Computerized dynamic posturography (CDP) has been used to detect balance and stability impairments in adults of all ages. The goal of the current pilot study was to evaluate balance in healthy older adults after a middle-of-the-night awakening and to assess the ability of CDP to measure effects of bedtime zolpidem administration.Two studies used CDP to evaluate balance in healthy older adults (≥ 65 years) during middle-of-the-night awakenings. The first study used a drug-free, single-period, within-subject, repeated measures study design. Subjects were evaluated during the day, pre-sleep, and 2 hours after bedtime for dynamic standing balance using the NeuroCom EquiTest Sensory Organization Test (SOT). Pairwise comparisons were made using one-way ANOVA. The second study was a single-blind, randomized, placebo-controlled, crossover study evaluating the ability of the SOT to measure medication-induced dynamic standing balance impairments using the commonly prescribed sleep medication, zolpidem 10 mg, as a test medication. Assessments were performed at night before zolpidem administration and then again 2 hours after bedtime. Comparisons were made between the 2 groups using an ANCOVA model.P = 0.439). Eleven older adults (mean age 68.9 years) were evaluated in the second study. Zolpidem administration significantly decreased the SOT composite score after a middle-of-the-night awakening compared with placebo (P < 0.001).Twelve older adults (mean age 68.4 years) were evaluated in the first study. There was no significant difference between pre-sleep and middle-of-the-night assessments for the SOT composite score (In healthy older adults, getting up in the middle of the night did not have a significant effect on dynamic standing balance; however, bedtime administration of zolpidem 10 mg did lead to significant impairments. Thus, the SOT was able to measure medication-induced dynamic standing balance impairments and may be useful for future studies comparing balance effects of medications. Older adults often take medications to treat a variety of conditions, many of which can profoundly affect balance and stability. ImpairedSeveral classes of medication commonly prescribed to older adults have demonstrated potential effects on balance, including the sedative-hypnotics (for insomnia), antidepressants (for depression and anxiety), and anticonvulsants (for seizure disorders). In older adults, several studies have suggested that sedative-hypnotics may be associated with impaired balance and an increased risk of falls and hip fractures. -9 A retrMost medications are not routinely assessed for the possibility of affecting balance and stability; however, as stated previously, the consequences of impaired balance can be severe in older adults. A test is needed that can detect a medication's relative risk of balance impairment in older adults. This would help to better identify appropriate medications for those patients at particular risk for complications due to potential mobility issues.Computerized dynamic posturography (CDP) has been used previously to assess balance in a variety of conditions. -14 One sIn the current analysis, 2 separate pilot studies used the SOT assessment to evaluate dynamic standing balance in older adults during middle-of-the-night awakenings. In the first study, normal healthy older adults were evaluated after a middle-of-the-night awakening. The goal was to establish a comparative normal balance baseline for further studies in older adults using the SOT assessment and to determine if getting out of bed in the middle of the night was itself a risk factor for balance impairment. The second study was placebo-controlled and evaluated the ability of the SOT to determine medication-induced dynamic standing balance impairments. Zolpidem is a commonly prescribed medication that has been previously determined to alter balance up to 5 hours after administration, and was All studies were conducted in compliance with Good Clinical Practices and applicable Federal and local laws with Institutional Review Board approval. Informed consent was obtained from all subjects prior to enrollment in the study.max - θmin)}/12.5° × 100%. In this formula, θmax indicates the greatest anterior-posterior sway displayed by the subject and θmin indicates the least anterior-posterior sway. A score of 100 represents perfect balance (no sway), and a score of 0 represents a potential fall (sway exceeds limits of stability). If at any time during the test, the subject takes a step or requires the assistance of the safety harness, the subject scores a 0 for that test. An average score is calculated for each of the 6 conditions and a composite equilibrium score (CES) is calculated as a weighted average of all 6 individual scores with each of the first 2 conditions carrying a weight of 1/14 and each of the other 4 conditions carrying a weight of 3/14. These weights were specified by the manufacture to reflect the difficulty levels of the 6 tasks. Sensory analysis ratios also are used to identify possible impairments of individual sensory systems. The somatosensory ratio (condition 2/condition 1), visual ratio (condition 4/condition 1), and vestibular ratio (condition 5/condition 1) assess the ability to use input from each sensory system to control balance. The preference ratio (condition 3 + 6/condition 2 + 5) assesses the extent upon which a subject relies on visual input to control balance, even when the visual information is incorrect. The SOT has been used in many previous studies and has been shown to be a valid assessment of balance impairment in older adults and accurately predicts fall risk.[The NeuroCom EquiTest SOT  assesses postural stability and the potential for falls by identifying abnormalities in the subject's use of the somatosensory, visual, and vestibular sensory systems that contribute to balance. The SOT procedure requires subjects to stand on a pressure-sensitive, dynamic tilted force plate facing a sway-referenced visual surround while strapped into a safety harness to prevent injury in the event of a loss of balance. Each test comprises 3 trials for each of 6 conditions representing different aspects of balance Figure . For conall risk.,14,17This study evaluated CDP balance assessments using a drug-free, single-period, within-subject, repeated measures study design. Subjects eligible for the study were healthy adults, aged 65 or older, with no history of insomnia, psychiatric disorder, significant medical condition , or balance impairment. Subjects were asked about any history of recurrent falls, recent serious head injuries, presence of an inner ear or vestibular system disorder, history of dizziness, or any significant visual acuity or field abnormalities not improved with the use of corrective lenses to detect any possible undiagnosed balance impairments. Subjects were not allowed to participate if they had taken any medications known to affect sleep-wake functions or alter the central nervous system, or if they participated in a clinical trial of an investigational drug within 1 week  of the start of the study. Subjects were screened 4–10 days prior to testing, which included documentation of medical history, sleep history , and a physical examination. Subjects were also exposed to the EquiTest CDP tests to acclimate them to the testing procedures and assess their daytime SOT scores. On the day of testing, subjects reported to the clinic 2 hours prior to habitual bedtime and were allowed to relax in a private room. After 30 minutes, a pre-sleep assessment of the SOT was performed as described above. Subjects went to sleep at their habitual bedtime. After 2 hours, subjects were awakened and completed the SOT procedure again. Each SOT assessment consisted of 3 trials for each condition. Following middle-of-the-night testing, subjects returned to bed and were allowed to sleep until morning (8 hours after bedtime). Subjects left the clinic in the morning after a brief neurological exam.P value less than 0.05 was considered statistically significant.Descriptive statistics were used to summarize the SOT results at daytime, pre-sleep, and after the middle-of-the-night awakening. Pairwise comparisons were made using one-way ANOVA with the time of day as a class effect. A This study evaluated the ability of CDP to detect medication-induced balance impairments using a single-blind, randomized, placebo-controlled, crossover study design with zolpidem as the test medication. Healthy older adults (≥ 65 years of age) with no history of sleep or balance disorders were included in this study. The subjects enrolled in this study were not the same as those enrolled in the first, drug-free study. Subjects were excluded from the study if they had a history of a psychiatric disorder or significant medical condition , had taken any medications known to affect sleep-wake functions or alter the central nervous system, or if they participated in a clinical trial of an investigational drug within 1 week  of the start of the study. Subjects with a history of recurrent falls, recent serious head injuries, presence of an inner ear or vestibular system disorder, history of dizziness, or any significant visual acuity or field abnormalities not improved with the use of corrective lenses were excluded from the study. At initial screening (between Days -7 and -1), medical histories were obtained, and subjects underwent a physical exam, 12-lead electrocardiogram, and CDP assessments. On the first crossover treatment day (Day 1), subjects arrived at the clinic 2 hours before habitual bedtime, and a pre-sleep assessment for the SOT was obtained. Immediately prior to bedtime, subjects were randomly assigned to receive either zolpidem 10 mg or placebo. Subjects were awakened after 2 hours for middle-of-the-night SOT balance assessment. After balance testing, subjects were allowed to go back to sleep until morning (8 hours after bedtime). Subjects were required to remain at the clinic until noon to assess potential residual sedation. On the second crossover treatment day (between Days 4 and 7), subjects followed the same procedures and were assigned to receive the opposite treatment as their first visit (zolpidem 10 mg or placebo). Adverse events (AEs) were monitored throughout the study.P value less than 0.05 was considered statistically significant.Descriptive statistics were used to summarize the SOT results for the placebo and zolpidem 10 mg groups. Comparisons were made between the 2 groups using an ANCOVA with sequence, period, and treatment group as class effects and the pre-treatment baseline measurement as a continuous covariate. A A total of 12 older adults  completed the study. Results for the SOT assessment are shown in Table P > 0.05 for both) and were within normal limits for older adults. The differences between the placebo and zolpidem middle-of-the-night SOT assessment is shown in Table P < 0.001). The vestibular ratio was also significantly decreased after zolpidem treatment compared with placebo (P < 0.002).A total of 11 older adults  were enrolled in the crossover study, with 1 subject discontinuing after the first treatment period (only completed zolpidem 10 mg treatment). Pre-sleep baseline assessments (before medication administration) showed no significant differences between the placebo and zolpidem groups on any of the SOT equilibrium scores or