostatic responses to seizure activity that may be protective in epilepsy. Analysis of differential expression showed that the transcriptional response to seizures in the non-lesioned hippocampus was large and essentially equivalent to the response to seizures in the lesioned hippocampus. Although it intuitively makes sense that homeostatic responses to chronic, generalized seizures would affect both hippocampi equally, there are few studies that examine this phenomenon. For example, it has been shown that the expression of several neuropeptides is changed in non-lesioned hemisphere in the intrahippocampal KA animal model Arf1, which is a small GTPase that is involved in synaptic vesicle formation and trafficking Arf1 is known to interact with the AP-3 complex and stimulate vesicle budding from the endosome Arf1 is thought to be responsible for one of these endocytic processes Arf1 and AP-3 have been shown to have unique properties and have altered release characteristics Arf1) and module were conserved but that the connectivity of many genes was increased in the epileptic module, suggesting that genes present in the blue module have gained new importance in the neurons of epileptic animals. These data lead to the hypothesis that granule cells in the dentate gyrus have altered synaptic vesicle retrieval because of an increased reliance on the Arf1 and AP-3 dependent endocytic pathway, which could lead to changes in neurotransmission.To examine this phenomenon further, we investigated the blue module that was related to chronic seizures in both hippocampi, although there was substantial variability between animals, which could have been caused by a number of variables . This module was enriched in genes involved in synaptic transmission, and the most highly connected gene within this module was Bdnf has two known polyadenylation sites, leading to two populations of mRNA with different lengths Several molecules and pathways may be involved in the underlying regulation of the genes within the blue module, but we identified one RNA binding protein, HuD, that may contribute significantly to these processes. However, we cannot discount other members of the Hu family. In fact, one previous study identified several hundred mRNAs that bind to HuR after pentylenetetrazol induced seizures Sv2a, is the target of the anti-epileptic drug levetiracetam and disruption of this gene causes seizures in mice Sv2a is not completely understood, it is localized to synaptic vesicles and influences neurotransmitter release by playing a role in vesicle priming in vivo when hub genes are disrupted Necap1, a gene involved in synaptic vesicle endocytosis Necap1 and other hub genes within the epileptic network are plausible therapeutic targets for inhibiting epileptiform activity in MTLE.Identification of gene expression changes caused by epilepsy and unassociated with epileptogenicity could provide important insights into natural neuronal mechanisms that alter neurotransmission, and these changes could represent a homeostatic, anti-epileptic response that limits seizure activity. Our data suggest that genes upregulated due to seizures may regulate neuronal excitability through effects on vesicular trafficking. Indeed, one of the hub genes within this module, Sv2a. Therefore, we hypothesize that these pathways associated with synaptic function modulate epileptiform activity and that genes identified in these analyses that are central to these processes represent plausible anti-epileptic drug targets  that are isolated using an automatic module detection algorithm and assigned a color, which is shown on the horizontal bar below the dendrogram.(TIF)Click here for additional data file.Figure S2In situ hybridizations of control sense probes. Sense probes for Arf1, Ppp2ca, Ywhag, and Ppm1a1 were used for in situ hybridization to show specificity of expression. In situ hybridizations using sense probes were carried out on adjacent sections to those that were used for antisense probes. Sense and antisense probes were hybridized to sections under the same conditions at the same time.(TIF)Click here for additional data file.Table S1Primers used for quantitative RT-PCR. This table shows the primers that were used for quantitative RT-PCR confirmation. The first column shows the gene symbol. The second column shows the genomic location that was targeted. The third column represents the forward primer in 5′→3′ direction. The fourth column represents the reverse primer in 5′→3′ direction.(XLS)Click here for additional data file.Table S2Primer and probe sequences used for in situ hybridization. This table shows the primer and probe sequences that were used for in situ hybridization. The first column shows the gene symbol. The second column shows the genomic location that was targeted. The third column shows the primers in 5′→3′ direction that were used to clone the probe sequences. The fourth column shows the entire probe sequence that was used for hybridization.