SOT sensory ratios  and 2 AEs occurred during the zolpidem phase (upper respiratory infection and headache). The 2 occurrences of headache were deemed possibly related to study drug while the upper respiratory infection was deemed unrelated to study drug. All AEs were considered mild.The majority of balance studies in older adults have focused on daytime assessments of stability and fall risk.,19 The cUsing the NeuroCom EquiTest SOT balance assessment, healthy older adults exhibited no significant impairments after a middle-of-the-night awakening compared with pre-sleep measurements. This suggests that getting out of bed in the middle of the night, by itself, does not affect dynamic standing balance in healthy older adults with no history of impaired balance. Any increase in balance problems or falls during the night in older adults might therefore indicate an underlying medical disorder or a symptom of nighttime medication effects on balance rather than a symptom of aging. However, the small sample size in this study may have prevented the detection of small balance impairments. Also, the SOT is only designed to measure postural control and the sensory systems associated with standing balance. It is possible that other aspects of balance may be affected after a middle-of-the-night awakening. The daytime assessments were also obtained during the screening period and may be lower due to unfamiliarity with the SOT procedures. Further assessments comparing daytime and nighttime balance may be needed to fully understand the time-of-day effects on balance in older adults.In contrast to the first pilot study, dynamic standing balance was significantly impaired during the middle of the night after a bedtime dose of zolpidem 10 mg in the second study. This suggests that the SOT has the ability to measure medication-induced dynamic standing balance impairments, since previous studies using other measures of balance have shown significant balance impairments and increased fall risk after zolpidem administration.,9,20 EveBalance was evaluated in the current study using the NeuroCom EquiTest SOT assessment. Several studies have demonstrated the association between impaired SOT equilibrium scores and the incidence of falls in adults of all ages.,14,21,22In healthy older adults, getting up in the middle of the night did not have any significant effect on dynamic standing balance; however, bedtime administration of zolpidem 10 mg did lead to significant deficits. The SOT assessment successfully detected dynamic standing balance impairments due to zolpidem and may be useful for future studies comparing balance effects of medications. However, additional measures may be useful in assessing other aspects of balance to gain a fuller understanding of the effects of medications on balance.This study was supported by the Takeda Pharmaceutical Company, Ltd., Osaka, Japan. SWW and XP are employees of the Takeda Global Research and Development Center. GZ has received grants/research support or worked as a consultant for Ancile Pharmaceuticals, Arena, Aventis, Biovail, Boehringer-Ingelheim, Cephalon Inc., Elan, Eli Lilly, Epix, Evotec, Forest, Glaxo Smith Kline, Jazz, King Pharmaceuticals, Ligand, H. Lundbeck A/S, McNeil, Merck and Co., National Institutes of Health (NIH), Neurim, Neurocrine Biosciences, Neurogen, Organon, Orphan Medical, Pfizer, Renovis, Respironics, Sanofi-Aventis, Sanofi-Synthelabo, Schering-Plough, Select Comfort, Sepracor, Shire, Somaxon, Somnus, Takeda Pharmaceuticals North America, Transcept, UCB Pharma, Predix, Vanda, Vela, and Wyeth-Ayerst Research. GZ is also a shareholder of Clinilabs, Inc.SW–W and XP are employees of Takeda Global Research and Development Center and participated in the study design, data analysis, and interpretation of results. GZ was involved in the study design, data analysis, and interpretation of results. GZ, SW–W, and XP all participated fully in the writing and review of the manuscript.The pre-publication history for this paper can be accessed here:


The aim of this study was to determine the long-term prevalence of post-traumatic stress disorder (PTSD) symptomology in patients following secondary peritonitis and to determine whether the prevalence of PTSD-related symptoms differed between patients admitted to the intensive care unit (ICU) and patients admitted only to the surgical ward.A retrospective cohort of consecutive patients treated for secondary peritonitis was sent a postal survey containing a self-report questionnaire, namely the Post-traumatic Stress Syndrome 10-question inventory (PTSS-10). From a database of 278 patients undergoing surgery for secondary peritonitis between 1994 and 2000, 131 patients were long-term survivors (follow-up period at least four years) and were eligible for inclusion in our study, conducted at a tertiary referral hospital in Amsterdam, The Netherlands.The response rate was 86%, yielding a cohort of 100 patients; 61% of these patients had been admitted to the ICU. PTSD-related symptoms were found in 24%  of patients when a PTSS-10 score of 35 was chosen as the cutoff, whereas the prevalence of PTSD symptomology when borderline patients scoring 27 points or more were included was 38% . In a multivariate analyses controlling for age, sex, Acute Physiology and Chronic Health Evaluation II (APACHE II) score, number of relaparotomies and length of hospital stay, the likelihood of ICU-admitted patients having PTSD symptomology was 4.3 times higher  than patients not admitted to the ICU, using a PTSS-10 score cutoff of 35 or greater. Older patients and males were less likely to report PTSD symptoms.Nearly a quarter of patients receiving surgical treatment for secondary peritonitis developed PTSD symptoms. Patients admitted to the ICU were at significantly greater risk for having PTSD symptoms after adjusting for baseline differences, in particular age. Peritonitis or abdominal sepsis is a severe disease with high mortality (approximately 30%) ,2. IntenMany survivors of critical illness and its treatment suffer from continuous traumatic memories and re-live adverse experiences from their illness, such as respiratory distress, anxiety, pain and loss of control, which are all associated with an increased risk for development of PTSD ,6. StudiIn addition, data on the prevalence of PTSD-related symptoms following secondary peritonitis are lacking. It is unknown whether the prevalence of symptoms related to PTSD or memories of traumatic experiences differ between peritonitis patients after ICU admission  and patients without ICU admission .The aim of the present study was first to determine the long-term prevalence of PTSD symptomology in patients 4 to 10 years after secondary peritonitis based on a self-report questionnaire. We also aimed to compare the prevalence of PTSD-related symptoms between patients admitted to the ICU and patients admitted only to the surgical ward. Finally, we examined whether the prevalence of PTSD symptomology in these patients was increased because of the traumatic memories that patients had during their ICU and/or hospital stay  II score before surgery and Mannheim Peritonitis Index (MPI). Disease and surgical characteristics recorded contained aetiology of peritonitis, origin of peritonitis, surgical treatment strategy and number of relaparotomies. Postoperative characteristics recorded included the number of days spent in hospital, the number of days spent in the ICU, days of mechanical ventilation, 'open abdomen' (laparostomy) during admission, number and type of complications, number of readmissions and the mean follow-up time. Patient recall was checked using the hospital information and medication system to check readmission and use of medication. Details regarding out-of-hospital medications, such as those prescribed by the family physician, were obtained only by questionnaire.The PTSS-10 was originally designed to diagnose PTSD, according to Diagnosis and Statistical Manual of Mental Disorders (DSM)-II criteria, in victims of natural disasters , and it The questionnaire consists of 10 items, each with a Likert scale ranging from 1 ('never') to 7 . A summated score with a range between 10 and 70 is calculated, with higher scores indicating more PTSD-related symptoms. A score of 35 or greater is considered an adequate cutoff for PTSD-related symptomology ,17-19, wThe four-item Adverse Experiences Questionnaire assesses the presence of four types of traumatic memories during a stay in the ICU or hospital ward : anxiety2, Student's t, or Mann-Whitney U tests.Ninety-five per cent confidence intervals around estimates of prevalence were calculated using the method of Wilson . ClinicaP < 0.1) with the dependent factor (PTSD symptomology) in our study . If factors were highly correlated, we selected only one of the correlated factors in the multivariate model to avoid the problem of co-linearity. Odds ratios with 95% confidence intervals (CIs) were used to quantify the strength of the association. To determine the fit of the final multivariate logistic model, we calculated the area under the receiver operating characteristic curve, also known as the concordance statistic, and performed the Hosmer-Lemeshow goodness-of-fit test.We built multivariate logistic regression models to assess the association between ICU stay and the presence of PTSD symptomology (PTSS-10 score >35) after adjusting for other factors. We adjusted for factors related to patient characteristics (age [continuous] and sex), disease characteristics (APACHE-II score at baseline [continuous] and whether patients had undergone one or more relaparotomies [yes/no]) and postoperative characteristics  . These f2 test for trend to examine this relation.To determine whether traumatic memories acquired during the stay in hospital or the ICU played a role in the development of PTSD symptomology, we examined the percentage of patients with PTSD symptomology within each level of response on the traumatic memories questions. Because of the ordered response on the traumatic memories questions, we used the χP < 0.05 were considered statistically significant.n = 100) and eligible patients still alive who did not respond (n = 32). However, comparison of patient characteristics between the two groups revealed that patients in the nonresponding group were younger , presented with fewer comorbidities  at initial surgery, and had lower APACHE II scores  and MPI scores  than did patients in the responder group. There was no difference between responding patients and nonresponders in ICU admittance.From the initial cohort of 278 patients with secondary peritonitis , 118 patIn responding patients the average interval between index operation and follow up was 5.3 years for ICU and non-ICU patients. Comorbidity was present in 65% of patients, and nearly 80% of patients were on some type of medication Table . The APAThe median PTSS-10 score among all patients was 22, with 25% of the patients having a score below 13 and 25% of patients with a score above 33. Using the recommended cutoff value for PTSD symptomology of 35 points on the PTSS-10 questionnaire ,19,22,23P = 0.011). ICU patients also had higher APACHE II score  and MPI score . Of patients who had had an ICU stay 36% underwent laparostomy (open abdomen), whereas only 8% of the ward patients underwent laparostomy . A relaparotomy was significantly more common