(XLS)Click here for additional data file.Table S3Summary of tissue samples used for microarrays. This table summarizes the tissue samples that were used for gene expression studies. The first column shows the animal identifier. The second column denotes the results of the video monitoring. The third column shows the hemisphere that the sample was taken from. The fourth column shows the number of sections that were used for mRNA extraction. The fifth column shows thickness of the sections that were used. The sixth column represents the concentration of mRNA that was extracted. The seventh column shows the 260/280 ratio of the extracted mRNA. The eighth column shows the results from the Agilent Bioanalyzer.(XLS)Click here for additional data file.Table S4Differentially expressed genes due to the lesion in animals without seizures. This table summarizes the genes that are differentially expressed between the lesioned and non-lesioned hippocampi in animals without seizures. The gene symbols, gene names, and Refseq identifiers are shown in the first column. The probe on the Agilent platform and the fold change and differential expression p-value for that probe is shown in the fourth, fifth, and sixth columns. The probe on the Codelink platform and the fold change and differential expression p-value for that probe is shown in the seventh, eighth, and ninth columns.(XLS)Click here for additional data file.Table S5Differentially expressed genes due to the lesion in animals with seizures. This table summarizes the genes that are differentially expressed between the lesioned and non-lesioned hippocampi in animals with seizures. The gene symbols, gene names, and Refseq identifiers are shown in the first column. The probe on the Agilent platform and the fold change and differential expression p-value for that probe is shown in the fourth, fifth, and sixth columns. The probe on the Codelink platform and the fold change and differential expression p-value for that probe is shown in the seventh, eighth, and ninth columns.(XLS)Click here for additional data file.Table S6Differentially expressed genes due to seizures in the non-lesioned hippocampus. This table summarizes the genes that are differentially expressed between lesioned hippocampi in animals with and without seizures. The gene symbols, gene names, and Refseq identifiers are shown in the first column. The probe on the Agilent platform and the fold change and differential expression p-value for that probe is shown in the fourth, fifth, and sixth columns. The probe on the Codelink platform and the fold change and differential expression p-value for that probe is shown in the seventh, eighth, and ninth columns.(XLS)Click here for additional data file.Table S7Differentially expressed genes due to seizures in the lesioned hippocampus. This table summarizes the genes that are differentially expressed between the non-lesioned hippocampi in animals with and without seizures. The gene symbols, gene names, and Refseq identifiers are shown in the first column. The probe on the Agilent platform and the fold change and differential expression p-value for that probe is shown in the fourth, fifth, and sixth columns. The probe on the Codelink platform and the fold change and differential expression p-value for that probe is shown in the seventh, eighth, and ninth columns.(XLS)Click here for additional data file.Table S8Functional annotation of the module associated with epileptogenicity. Enriched GO categories for the genes present within the yellow module that was specific to the epileptogenic region. The first column shows whether the categories are associated with a biological process, cellular component, or molecular function. Specific, enriched functional categories are shown in the second column. The third and fourth columns show the number and percentage of genes in the module that are associated with the specific term. The fifth column shows enrichment p-value.(XLS)Click here for additional data file.Table S9Functional annotation of the module associated with the effect of seizures. Enriched GO categories for the genes present within the blue module that corresponded to both hippocampi that were subject to seizures. The first column shows whether the categories are associated with a biological process, cellular component, or molecular function. Specific, enriched functional categories are shown in the second column. The third and fourth columns show the number and percentage of genes in the module that are associated with the specific term. The fifth column shows enrichment p-value.(XLS)Click here for additional data file.


The healthy worker effect (HWE) is a well-known phenomenon. In this study we used the extensive registration of all Danish citizens to describe the magnitude of HWE among all Danish electricians and evaluated strategies for minimizing HWE bias of the association between occupation and mortality.All Danish male citizens aged 26-56 years in the period 1984-1992 were followed for three years in several registers. We evaluated HWE bias among electricians because they were unexposed to detrimental occupational exposures. We compared electricians to three reference groups  and utilized analytical methods for minimizing HWE bias .The mortality rate was higher among electricians, who the year following active employment received incapacity benefits or were on long-term sick leave. Electricians receiving incapacity benefits, on long-term sick leave, unemployed, or with increased comorbidity index had lower odds of re-employment. Electricians had lower mortality rate  compared to the general population, while electricians leaving employment had increased mortality . Adjusting for several social events slightly attenuated the estimates, while the marginal structural model did not minimize bias. Electricians had the same mortality as the construction industry and carpenters/brick layers. Mortality was comparable to the general population after three or more years of lag time.In this nationwide study, employment as electricians had marked effect on mortality. Appropriate reference selection and lag time analyses minimized the HWE bias. Since 1885 ,3. This vor bias . Severalvor bias , mostly HWE has previously been reported to be strongest for young age groups and at the beginning of employment . It has We often wish to compare persons with high exposure  with persons with no or low exposure, e.g. persons in the reference population or persons in the same occupation but with lower exposure. In other words, we wish to estimate the influence of the cumulative exposure in one analysis.In this study we follow a nationwide cohort of electricians for three years to evaluate the influence of employment in year 0 and 2 on all-cause mortality in year 3, while taking account of covariates in year 1. We study these effects among electricians because, at least to our knowledge, electricians are a trade without any generally acknowledged occupational diseases . We would therefore expect similar mortality among electricians as selected reference groups if the bias caused by HWE (HWE bias) is minimized. We use the extensive registration of all Danish citizens in registers to describe the magnitude of HWE by estimating the influence of termination of employment on subsequent mortality and the probability of re-employment among persons with terminated employments (selection out of and into the working force). We utilize analytical methods to minimize HWE bias offered by a number of authors ,9,12-15,In this study we follow a nationwide cohort of electricians for three years (baseline in year 0 with follow-up until year 3) to evaluate the influence of employment in year 0 and 2 (proxy measure of cumulative exposure) on the outcome in year 3 , while taking account of covariates in year 1  as a proxy measure of exposure, which, to make things simple, is assumed to be unrelated with outcome , shown by no arrow from Elec0 and Elec2 to Mort3 in Figure st from 1984 to 1992, who survived until January 1st next year. The same person could therefore contribute during several years. We only included males, since only 200 women were electricians during this period compared to 29,613 men in the same period. Each Danish citizen has a unique personal identification number, which makes linkage on individual level between several registers possible.We included all Danish male citizens aged 26-56 years on January 1We obtained information on employment from the Integrated Databank on Research in the Labor Market , which igeneral Danish male population in the same age-span as specified above, (2) the construction industry defined as persons employed in the building and construction sector or the timber, paper, stone, clay or glass industries excluding bachelors or masters of engineering and building technicians, and (3) carpenters and brick layers defined as persons employed as carpenter or brick layer with highest educational level as skilled carpenter or brick layer (Table We included three comparison groups (reference groups): 1) The  The geneWe followed each Danish citizen in Statistics on Social Benefits , which cFurthermore, we included information on unemployment measured as number of weeks unemployed divided by number of weeks during the year resulting in a number between 0% and 100%. Short and long term unemployment was characterized as 21-80% or more than 80% and not having received sickness benefits more than 13 days during that specific year.In the following we use the term social event for persons who received incapacity benefits, were on sick leave or were unemployed. A person was considered employed in a specific year if he did not receive incapacity benefits, was employed at least 20% of the year and had less than 91 days of sick leave.Information on comorbidity was given by the Danish National Patient Register, which is a register of all inpatient hospitalizations in Denmark since 1977 . For eacInformation on mortality, emigration and disappearance were obtained from the Danish Civil Registration System .and year 2 (Elec0 and Elec2) and persons employed as electricians in year 0 or 2 with persons not employed as electricians. The