in the ICU group than in the non-ICU group .Patient, disease and operative characteristics for ICU patients 61%) and non-ICU patients (39%) are presented in Table % and nonP = 0.001) and suffered more nonsurgical complications . Fifty-four (89%) patients required mechanical ventilation during their ICU stay. These patients were ventilated for a median of 11 days. Four of the ICU-admitted patients suffered early ARDS (within 96 hours) following peritonitis.With respect to postoperative characteristics, patients had a median stay in hospital of 37 days. ICU survivors had a longer hospital stay than did non-ICU survivors . We examined several factors to determine whether they confounded the strength of the relationship between ICU stay and the probability of having relevant PTSD symptomology  in the non-ICU group and 28% (17/61) in the ICU group . With every one-year decrease in age, the likelihood of developing PTSD symptoms decreased . Finally, with every point increase in APACHE-II score, the chances of developing PTSD symptoms increased . Therefore, the main reason for finding a stronger relation between ICU stay and PTSD symptomology in the multivariate model is that older patients are less likely to develop PTSD symptoms. Because ICU patients on average were older than non-ICU patents, the unadjusted relationship underestimated the effect of ICU on PTSD symptoms. Males were also less likely to report PTSD symptomology , but because of the comparable sex distribution in ICU and non-ICU patients it did not confound the relation between ICU stay and PTSD symptomology .In the total study population, traumatic memories were associated with more PTSD symptomology Table . PatientOur cohort of patients experiencing the same acute disease includes both patients who have been admitted to the ICU and those who were treated on the surgical ward only. This enabled us to conduct a detailed analysis of the impact of ICU stay on long-term PTSD symptomology. We found a high overall prevalence of long-term PTSD symptomology, as indicated by the PTSS-10 questionnaire, many years after surgical treatment for secondary peritonitis. The proportion of patients scoring above the 35-point threshold on PTSS-10 was 24%.The PTSS-10 is an instrument specifically designed to identify PTSD symptoms in ICU patients. The prevalence of PTSD symptoms in our patients was similar to that in a retrospective study conducted in ARDS patients in 1998 using the PTSS-10 , and it n = 100). However, our patient group had an overall lower mean APACHE II score than that reported in other ICU populations with similar prevalence of PTSD symptoms. Although the APACHE II scores of patients admitted to the ICU in our study are lower than those in other studies on PTSD symptoms using the PTSS-10 questionnaire [We found that patients who responded to the PTSS-10 questionnaire exhibited higher APACHE II scores and MPI scores, and increased comorbidity than did patients who did not respond to the questionnaire. These differences might have led to a small overestimation of the prevalence of PTSD symptoms  and non-ICU patients (18%) on the PTSS-10. However, ICU stay was independently associated with PTSD symptomology after adjusting for other factors related to PTSD, in particular age. As expected, when comparing ICU patients with non-ICU patients, differences were found in patient, disease, operative and postoperative characteristics. ICU patients were older and had more severe disease (based on the recorded APACHE II score), more surgical interventions and longer hospital stay, all of which could have affected their eventual PTSD symptomology. To control for these differences and to determine whether ICU was an independent factor for PTSD, we created a multivariate model. When controlling for age, sex, APACHE II score, having undergone one or more relaparotomy, and length of hospital stay in the postoperative period, we found a significant difference in the prevalence of PTSD symptomology (based on PTSS-10 score) between patients with and without an ICU stay. Older age and being male had a protective role, whereas higher APACHE II scores led to more PTSD symptoms. These findings are in contrast to earlier data, in which no associations between higher APACHE II score and greater probability of developing of PTSD symptoms were identified ,5. It isBecause mechanical ventilation has previously been associated with development of more PTSD-like symptoms after ICU treatment , this miBecause of the retrospective nature of the study, details concerning the severity of sepsis (such as septic shock status on admission and hydrocortisone use during the ICU stay) could not be ascertained as risk factors in all patients ,23. ThesThere were no differences in the number of traumatic memories reported between ICU patients and the patients managed on the surgical ward only, although we found a clear positive linear association between more traumatic memories and higher scores on the PTSS-10. This relation between traumatic memories and the PTSS-10 score was also found in two earlier studies conducted in ICU patients ,30. We aIdeally, PTSD is diagnosed using a SCID , in accoRecognition of the distinction between PTSD symptoms captured by the PTSS-10 and a PTSD diagnosis is vital, because this questionnaire does not give a DSM-IV diagnosis but only an indication of the level of PTSD symptomology. Clinically, a score on the PTSS-10 above the cutoff should prompt the attending physician to refer the patient to a psychologist to conduct a SCID .Our results suggest that the (persisting) presence of traumatic memories is likely to be relevant to development of PTSD-related symptoms following a traumatic event, and not the ICU stay alone, because we observed a strong linear relationship between traumatic memories and PTSS-10 score. We assessed these traumatic memories (or adverse experiences) in accordance with patients' recollections. This may limit the conclusions one can make, because it is possible that perception of a traumatic experience may contribute to long-term PTSD symptomology, hence making a causal conclusion impossible.Information concerning other unrelated traumatic experiences or life events that may have occurred after hospital admission was not collected. Therefore, the influence of superimposed trauma cannot be ruled out . Also, bNearly a quarter of patients receiving surgical treatment for secondary peritonitis developed PTSD symptoms. Considering the high long-term prevalence of PTSD, patients admitted to the ICU had a higher risk for PTSD symptoms but only after taking their higher age into account. Early detection of PTSD in peritonitis patients by questionnaires such as the PTSS-10 deserves attention.• In a cohort of 100 patients with secondary peritonitis, of whom 61 were admitted to the ICU and 39 were not , the overall prevalence of long-term PTSD symptomology using the PTSS-10 questionnaire was 24%.• In a univariate analysis we found no differences in PTSS-10 scores between ICU and non-ICU patients, but ICU stay was significantly associated with PTSD symptomology after adjusting for other factors related to PTSD, in particular age.• There were no differences in the number of traumatic memories reported between ICU patients and patients managed on the surgical ward only, although we found a clear positive linear association between more traumatic memories and higher scores on the PTSS-10.APACHE = Acute Physiology and Chronic Health Evaluation; ARDS = acute respiratory distress syndrome; CI = confidence interval; DSM = Diagnostic and Statistical Manual of Mental Disorders; ICU = intensive care unit; MPI = Mannheim Peritonitis Scale; OR = odds ratio; PTSD = post-traumatic stress disorder; PTSS-10 = Post-traumatic Stress Syndrome 10-question inventory; SCID = Structured Clinical Interview for DSM-IV Axis II Personality Disorders.The authors declare that they have no competing interests.MB, DG, MV and BL designed the study and advised on surgical and ICU information; all information pertaining to surgical procedures and ICU stay for the final manuscript were considered by MB and BL. CM, BL and CU were responsible for the coordination of the study. CU and CM contacted patients, and collected and entered data. MS and CB advised for all quality of life and PTSD issues. KB, HR and MB analyzed data, and KB was responsible for the final manuscript. KB, CB, HR, MS and MB interpreted and discussed all data. All authors read and approved the final manuscript.


The diagnostic potential of breast MRI can be fully utilized only when it is possible to biopsy lesions detected on MRI, especially when they are not visible on mammography or USG. We would like to describe our experience with MRI-guided wire localization and biopsy. Dynamic contrast-enhanced MRI of the breast (DCE-MRI) is a sensitive modality to pick up small cancers in the breast. NevertheThe MRI-guided breast biopsies were performed on a 1.5-T scanner  with a dedicated 4-channel breast biopsy coil and positioning device  which can either be a grid or pillar and post positioning device Figures –c. The pAn MRI-compatible 13-gauge coaxial needle  was usedBoth un-enhanced and contrast-enhanced axial images were obtained to confirm the persistence of the enhancing lesion seen on a previous diagnostic MRI done at our institute or elsewhere , and theThe procedure for wire placement was the same as that for lesion localization. After localizing the lesion, a cannula with a wire loaded into it was inserted to the required depth. After confirming the position on axial images, the wire was placed into the lesion and the position was verified with a repeat axial scan. The wire gives a thin linear magnetic susceptibility artifact, and the lesion can be seen near the tip of the wire . It was 15 patients underwent MRI-guided interventions for MRI-only lesions. Seven patients underwent MRI-guided breast biopsy and eight patients underwent MRI-guided wire localization followed by surgery. The average lesion size was 1.2 cm . The average procedure time for biopsy  was 40-45 minutes and for wire localization, approximately 30 minutes.With the pillar-and-post technique used, 1-2 cores of at least a centimeter length were obtained that were adequate for histology. Wire localization was satisfactorily done in all cases with no report of any dislodgement. 5/15 (33.3%) patients were found to have malignancy on histopathology. There were no immediate or delayed complications of the procedures.Compression grid or pillar-and-post systems to immobilize the breast with the patient lying prone are the commonly used techniques, though free-hand MRI-guided interventions and supiet al.,[et al,[et al.,[Fischer et al., in 1994 et al., in 2001,.,[et al, reported,[et al., the rateOur series is small and represents our initial experience with 15 patients, 7 of whom underwent biopsy and 8, wire localization. There was no immediate or delayed complication. Adequate tissue samples could be obtained. No wire dislodgement was reported. The rate of malignancy in our limited study was 33.3%, which conforms to the previously reported findings.14The difficulties we faced in our experience were as follows:The prone position is sometimes difficult for the patient.Only one breast can be biopsied at a time.Deep posteriorly placed lesions and retroareolar lesions are difficult to target.


The introduction of the highly active antiretroviral therapy (HAART) has greatly improved survival. However, these treatments fail to definitively cure the patients and unveil the presence of quiescent HIV-1 reservoirs like cells from monocyte-macrophage lineage. A purge, or at least a significant reduction of these long lived HIV-1 reservoirs will be needed to raise the hope of the viral eradication. This review focuses on the molecular mechanisms responsible for viral persistence in cells of the monocyte-macrophage lineage. Controversy on latency and/or cryptic chronic replication will be specifically evoked. In addition, since HIV-1 infected monocyte-macrophage cells appear to be more resistant to apoptosis, this obstacle to the viral eradication will be discussed. Understanding the intimate mechanisms of HIV-1 persistence is a prerequisite to devise new and original therapies aiming to achieve viral eradication. Human immunodeficiency 1 (HIV-1), identified in 1983 . HoweverResting memory CD4+ T cells are the major cellular and the best characterized reservoir in the natural host ,10-13. TIn this review, we focus on the molecular mechanisms responsible for viral persistence in cells of the monocyte-macrophage lineage since they are believed to be an important source of HIV-1 ,19. SeveCells of myeloid lineage including monocytes, macrophages and dendritic cells figure  play an Infected monocytes have been recovered from the blood of HIV-1 infected patients, even from those on HAART and with a viral load below detectable limits ,31. EarlDendritic cells are also involved in the dissemination of HIV-1 following primary infection . After cin vivo reactivation of these infected macrophages in response to opportunistic infections is in favor of macrophages as HIV-1 reservoirs [Macrophages harboring the CD4 receptor and CCR5 coreceptor are now recognized as early cellular targets for HIV-1 . These cservoirs ,47. Finaservoirs ,49. Fourservoirs . Among tservoirs ,50.Haematopoïtic cells (HPC) have also been proposed to serve as a viral reservoir, since a subpopulation of CD34+ HPCs express CD4 and CCR5 and/or CXCR4 and these cells are susceptible to HIV-1 infection -54. Furtstricto sensu as previously defined, but in a larger sense which includes true latency and low ongoing replication. Contrary to the CD4+ T cells, in which the mechanisms of the establishment and the maintenance of true latency have been well described [Following fusion-mediated entry into the host cell, the virus is uncoated, the virus genome is reverse transcribed and the pre-integration complex enters the nucleus where the proviral DNA is integrated into the host cell genome. In productive cells, the transcription of the provirus DNA is regulated by the interplay of a combination of viral and cellular transcription factors -63. Howeescribed , our knoPre-integration latency is frequently observed in CD4+ T cells. This form of latency has a very limited contribution to viral persistence since the half lives of the cells is very short (1 day). On the contrary, this form of latency in the monocyte-macrophage lineage may contribute to the formation of reservoirs to a larger extent and may participate in viral dissemination. This form of latency is characterized by a poor reverse transcriptase activity and therefore it is unable to synthesize the provirus DNA. Various mechanisms are involved in this form of latency, such as hypermutation of the DNA induced by the restriction factor APOBEC3, a low level of dNTP pool and an impaired nuclear importation of the pre-integration complex associated to a low level of ATP pool -68. SevePost-integration latency occurs once the viral genome has been reverse transcribed and has been stably integrated into the host genome. At that moment, the level of transcription is very low with a no or a low level of virus replication. Mechanisms generating HIV latency in the CD4+ T cells are well described ,76. ViraIt has been proposed that restriction of the integrated HIV-1 genome transcription is due to the lack of Tat transactivation. The recruitment of the positive transcription elongation factor (pTEFb), which is composed of two proteins, cyclin T1 (CycT1) and cyclin dependant kinase 9 (Cdk9) -82 makesThe lack of host transcriptional activators or the presence of host transcriptional repressors may also explain latency in these cells. It has been reported that distal LTR binding sites upstream of the NF-KB binding site are essential for the efficient transcription in monocytes and macrophages. In addition to NF-KB and Sp1 binding, NF-IL6 and/or USF protein binding to the LTR modulatory region are essential for HIV-1 transcription -87. In cThe inhibiting form of C/EBPβ/NF-IL6 (LIP), a 16 kDa inhibitory isoform that is structuraly close to C/EBPγ, is expressed in macrophages during differentiation. LIP expression is linked to the suppression of HIV-1 replication . AlthougThe zinc-finger protein OKT18, which is produced during HIV-1 infection of macrophages, suppresses HIV-1 transcription through the viral LTR ,93. ThisHIV-1 transcription is also modulated by proteins of the Sp1 family which differ in the nature of the Sp protein bound to the LTR and of the cell type. Indeed, Sp1 and Sp3 are both expressed in microglial cells, unlike CD4+ T-cells, which express only Sp1. In microglial cells, although Sp1 acts as an activator of HIV-1 transcription, the Sp3 protein represses the HIV-1 promoter activity . Some faIt is now well established that viral promoter activity depends on the chromatin environment . Nucleoswaf (unpublished data). This latter factor has been recently described as a pivotal facilitator of the HIV-1 life cycle in macrophages [The transcription factor COUP-TF interacting protein 2 (CTIP2) has been reported  to play rophages ,117. Indrophages . CTIP2 rrophages . Howeverrophages . Indeed,rophages . As discThe host antiviral processes using microRNA (miRNA) as a defense mechanism are now considered as fundamental for regulation of animal and plant gene expression . Indeed Finally, it has been proposed that the establishment of latently infected macrophages occurs in a suppressive microenvironment made of apoptotic cells . ApoptotAnother strategy developed by the virus in order to persist in infected cells is to render them resistant to apoptosis.Several reports have pointed out that NF-kB activity prevents cells to undergo apoptosis ,133 The in vivo establishment of the HIV-1 macrophage reservoirs. Macrophage express 10-times lower numbers of cell surface CD4 than CD4+ T cells [+ T cells, thereby allowing macrophage to form long-lasting virus reservoirs [The protein Nef is a regulating protein expressed early and abundantly throughout the course of HIV-1 infection. This protein has dual effects depending on the stage of infection. In the early stage, Nef contributes to the constitution of reservoirs with sustained virus production. It mimics the action of TNFα with subsequent activation of NF-kB and MAPK ,141. In  T cells , and the T cells , low levservoirs ,149.Tat and gp120 have also dual effects on apoptosis depending of the cell type. In the central nervous system, HIV-1 triggers apoptosis in neurons. This is also seen when neurons are exposed to extracellular Tat or gp120 . On the Over-expression of CTIP2, described as an anti-apoptotic factor , in micrThe protein gp120 produced by monocyte-macrophages inhibits TRAIL-mediated cell death by inducing the expression of macrophage-stimulating factor (M-CSF). This envelope protein also up-regulates expression of several anti-apoptotic genes such as Bfl-1 and Mcl-1 . A stablFinally, a new mechanism has been proposed that protect cells from apoptosis and therefore extend the lifespan of infected cells. This mechanism is based on the suppression of the cell's RNAi activity by synthesis of a TAR miRNA, a small hairpin RNA. This RNA is capable to sequester the miRNA processing machinery of the cell and therefore impedes the functioning of cellular antiviral miRNAs . MoreoveOur knowledge of the mechanisms involved in apoptosis resistance is far from complete. The diversity of strategies used by HIV-1 to manipulate the apoptotic pathway emphasizes the capacity this virus possesses to survive in its host. We note that mechanisms involved in apoptosis resistance, at least the mechanisms involving the TNF-signaling pathway, are also involved in virus production. It seems that the nature of this reservoir is rather different from the latent reservoir. The therapeutical implications are therefore important since stimuli such as phorbol esters will not be suitable to purge the reservoir. Indeed, this treatment will reactivate the expression of HIV-1 in latent reservoir but may increase the resistance to apoptosis in viral reservoir that exhibit a sustained production of virions. The survival of viral reservoirs is of great importance since it is also an obstacle to HIV-1 eradication. The mechanisms underlying this apoptosis resistance are essential for devising new and original therapeutic strategies to purge the reservoirs, but are far from being completely known.The introduction of HAART in 1996 has greatly improved survival, but it has been unable to eradicate the virus from latently infected reservoirs. A principle cause may be that besides the best characterized cellular reservoir of memory CD4+ T-cells there are other reservoirs, such as the monocyte-macrophage reservoir. Moreover, these cells are often found in tissue sanctuary sites, like the brain, that are protected from drug penetration -168. FurAt present the therapy of HIV-1-infected patients is based on a combination of HIV gp41, reverse transcriptase and protease inhibitors. We believe that new drugs should target other steps of the HIV-1 cycle. For example, they could be directed against proteins involved in the transcription of the inserted virus genome. Tat has a critical role in transcription, and constitutes a major target in therapeutic intervention in the HIV replicative cycle -174. MorFinally, new drugs must be designed with properties that allow them to penetrate tissue-sanctuary sites such as the brain .Recently, a new and original strategy has been proposed to eradicate the virus from infected patients. The main idea is to facilitate the reactivation of viruses from latent reservoirs, which are then destroyed by HAART figure . Many faIncreasing the susceptibility of the infected cells to apoptosis is also of great interest. Essentially, the HIV-1 proteins Tat, Nef and the envelope protein gp120 should be targeted, as these proteins have a crucial function in different steps of the virus cycle and also in the acquired resistance to apoptosis. A better comprehension of mechanisms involved in resistance to apoptosis has also allowed to devise drugs against host factors which render cells susceptible to die. For example, molecules which interfere with the chemokine receptor CCR5 are promising since these molecules are both involved in virus entry and in apoptosis resistance. Several CCR5 antagonists are already used in clinical trials ,189. A cA dramatic reduction of monocyte-macrophage reservoirs might be achieved by strategic interventions targeting both the resistance of infected cells to apoptosis and the reactivation of latently-infected cells associated with a reinforced antiretroviral therapy.Cells from monocyte-macrophage lineage are an ideal reservoir for HIV-1. This is due to several features of these cells including long lifespan, absence of direct cytopathic effect, apoptosis resistance of infected cells, existence of latently infected cells (with low or no virus expression) and their localization in sanctuaries. The purging of this reservoir is therefore crucial since it constitutes one of the major obstacles to HIV-1 eradication from infected patients. A drastic reduction of this reservoir might be achieved by the development of new and original therapies which target specifically all the reservoirs, including the monocyte-macrophages. These treatments might not lead to a complete eradication of HIV-1 but there is hope that a drastic reduction could be achieved with levels of expression comparable to those in elite non progressors patients who remain asymptomatic without an antiretroviral therapy.The authors declare that they have no competing interests.CS conceived and wrote the manuscript. VLD, GH and OR contributed to the writing of the manuscript. VLD created figures