first group had 'high exposure' (electrician both in year 0 and 2). The second group had 'low exposure' (electrician only in year 0 or year 2), which was divided into persons only employed as electricians in year 0 and persons only employed as electricians in year 2. The last group was 'not exposed' (not employed as electrician). When using the construction industry and carpenters/brick layers as reference groups, we made pair wise comparisons between electricians and reference group with the same employment, e.g. persons employed as electricians only in year 0 were compared with construction industry workers only employed in year 0.When using the general population as a reference group, we compared persons employed as electricians in year 0 We performed two groups of analyses: Firstly, we described HWE by estimating the mortality rates among electricians in year 0 and year 1 compared to electricians in year 0, who were not employed as electricians in year 1, e.g. persons unemployed or persons receiving incapacity benefits. Furthermore, we estimated the odds ratio of returning to work as electrician in year 2 among electricians in year 0 after experiencing any social event or having increased comorbidity index in year 1.Secondly, we utilized analytical suggestions to minimize HWE bias of the association between employment as electrician and mortality. We used three different reference groups , adjusteSince employment in year 1 (Emp1) may both determine subsequent employment as electrician (Elec2) and be determined by previous employment as electrician (Elec0) . Mortality rate ratios (MRR), odds ratios (OR) and 95% confidence intervals (95%CI) were calculated.st 1988 who survived until January 1st 1989 was 1,143,018 including 13,982 electricians . The number of men on January 1Among electricians at baseline (year 0) not employed as electricians in year 1, the mortality rate in year 2 was significantly higher than persons employed as electricians both in year 0 and 1  and very high for persons on long-term sick leave  and persons receiving incapacity benefits   Table . PersonsThe tracking of being an electrician in year 0 (1984-92), to social events in year 1 1985-93) and back to being an electrician in year 2 (1986-94) is shown in Figure  and backThe mortality rates (MR) were lowest for electricians in year 2 and highest for persons who were electricians only in year 0 Table . In ModeIn the multivariable adjusted model (Model 2) an attenuation of risk estimates was observed, but significantly decreased MRR for electricians in year 0 and 2 and increased MRR for electricians only in year 0 was still observed. In Model 3, only persons employed in year 2 were included, showing stronger risk estimates for electricians only in year 0  and electricians in year 0 and 2 still had significantly decreased mortality rate  (Table 5 p-yrs) - Table 5 p-yrs - Table The mortality rate among persons employed in the construction industry was slightly lower  and approached unity for the oldest age group 50-59 years, MRR = 1.00 (0.81-1.23) Table . The sigWhen inferring lag time the mortality rates among electricians were comparable to the general population three and more years after exposure, e.g. MRR among electricians in year 0 and 2 for three years of lag time was 0.92 0.82-1.04) . The number of outcomes was small in this analysis though. When restricting the analysis to long-term electricians, the MRR showed increased risk for electricians only in year 0 1.90; 1.49-2.42) and decreased MRR for electricians both in year 0 and 2 . These results were similar to the results in Table 9-2.42 anIn this study we characterized HWE in a nation-wide cohort of electricians as the selection out of and into the working force and evaluated different methods for minimizing the influence of HWE bias. There was a strong selection out of the cohort in that increased mortality among electricians leaving the working force was observed especially among persons on long-term sick leave and persons receiving incapacity benefits. The odds ratio of re-entering the electrician work force was low for persons on long-term sick leave, unemployed, receiving incapacity benefits and with increased comorbidity index. A decreased mortality rate was observed among electricians compared to the general population. Adjustment for several social variables and comorbidity index slightly attenuated this difference. When including relevant reference groups (construction industry and carpenters/brick layers) the differences attenuated although electricians who left employment had increased mortality compared to workers in the construction industry and electricians who entered employment had lowered mortality compared to carpenters/brick layers. Electricians had the same mortality rate as the general population when inferring lag time of three or more years. Our findings that appropriate reference group selection and lag time analyses may minimize HWE bias should be considered in other occupational studies.Other occupational cohort studies have shown lower mortality among persons employed compared to the general population. The standardized mortality ratio was often reported as approximately 80 , from 30We evaluated different methods for minimizing the influence of HWE bias and found that selection of an appropriate reference group and latency analyses were effective in minimizing the lower mortality among electricians and the increased mortality among electricians who stopped being electricians. Selection of an appropriate reference group has been suggested