Gamma-band oscillations recorded from human electrophysiological recordings, which may be associated with perceptual binding and neuronal connectivity, have been shown to be altered in people with autism. Transient auditory gamma-band responses, however, have not yet been investigated in autism or in the first-degree relatives of persons with the autism.We measured transient evoked and induced magnetic gamma-band power and inter-trial phase-locking consistency in the magnetoencephalographic recordings of 16 parents of children with autism, 11 adults with autism and 16 control participants. Source space projection was used to separate left and right hemisphere transient gamma-band measures of power and phase-locking.Induced gamma-power at 40 Hz was significantly higher in the parent and autism groups than in controls, while evoked gamma-band power was reduced compared to controls. The phase-locking factor, a measure of phase consistency of neuronal responses with external stimuli, was significantly lower in the subjects with autism and the autism parent group, potentially explaining the difference between the evoked and induced power results.These findings, especially in first degree relatives, suggest that gamma-band phase consistency and changes in induced versus induced power may be potentially useful endophenotypes for autism, particularly given emerging molecular mechanisms concerning the generation of gamma-band signals. The gamma-band range of oscillatory EEG activity 30–80 Hz) has received significant attention in recent years because of its long postulated association with perceptual binding and connectivity ,2, relat0–80 Hz hChanges in oscillatory activity such as those described for the gamma-band may be subdivided into induced changes  and evoked changes , plus gWe undertook the current study to ascertain whether people with autism and first-degree relatives of people with autism exhibit changes in transient gamma-band power. Autism is highly heritable, with monozygotic concordance rates ranging from 36% to 91% and overall autism spectrum heritability rates estimated to be as high as 90% -15. Few We recruited adults with autism and parents of children with autism to participate in the study and hypothesized that phase-locked auditory evoked gamma-power would be lower in the autism group and the parent group than in a healthy comparison sample, while non-phase-locked power would be significantly higher in the parent group. We further speculated that a measure of inter-trial phase-consistency for gamma oscillations, the phase-locking factor, would be reduced in the autism and parent groups relative to control subjects as a potential explanation for the proposed power changes.rd edition  who are parents of a child with autistic disorder were recruited to participate in the experiment. Each parent had just one child who met DSM-IV criteria for autism, as determined by consensus of the Autism Diagnostic Observation Schedule , the AutAIS-III: ). SixteeMEG recordings were made to monaural, contralateral ear stimulus presentations of 1 kHz sine-wave stimuli . Stimuli were delivered via foam insert earphones . At least 150 discrete stimulus trials  were delivered per ear.MEG data were acquired with a 4D Neuroimaging  Magnes I neuromagnetometer system with 37 axially-wound, first-order gradiometers (5.1 cm baselines and 2 cm coil diameters). Recordings were made inside a custom-built magnetically-shielded room with 35 dB reduction at DC, and 55 dB reduction at 60 Hz. Participants were seated comfortably in a non-magnetic recording chair, and were allowed to view a silent video of their choice during recording sessions on a monitor located 4 m outside the room. Each hemisphere was recorded separately and the order of recording was random for each participant. The dewar was positioned over each side of the head so that the center channel of the hexagonal array would be centered between the minima and maxima for the M100 .Prior to MEG recordings, the location and orientation of the MEG coils relative to each subject's head were determined by digitizing a set of fiducial reference points on the head using a magnetic digitizer (Polhemus 3SPACE). The left and right preauricular points (LPA and RPA) and the nasion, as defined in the EEG 10–20 electrode system, were used to establish a right-handed Cartesian coordinate system, where the line between LPA and RPA is the x-axis (positive x out the left ear). The y-axis is the line normal to the x-axis at the midpoint (origin), with positive y exiting through the front of the head at the nasion, and the z-axis is normal to x and y at the origin (positive z exits at the top of the head). After digitizing the reference points, the shape of each subject's head under the recording surface of the MEG system was digitized for use in constructing a volume conductor model for MEG source localizations (see below).MEG data were digitized at 16-bits quantization at a sampling rate of 1041.7 Hz within an epoch window of 450 milliseconds (200 ms pre-stimulus and 250 ms post-stimulus) triggered by the onset of each sound. An analog pass band between 1 and 200 Hz was used during acquisition.After acquisition, all data epochs with values exceeding ± 2500 fT were rejected from further analysis to exclude trials with eye-blinks and movement artifacts. The remaining epochs were signal averaged separately for each hemisphere to produce an averaged auditory evoked magnetic field for each of the 37 sensors. Averages were then baseline corrected using the -200 to 0 ms window and were digitally band-pass filtered  at between 35 and 45 Hz for source analyses of the transient gamma-band response.Source analyses of the averaged data were conducted using BESA 5.1 software . A best fitting conductive sphere model was constructed from the intracranial surfaces of the T1-weighted MRI scans of subjects enrolled in the study  was then fit separately for left and right hemispheres using a spatiotemporal model to the observed data in the post-stimulus window between 40–80 ms, yielding parameter estimates of the x, y, and z ECD position information, as well as dipole orientation and magnitude over time . SSP isThe SSP channel timeseries, q(t), for the left and right hemispheres were then transformed to the frequency domain using the Morlet wavelet  decomposition . For eacIn addition to evoked source strength, a more direct measure of phase consistency across trials at some specified time slice was also calculated. If there were no phase consistency in response to the stimulus across trials (at some particular time slice), then the distribution of phases would be approximately uniform. Accordingly, some dominant consistent phase angle across trials would result in a unimodal distribution. The circular variance of this distribution would then provide one method of quantifying phase consistency and is defined as :θI is the phase angle at the ith trial. The second term in the above equation is often described in the literature . The liThe findings of this study are interesting to consider together with our recently reported findings of reduced steady-state evoked gamma-band power in children and adolescents with autism . In doinSecond, the previous study employed steady-state stimulation, which directly entrains a particular frequency  in the EEG or MEG response. Our own unpublished observations suggest that this type of stimulation may accentuate group differences in gamma-power and phase-locking between groups over pure tone or unmodulated stimuli. For this study, a transient rather than driving stimulus was chosen because we were specifically interested in the obligatory, transient gamma-band response rather than the steady-state response. Although we believe that the overall result of reduced phase-locked power is consistent with the earlier proband paper, a direct comparison between probands, first-degree relatives and control subjects using both transient and steady-state stimuli will be needed to establish which stimulation method, if any, produces more robust results. A final comment regarding our earlier finding is that we did not examine either phase-locking or induced power in the previous paper. The inclusion of the adult autism sample in the current experiment would seem to suggest some continuity of the finding of reduced evoked power across two independent samples of persons with autism at much different developmental stages, but future studies should examine more thoroughly the relationships between phase-locked and non-phase-locked power in persons with autism spectrum conditions.A-receptor mediated neurotransmission , but noa et al. ,47).The reduced anterior-posterior asymmetry of the ECD coordinates for the autism and autism parent groups may be a reflection of a general neurodevelopmental disturbance in the asymmetry of the brain. Previous work in our own lab -51, replThe use of endophenotypes, whether behavioral or physiological, may increase the reliability of such genetic studies. The potential utility of this approach with respect to GABA was demonstrated by a study reporting that a symptom-level approach to creating family subsets based on the presence of savant skills improved genetic linkage to 15q11-13 . Few stuThe authors declare that they have no competing interests.DCR conceived the study, including the data analytic strategy and statistical analyses and drafted the manuscript. KM and PT made important contributions concerning the time-frequency analyses and source space projection approaches. SJR made a contribution to the adult autism recruitment and diagnosis as well as to the proband assessments necessary for recruiting the parent sample for the manuscript.The pre-publication history for this paper can be accessed here:


It was determined in an earlier study that ferritin-heavy (H) and -light (L) chains in lens fiber cells are modified in comparison to those in lens epithelial cells. The purpose of the present study was to determine whether changes in ferritin chain characteristics are developmental, age-related, or associated with cataractogenesis, by analyzing the distribution of modified chains throughout the lens fiber mass.59Fe.After removing the capsule, noncataractous and cataractous lenses were separated into six layers of fiber cells. The content of ferritin H and L chains in each layer was determined by western blotting with chain-specific antibodies. The level of ferritin complex (450 kDa protein made up of assembled L and H chains) was determined using the enzyme-linked immunosorbent assay. The ability of ferritin complex to bind iron was assessed by in vitro labeling with Fiber cell ferritin L chains were 30 kDa , and were present at the highest level in the outermost layers of both cataractous and non-cataractous lenses. The amount of modified L chains decreased gradually in the inner layers of the fiber mass, and was undetectable in the inner two layers of cataractous lenses. The ferritin H chains were also modified to 12 kDa  in both cataractous and non-cataractous lenses. Similar levels of this modified H chain were found throughout the normal lens. Interestingly, in cataractous lenses, the modified H chains were found in decreasing amounts towards the interior of the lens, and were undetectable in the nucleus. However, in these cataractous lenses, the normal-sized ferritin H chains (21 kDA) appear in small quantities in the outer fiber layers, and increase in quantity and size (to 29 kDa) in the inner layers. This observation was best seen and demonstrated in advanced cataracts. Ferritin, which can bind iron, was found mainly in the outer layers of the lens fiber mass of normal lenses, but was more evenly distributed in fiber layers from cataractous lenses.Both ferritin H and L chains were modified in lens fiber cells from normal and cataractous canine lenses. These modifications were not age-related, and most likely occur during the differentiation of epithelial cells to fiber cells, since only normal-sized chains have been found in lens epithelial cells. In addition, there was a specific and distinct distribution of these modified chains throughout the lens fiber mass. The most striking differences between normal and cataractous lenses fiber cells were the appearance of normal-sized ferritin H chains and the relatively even distribution of iron binding capacity throughout the fiber mass of the cataractous lenses. These differences may reflect a response of the lens to increased oxidative stress during cataractogenesis. Aging lenses develop a barrier preventing the flow of antioxidants from the metabolically active cortex to the lens nucleus . As a reFerritin is present throughout the entire noncataractous and cataractous lens ,7. HowevAnalysis of the distribution of ferritin and ferritin chains in different layers would provide important information about the processing of these proteins and the roles they play in the lens. This includes how ferritin and ferritin chains are modified during differentiation from epithelial cells to fibers, during the process of aging, and, more specifically, whether these modifications are related to aging and/or cataractogenesis. Furthermore, these studies will help to determine if there are unmodified ferritin chains in specific layers of fiber cells that cannot be detected in protein-rich homogenates of whole lenses. A clearer understanding of the distribution of ferritin and ferritin chains in different lens layers would provide important information on the possible involvement of ferritin in the prevention of lens opacification caused by iron-induced oxidative damage.Eyes were obtained postmortem from mixed-breed dogs euthanized at the Johnston County, North Carolina Animal Shelter. Five normal lenses were from 1- to 10-year-old dogs. Three cataractous lenses were from 8- to 10-year-old dogs. They were categorized as early-cataract (2 lenses) and advanced-cataract (1 lens), according to the severity of visible opacities.The lens capsule with epithelial cells was removed. The diameter of the total lens was measured and layers of fibers were peeled off using forceps under a dissecting microscope. The diameter of the remaining part of the lens was measured after each layer of fiber was removed. Each lens was dissected into six similar-size regions, and labeled in the order in which they were isolated . The fibers of each lens region were collected in a small volume of phosphate buffered saline.Lens fiber fractions were sonicated in 10 mM Tris/HCl buffer, pH 7.4, containing 2% SDS and protease inhibitor cocktail for mammalian cells . The concentration of protein in the homogenates was determined using the BCA Protein Assay Kit .. Chain-specific antibodies and TrueBlot HRP-anti-rabbit IgG antibodies  were used for immunodetection of ferritin chains, as described previously [Samples containing 50 µg of protein were separated by 15% SDS-PAGE. Proteins were transferred to nitrocellulose membranes  by semidry blotting at 20 V for 20-30 mineviously . We desi59 Fe as ferric chloride  or as ferrous sulfate  for 1 h at room temperature. Labeled proteins were separated by 8% SDS-PAGE under non-reducing conditions. Ferritin binding of 59Fe was measured in an Instant Imager . There was no difference between labeling ferritin with radioactive ferrous or ferric iron.Samples containing 1-1.5 mg of protein were incubated with Ferritin concentration was measured by a sandwich enzyme-linked immunosorbent assay (ELISA) using goat anti-horse ferritin and HRP-labeled goat anti-horse ferritin antibodies (Bethyl Laboratories Inc.), as described previously .The lenses with early cataracts showed central opacities and a transparent rim. The more advanced, mature cataractous lens was opaque throughout . The thiThe SDS-PAGE total protein profiles of homogenates of fiber layers were used to assess the separation of the lens into different regions. There were differences in the protein profiles of fiber cell lysates originating from different layers of lens. The differences included gradual elimination of higher molecular weight proteins (~ 90 kDa) from the lysates of layers 3-6 and visibly lower resolution of  lower weight proteins ( ~ 20 kDa) in layers 5 and 6 . The anaIn fiber cell homogenates of noncataractous and cataractous lenses, anti-ferritin L-chain antibodies detected a 30 kDa protein that was larger than the liver ferritin L-chain (19 kDa) used as a standard . These rAntibodies against ferritin H chains detected a 12 kDa protein in fiber cells from noncataractous and cataractous lenses . This 12The distribution and content of ferritin throughout the lens sections were measured by ELISA using the antibodies that predominantly recognize the whole assembled ferritin molecule . They had a low affinity for unassembled free ferritin chains, as previously reported ,8. The r59Fe, that lens fiber cell homogenates contain assembled ferritin (450 kDa) of a mobility equal to that of holoferritin from canine livers [59FeCl3) and ferrous (59Fe2SO4), which must undergo oxidation before incorporation into ferritin. There was no difference between in vitro labeling of ferritin with radioactive ferrous or ferric iron.In our previous studies, we showed, by labeling with e livers ,8. In orNoncataractous lenses contained more iron-labeled ferritin in the three outer layers of fiber cells and significantly less in the inner regions of the lens . The disThis study also confirmed our previous findings that assembled ferritin from cataractous lenses binds more iron than does ferritin from noncataractous lenses .Long-lived lens proteins undergo little turnover but show a high degree of enzymatic and non-enzymatic posttranslational modification. These modifications start early in life and continue as the lens ages, resulting in the accumulation of modified proteins . ChangesAnalysis of the distribution pattern and characteristics of ferritin chains in lens fiber cells revealed similar changes to those described for lens crystallins, and consisted of size alteration, acidification, and polymorphism . The modFerritin H chains identified in human and canine lens fiber cells, regardless of age, were smaller—and most likely truncated —than those found in lens epithelial cells, which contain the well-characterized 21 kDa form . This mo59Fe, iron-binding ferritin was detected in all layers of fiber cells, although there was a trend to lower levels in the nuclear region , and is close to the detection limit of this technique [59Fe labeling may detect the presence of ferritin at a concentration below the ELISA’s detection level, but only if ferritin is functional—that is, capable of binding iron. Based upon these results, we hypothesized that the lens nucleus contains mainly improperly assembled ferritin, which retained its antigenity and therefore was measured by ELISA but has limited ability to bind iron. Indeed, ferritin associated with the water-insoluble fraction of lens protein homogenates has been reported in the nucleus of human lenses with age-related nuclear cataracts [In order to determine whether the distribution of ferritin chains coincided with that of assembled ferritin, ferritin was measured throughout the lens by ELISA, using polyclonal anti-ferritin antibodies that preferentially recognized assembled ferritin but had a low affinity for ferritin L and H chains. In both cataractous and noncataractous lenses, almost all ferritin detected by ELISA was located in the lens nucleus . Howeverr region . Such a echnique ,10. Therataracts . The abnWe have previously shown that ferritin from cataractous lenses incorporates more iron than that from noncataractous lenses . The preBased on our previous and current investigations, we hypothesized that L and H ferritin chains are modified during the differentiation of lens epithelial cells into fiber cells. As the lens ages, the chains undergo additional conformational changes, which renders them unrecognizable by anti-ferritin-chain antibodies, and/or degradable by proteolytic enzymes. The activity of proteolytic systems declines in mature fiber cells , which mThe distribution and chain content of the assembled ferritin reflect the need for lenticular tissue as protection against iron-catalyzed oxidative damage. Since both transparent and cataractous lenses contained iron-binding ferritin, the modified and normal-sized ferritin chains apparently assembled into functional ferritin complexes . Lenses