elsewhere ,12 assumGilbert recommended that a lag of at least one or two years would seem essential in any analysis and for certain chronic nonmalignant diseases longer lags may be necessary , which iContrary, restricting the analysis to persons actively employed ,14,24 diOther authors suggest restriction to newly employed workers ,9, whichFrom a methodological standpoint, selection bias and confounding are two concepts that often overlap , which iOur study has several strengths, e.g. we have information on all Danish men aged 26-56 years at baseline and we have complete follow-up until death or emigration of all Danish citizens. The results of the study are therefore not influenced by missing outcome information on those individuals who leave the work force before retirement -33 or diWe also wish to emphasize the limitations of our study. We used administrative registers, which primarily contains information relevant to public sector administrators, e.g. when using information on sick leave, we only had information on the number of days of sick leave compensation and e.g. not the reasons for the compensation . AnotherWe observed a strong selection out of the cohort with increased mortality among electricians leaving the work force. The odds ratio of re-entering was low for persons on long-term sick leave, unemployment periods, incapacity benefits and with increased comorbidity index. The HWE was strong with 40% decreased mortality among active employed electricians compared to the general population; adjustment for several social variables and comorbidity index slightly attenuated this result. When including relevant reference groups and inferring lag time these differences were minimized. We conclude that HWE is a strong selection process in occupational cohorts and that the bias that arises due to this phenomenon can be minimized by appropriate reference selection and lag time analyses.95%CI: 95% confidence intervals; Emp1: employment in any occupation in year 1; Elec0: employment as electrician in year 0; Elec2: employment as electrician in year 2; HWE: healthy worker effect; HWE bias: bias caused by the healthy worker effect; Mort3: mortality in year 3; MR: mortality rate; MRR: mortality rate ratios; MSM: marginal structural model; OR: odds ratio; UH1: underlying health in year 1.The authors decleare that they have no competing interests.LCT designed the study and participated in analysis and interpretation of data, drafting of the manuscript, had full access to all of the data in the study and performed the statistical analysis. UAH participated in developing the study concept and design, in the analysis and interpretation of data and helped with critical revision of the manuscript for important intellectual content. SM and HBH both participated in the interpretation of data and helped with critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/571/prepub


The aim of this study was to compare two groups of patients with early stage cervical cancer who underwent either abdominal or vaginal surgery, in terms of post-operative findings and survival.55 patients with diagnosed cervical cancer were retrospectively selected for this study. They were preoperatively staged according to FIGO criteria. Forty four patients had disease between stages Ib and IIa with no evidence of extra-pelvic lymph node involvement and 10 patients had stage ≥ IIb.p=0.494) and parity of the patients (Median parity was 2 (range: 0-5) for Piver II group and 1 (range: 0-4) for Schauta group (p=0.607)) and about histotype and stage of the cervical cancer . Among the two groups there were significant statistical differences regarding the mean operative time  and the average hospital stay . Significant statistical difference was reported as regarding adjuvant RT, increased in the Piver II group with respect to the Schauta group . The survival rate at 5-years was without significant difference between the two groups .Of the 55 patients, 17 had been operated by Schauta-Amreich radical vaginal hysterectomy and 38 by Piver type II abdominal hysterectomy. No significant statistical differences have been found between two groups about age (median age was 49 for Schauta and 54 for Piver This study confirms the benefits of the Schauta-Amreich vaginal radical hysterectomy in terms of hospital stay, mean operative time and early complications.We believe that this surgery is a plausible alternative to radical abdominal hysterectomy and could be considered to be a valid approach for the treatment of patients with cervical neoplasms, but still randomized trials are needed on this topic with respect to the ethical issues involved. Improved overall survival rates combined with the best quality of life should be the primary targets in any invasive cancer treatment. At present, for tumors less than 4 cm, the standard treatment options for early stages of cervical cancer  are radical abdominal hysterectomy and pelvic lymphadenectomy, vaginal radical hysterectomy with pelvic lymphadenectomy or external pelvic irradiation plus brachytherapy, as adequate . With thFor many gynecologists primary surgical treatment is considered to be the method of choice for tumors that are less than < 4 cm . The benOther techniques include a laparoscopic retroperitoneal interiliac lymphadenectomy approach  and lapaThe aim of this retrospective study was to compare two groups of patients with early stage cervical cancer who underwent either abdominal or vaginal surgery in our Gynecological Department, in terms of post-operative findings and survival.st 2001 