The management of metastatic spinal disease is generally considered palliative, as the progression of systemic disease is likely to hinder survival. Although the occurrence of C1-C2 instability due to metastatic disease is not uncommon and thus treatment options have been well-defined, craniocervical instability due to lesions occurring at the junction of the occiput and atlas is more rare, and treatment for metastasis to this region is less well-defined.We present a patient with non-small-cell lung cancer metastatic to the atlanto-occipital facet joint complex. A drastic improvement in the presenting debilitating mechanical neck pain was noted following an occipitocervical fusion. A literature review of published cases of metastases to the occipitocervical junction was conducted along with treatment options.The atlanto-occipital facet joint is a rare site of metastatic disease. Destruction of this joint can lead to significant neck pain secondary to instability. Spinal fusion may afford significant and rapid resolution of these symptoms, and should be considered in the management of patients—even those with end-stage oncologic disease. Lung cancer is classified by the World Health Organization as the leading cause of cancer-related deaths in men and the second highest in women, claiming more than 1.2 million lives worldwide per year. In the U20The prognosis of patients diagnosed with lung cancer is poor, due to association with systemic, incurable disease. As of 20NSCLC metastasizes to a variety of locations within the body, including the brain, bone, liver, and adrenal glands, in order of decreasing prevalence. Of note,In this report, we present a patient with known NSCLC metastasis who presented with severe axial neck pain due to tumor infiltration and destruction of the atlanto-occiptal joint. Although the etiologies and treatment strategies for managing destructive lesions of the atlanto-axial (C1-C2) joint are known, to our knowledge, this is the first published case of an NSCLC lesion that metastasized to both the occiptal condyle and the atlas , causing instability and severe neck pain. We also review the literature and discuss current treatment options for bony metastasis to the occipitocervical junction.A 66-year-old female with a two-year history of NSCLC status post lobectomy, with metastasis to the right kidney, right femur and pelvis, presented with severe occipital headaches and debilitating neck pain. She was functionally independent, with a Karnofsky score of 90. She was neurologically intact except for a left homonymous hemionopsia due to an old posterior cerebral artery distribution infarct. Brain and cervical spine computed tomography (CT) and magnetic resonance imaging (MRI) scans revealed an old right occipital stroke, right C1 lateral mass and occipital condyle destructive lytic lesions, and a fracture of the right atlanto-occipital joint Figure –c. ThereThe patient underwent an occiput to C5 instrumented stabilization and fusion using an occipital plate connected to lateral mass screws at C3, C4, and C5 Figures  and 3. DDistant bony metastases of lung cancer have been associated with significant skeletal-related events (SREs), including pain , pathological fractures, hypercalcemia, and spinal cord compression. Of these68et al. reported four cases of metastatic cancer involving the atlanto-occiptal joint over a 20-year period from 1981-2001.[et al. described a patient with malignant fibrous histiocytoma invasion of the right craniovertebral joint who presented with intractable occipitalgia.[et al. described a patient with C0-C1 instability due to myeloma,[et al. described a case of metastatic follicular thyroid cancer to the occipitocervical region.[Metastatic involvement of the craniovertebral junction due to other primary tumors has been described in seven cases with documentation of involvement of the C0-C1 junction and ensuing instability . Specifi myeloma, and Laohl region. Thus, toGiven the poor prognosis of metastatic spine disease, especially late-stage NSCLC, treatment for such lesions are palliative in nature. Well-defined NSCLC lesions may be treated with radiotherapy and/or surgery. Specific3520et al. concluded that direct surgical decompression plus radiation therapy for metastatic epidural spinal cord compression improved functional status, relative to ambulation and continence, compared to radiation alone.[79Although survival of patients with metastatic spinal disease ultimately depends on the extent and control of the underlying systemic disease, quality of life can be substantially improved with surgical stabilization. In their landmark study, Patchel on alone. Althoughon alone.7914 In oIn most cases, patients with metastatic disease at the atlanto-occipital joint experience a reduction in headache or upper neck pain following occipitocervical fusion.714 Howev7This is the first reported case of NSCLC metastasizing to the atlanto-occipital joint causing severe sudden onset neck pain due to mechanical instability. Significant alleviation of pain was achieved via surgical stabilization . Although metastatic disease caused by NSCLC carries a poor prognosis, occipitocervical stabilization may provide excellent pain relief and improvements in quality of life in properly selected patients.


Retroperitoneal fibrosis (RPF) is a rare fibroinflammatory disease that leads to hydronephrosis and renal failure. In a case-control study, we have recently shown that asbestos exposure was the most important risk factor for RPF in the Finnish population. The aim of this study was to evaluate the relation of asbestos exposure to radiologically confirmed lung and pleural fibrosis among patients with RPF.Chest high-resolution computed tomography (HRCT) was performed on 16 unexposed and 22 asbestos-exposed RPF patients and 18 asbestos-exposed controls. Parietal pleural plaques (PPP), diffuse pleural thickening (DPT) and parenchymal fibrosis were scored separately.Most of the asbestos-exposed RPF patients and half of the asbestos-exposed controls had bilateral PPP, but only a few had lung fibrosis. Minor bilateral plaques were detected in two of the unexposed RPF patients, and none had lung fibrosis. DPT was most frequent and thickest in the asbestos-exposed RPF-patients. In three asbestos-exposed patients with RPF we observed exceptionally large pleural masses that were located anteriorly in the pleural space and continued into the anterior mediastinum.Asbestos exposure was associated with DPT in comparisons between RPF patients and controls  as well as among RPF patients .The most distinctive feature of the asbestos-exposed RPF patients was a thick DPT. An asbestos-related pleural finding was common in the asbestos-exposed RPF patients, but only a few of these patients had parenchymal lung fibrosis. RPF without asbestos exposure was not associated with pleural or lung fibrosis. The findings suggest a shared etiology for RPF and pleural fibrosis and furthermore possibly a similar pathogenetic mechanisms. Retroperitoneal fibrosis (RPF), or Ormond's disease, is a rare condition with fibrosis covering the abdominal aorta and the ureters. The etiology of RPF is generally unknown. It has been proposed that approximately one-third of RPF cases develop secondarily to aortic aneurysm, abdominal infections or surgery and as a side effect of several drugs, especially methysergide and other ergot derivates -3. AsbesThis material was part of our case-control study including 43 persons with RPF and 179 randomly assigned controls matched for year of birth, gender and central hospital district in Finland . The diaAll of the participants were interviewed for medical history and asbestos exposure. The cumulative exposure to asbestos dust was estimated using fiber-years (40-hour shift per week at an average dust level of 1 fiber/ml for 1 year) and graded as follows: no significant asbestos exposure; slight exposure (asbestos exposure <10 fiber-years) and moderate-to-high exposure (asbestos exposure ≥ 10 fiber-years). Exposure was assessed by an occupational health physician with special expertise in the evaluation of asbestos exposure, the physician was blinded in terms of the case-control status of the participants. The details of the data collection have been given in our previous report .We asked all of the unexposed and exposed patients with RPF and the controls with moderate-to-high asbestos exposure to participate in a study evaluating pleural and lung fibrosis with chest high-resolution computed tomography (HRCT). The Ethics Committee of the Tampere University Hospital approved the study protocol.Altogether 38 (88%) of the patients with RPF and 18 (86%) of the asbestos-exposed controls were willing to participate. None of the 5 RPF patients who refused to participate in the HRCT study had notable asbestos exposure, which was also the main reason for their refusal.The mean time since first asbestos exposure was 41.4 (SD 12.1) years for the RPF patients and 42.4 (8.9) years for the controls  were scored separately . The outcome variable was the radiological finding categorized into four classes. The results of the ordinal logistic regression analysis therefore indicated susceptibility to the development of fibrotic changes, given asbestos exposure. Statistical significance was assessed using the likelihood ratio test. In addition, the susceptibility for asbestos-related pleural fibrosis among the RPF patients was evaluated in a case-case setting  using loThe unexposed RPF patients had only minor pleural plaques ≤ class 2), and the differences between this group and the asbestos-exposed groups were statistically significant Figure , Table 2, and theMore than 60% of the asbestos-exposed RPF patients and half of the exposed controls had bilateral pleural plaques Figure , Table 2DPT occurred more frequently among the RPF patients with asbestos exposure than among the unexposed patients (P = 0.045). There were no differences between the asbestos-exposed RPF patients and controls P = 0.190) and none of the differences between the three groups reached statistical significance  mm for the 4 unexposed RPF patients, 9.8 (SD 5.1) mm for the 12 exposed patients with RPF and 5.1 (SD 2.7) mm for the 7 exposed controls , one asbestos-exposed control (6%) and five asbestos-exposed RPF patients (22%), three of whom had bilateral findings. No statistical difference was noted (p = 0.182).In the ordinal logistic regression analysis, the asbestos-exposed RPF patients had a nonsignificantly increased risk for the development of DPT when compared with that of the asbestos-exposed controls . Age at the time of the HRCT, smoking history, pleural plaques, and lung fibrosis grade had no influence on the development of DPT  of the unexposed RPF patients, six (27%) of the asbestos-exposed RPF patients and six (33%) of the asbestos-exposed controls did not have any of these changes.Using patient's medical files, we were able to evaluate the presence of pleural fibrosis in 14 out of 17 RPF asbestos-exposed patients having fibrosis in the current HRCT. At the time of the RPF diagnosis, eight of them had had fibrotic changes in their thoracic X-ray and 6 had not. The subjects having pleural fibrosis at the time of the RPF diagnosis had a higher mean score for both PPP (2.8 versus 1.5) and DPT (2.1 versus 1.0) compared with the ones who had developed pleural changes after the appearance of RPF. These differences were not of statistical significance.This study describes the association between RPF and asbestos-related lung diseases. We hope that our results help to identify persons who have developed RPF through occupational exposure to asbestos. Even though the findings of the current study alone are not enough to declare a causal association between asbestos exposure and RPF, they strengthen the validity of the results of our earlier case-control study and clarify the phenotype of asbestos-related RPF.To our knowledge, the literature contains only three reports describing asbestos-related pleural findings in altogether five RPF patients -16. In oOn the basis of our results, it can be argued that RPF is an independent risk factor for pleural fibrosis. The results of the case-control setting  occupational asbestos exposure of ≥ 10 fiber-years (OR 8.8) or (ii) occupational asbestos exposure of <10 fiber-years (OR 5.5) combined with bilateral pleural plaques or DPT or both pleural