and December 31st 2005 with diagnosed cervical cancer were retrospectively selected for this study. Patients were preoperatively staged using FIGO criteria. Forty four patients had disease between stages Ib and IIa with no evidence of extra-pelvic lymph node involvement and 10 patients had stage ≥IIb and had also received primary chemoradiation treatment to reduce tumor size before surgery . All patients had pre-operative clinical staging included the following specialized investigations: full physical examination, chest X-rays, CT/MRI scan, routine pre-operative blood tests, cystoscopy and sigmoidoscopy if symptomatic. All patients were operated in our Gynecological Department.Fifty five patients admitted in Fatebenefratelli Isola Tiberina Hospital in Rome between January 1et al. . Median parity was 2 (range: 0-5) for Piver II group and 1 (range: 0-4) for Schauta group (p=0.607). All cases were classified according to the FIGO staging and according to histological type as indicated in charts. There were no significant differences between the two groups per histotype or per stage  in the two groups . On the other side there was a difference for the necessity of transfusions in the two group, even if not statistically significative .Preoperative values of Hb were respectively 12.56 ± 1.33 g/dl for the abdominal surgery and 13.12 ± 1.01 g/dl for vaginal surgery; postoperative values were respectively 9.84 ± 1.71 for abdominal and 10.99 ± 1.38 g/dl for vaginal procedures. Hb delta variation was not significantly different among groups .The mean number of resected nodes was 18 (range 7-28) from the Piver II Group and 19 (range 8-25) from the Schauta Group, with no difference between the two groups of surgical treatment (p=0.048). No severe operative/post-operative complications occurred among the two groups  was experienced in 8 patients undergoing Piver type II operation compared with no patient belonging to the Schauta group  and 3 extrapelvic metastases only in Piver type II group.p=0.51). Stratificating survival rates on the basis of staging, and considering the IB stage, as more representative (23/54 patients), we obtained the same survival rates in the two groups  was undertaken more readily with some advocates.the indication for the Schauta operation must take the lymph node problem into account” and later he performed extraperitoneal pelvic lymphadenectomy, with the radical vaginal hysterectomy, as did Mitra , has become more popular. Querleu and Leblanc were credited with this approach .Evaluation of lymph node status is an integral part of the surgical treatment for women with gynecologic cancer. It’s not possible to evaluate the pelvic lymph nodes by imaging alone and due to their deep anatomical location, most gynecologic cancers are staged at the time of surgery. Lymphadenectomy is important to achieve an adequate central dissection around the cervical tumor during the operation, so that involves removal of tissue from the hypogastric vessels, from the obturator fossa and from the lower presacral region. To date, several studies use to report a large number of newer approaches for lymphadenectomy.The extraperitoneal pelvic lymphadenectomy can be considered an adequate technique to complement radical vaginal operation for cervical cancer since it represents the most cost-effective option due to shorter operating times, a greater number of nodes removed and reduced length of hospital stay. In a previous study, we compared the extraperitoneal lymphadenectomy versus laparoscopic technique in performing pelvic lymphadenectomy in a series of patients with locally advanced cervical cancer undergoing a radical vaginal hysterectomy . No diffvs vaginal surgery. The number of lymph nodes resected was similar between the groups of patients, but there were significant differences in the mean operative time between the two groups, with reduced operating times in the vaginal operation and fewer patients with postoperative infection. Additionally, average hospital stay was significantly lower in the vaginal radical operation. According to the literature, our series of data show that radical vaginal hysterectomy carried out using the Schauta-Amreich technique with extraperitoneal lymphadenectomy represents adequate surgical management of early stage cervical cancer. This study confirms the benefits of the vaginal operation in terms of hospital stay, mean operative time and early complications. One of the major criticism against vaginal surgery in gynaecological oncology is about radicality and effectiveness.The present study compares two different approaches for the surgical treatment of cervical cancer, namely abdominal We perfectly know that the present work is a poor retrospective observational study. Nonetheless we observed no differences between groups in terms of 5 years survival, even after stratification.We believe that this surgery is a plausible alternative to radical abdominal hysterectomy and could be considered to be a valid approach for the treatment of patients with cervical neoplasms, but still randomized trials are needed on this topic with respect to the ethical issues involved.Schauta operation is a poor technique indeed, with limited costs and limited impact on the woman’s body. Unfortunately the few surgeons able to do it in the past years, did not leave a flow school, did not let their coemployers to learn about this procedure. They had a rocket machine but they hide it as a personnel requirement, leaving spaces and open fields to the expensive laparoscopic and robotic procedures.