plaques and DPT. The presence of asbestosis  should not to be required for the diagnosis of asbestos-related RPF. Asbestos-related RPF, like asbestos-related pleurisy, should be a diagnosis of exclusion. Nevertheless, asbestos-related pleural findings should be taken into account also in the presence of other risk factors, such as ergotamine medication or abdominal aortic aneurysm.In conclusion, the majority of the asbestos-exposed patients with RPF had asbestos-related pleural fibrosis and it was more extensive than in the asbestos-exposed controls. Lung fibrosis was equally frequent among the asbestos-exposed RPF patients and the controls. RPF without asbestos exposure had no association with pleural or lung fibrosis.The findings suggest a shared etiology for RPF and pleural fibrosis and possibly similar pathogenetic mechanisms in some subjects. All RPF patients should be evaluated for asbestos exposure, and lung HRCT should be performed if appropriate.The authors declare that they have no competing interests.TU was responsible for the data collection, statistical analysis and preparation of the article; RJ and JH evaluated the CT scans; AA planned the data analysis; EH, KM and HS participated in the identification of the RPF patients. PR was the initiator of the study, JU acted as an expert consultant and PO evaluated the asbestos exposure. All of the investigators contributed to the study design and writing of the manuscript.Classification of the pleural findings.Click here for fileClassification of lung fibrosis.Click here for fileAdjusted odds ratios for potential risk factors for parietal pleural plaques, DPT and lung fibrosis in the asbestos-exposed subjects according to an ordinal regression analysis.Click here for file


Alcoholic liver injury is a significant medical problem throughout the world. Protein trafficking pathways, including endocytosis, appear to be especially susceptible to the deleterious effects of alcohol. Using the asialoglycoprotein receptor (ASGPR) as a model, we have studied ethanol-induced alterations in the process of receptor-mediated endocytosis (RME). The ASGPR, a hepatocyte-specific receptor, binds proteins that have lost their terminal sialic acid moiety and have exposed galactose or N-acetylgalactosamine moieties . During 125I-ASOR). We exposed rats in vivo to the labeled ligand, and then assessed both ligand and ASGPR content in two isolated liver endosome populations. The "early endosomes" (EE) consist of endosomes from the periphery of the cell, including endosomes just entering the pathway and those containing receptor and/or ligand being recycled back to the cell surface. The "late endosomes" (LE) contain more distal endosomes which are destined for degradation in lysosomes. The purpose of the work described here was to gather information on the viability of this method for isolating endosome fractions and for assessing both ligand and receptor content in the isolated endosome fractions. Ultimately, our goal is to use this method to further investigate the affect of ethanol on the trafficking of ligand and receptor between endosome fractions.Our previous studies using the ASGPR model have identified several ethanol-induced alterations during the process of RME. Altered receptor-ligand uncoupling and endosomal acidification as well as decreased ligand binding, internalization, and degradation are some of the observed alterations -9. For t– Texas Red–-X protein labeling kit . Male Wistar chow-fed rats (200–250 g) were used for the current studies. Labeled ligand was injected via the penile vein; and either 5 min or 25 min after injection, the liver was perfused with ice cold KRH/10 mM EDTA, pH 7.4 to remove any surface bound ligand, flush blood from the liver, and stop endocytosis. Livers were removed and homogenized in 5 volumes (wt/v) of ice cold homogenization buffer , pH 7.4 in the presence of Sigma protease inhibitor cocktail. Liver homogenates were processed to obtain a microsomal/endosomal pellet that was resuspended in 1.5 M sucrose and applied to the bottom of a discontinuous sucrose gradient. After centrifugation , two endosome fractions were isolated from the sucrose gradient. The fraction enriched in EE was obtained from the 0.86 M/1.15 M interface, and the LE enriched fraction was obtained from the 0.6 M/0.86 M interface. The amount of TR-ASOR or 125I-ASOR was quantified for each time point. For Western blot analysis, protein concentration was adjusted to 2–5 mg/ml in Laemmli sample buffer and fractionated on 10% SDS-PAGE gels. Protein was then transferred to nitrocellulose membrane, and the blots exposed to one of the primary antibodies. Primary antibodies included a monoclonal mouse anti-actin , polyclonal rabbit anti-Rab 4 , and a polyclonal rabbit anti-ASGPR produced in our laboratory [Desialyation and iodination of orosomucoid was conducted using techniques previously described and standard to our laboratory . LabelinFor the current study, we wanted to isolate two endosome populations: one enriched in "early endosomes" and the other in "late endosomes." Pol and coworkers -12 have in vivo injection of labeled ligand it is possible to determine the distribution pattern of ligand in two distinct endosome fractions. In addition, the distribution pattern of receptor can also be assessed in isolated endosomes.Separation of the endosome fractions was assessed using Western blot analysis for the early endosome markers, actin and Rab 4. The endosome samples exposed to the anti-actin antibody showed a strong presence of actin in the EE fractions compared to the LE fractions, where only negligible amounts were detected. The same pattern was observed when the endosome fractions were exposed to the anti-Rab 4 antibody. These results indicate that isolation of the endosomes into fractions enriched in either "early" or "late" endosomes was accomplished. As expected, more ligand was internalized at the 25 min post-injection time point compared to the 5 min post-injection time point. In order to compare the distribution patterns at the two post-injection time points, both ligand and ASGPR content of the different endosome fractions was assessed as a percentage of the total content in the whole endosome population. At both time points, we observed that the EE fraction had significantly more ligand compared to the LE fraction (p – 0.001), and the same endosomal distribution pattern was observed for the ASGPR. These results indicate that following an in vivo method to investigate the affect of ethanol on the trafficking of ligand and receptor between endosome fractions. Studies are currently underway using ethanol-fed animals and the method described above, and preliminary results indicate that exposure to ethanol alters the endosomal distribution of both ligand and ASGPR.Collectively, the results presented here indicate that the isolation of two distinct endosome populations, as well as the characterization of both the ligand and receptor content of the isolated endosomes is possible. Using whole cell hepatocyte preparations we have previously identified ethanol-induced impairments in ligand binding, internalization, and degradation, receptor recycling, and endosome acidification -6. Two d


Idiopathic portal hypertension (IPH) is a disorder of unknown etiology and is characterized clinically by portal hypertension, splenomegaly, and hypersplenism accompanied by pancytopenia. This study evaluates the pathogenic concept of the disease by a systematic review of the literature and illustrates novel pathologic and laboratory findings.We report the first case of uncontrolled splenic hyperperfusion and enlargement with subsequent hypersplenism leading to life-threatening complications of IPH in infancy and emergent splenectomy.Our results suggest that splenic NO and VCAM-1, rather than ET-1, have a significant impact on the development of IPH, even at a very early stage of disease. The success of surgical interventions targeting the splenic hyperperfusion suggests that the primary defect in the regulation of splenic blood flow seems to be crucial for the development of IPH. Thus, beside other treatment options splenectomy needs to be considered as a prime therapeutic option for IPH. Non-cirrhotic portal hypertension (NCPH) comprises a group of diseases with increased portal pressure in the absence of cirrhosis, most of which have portal hypertension as a late manifestation of the disease. Common causes of NCPH include extrahepatic portal venous obstruction, non-cirrhotic portal fibrosis or idiopathic portal hypertension (IPH) . IPH is The underlying etiology and pathogenesis are poorly understood. It has been proposed that infectious, toxic , immunological and immunogenetic factors may play a role . As hepaTill this time, no cases have been described in children under the age of 10 years. Here we report of a boy who developed mild hepatomegaly and severe splenomegaly at 8 months of age. Following emergency splenectomy the child improved rapidly, and all symptoms of the previous illness disappeared including signs of portal hypertension. This is the first case of catastrophic IPH in early childhood, and to our knowledge the first report of disease remission in childhood by surgical means. This points to the spleen as the origin for disease progression in IPH patients. We present a detailed diagnostic work-up for this unusual case and provide a comprehensive review of the literature.A progressive splenomegaly was observed in an otherwise healthy 8 months-old Turkish infant during a pediatric routine examination. The boy's parents are non-consanguineous and healthy, as is his 6-year old brother. The patient's perinatal and neonatal history was unremarkable. An acute infection was initially suspected due to elevated EBV and CMV antibody titers, but a worsening clinical condition with progressive pancytopenia and fever prompted re-evaluation at the age of 13 months. An open liver biopsy, standard bone marrow smear and trephine biopsy demonstrated no features of a metabolic disorder, hematological or immunological disease, infection, hepatic disorder (except for a mild secondary hepatitis) or malignancy.Over the following 5 months, the boy was not thriving and progression of splenomegaly was obvious by palpation and increasing abdominal girth. Abdominal ultrasound confirmed these findings: the liver size was 13.7 cm and the spleen measured 19.1 cm without any findings of abnormal vessel variations and patent hepatic veins. The portal vein was relatively prominent with a width of 1 cm, which in combination with markedly increased blood flow indicates a hyperdynamic situation Figure . We concin situ  with no signs for inflammation, severe pancytopenia without adequate response to periodic transfusions , hemorrhagic diathesis (prolonged bleeding time) and symptoms due to compression of the bordering organs . Due to his life-threatening condition splenectomy was performed, which passed without any complications. During the surgical procedure a second liver biopsy was taken, the spleen was explanted, and small accessory spleen was left u Figure . The posHistopathologic examination of the spleen and liver revealed extreme hyperplasia of the lymphatic tissue within the white pulp of the spleen (splenadenoma), sinusoid ectasia especially in the spleen and expansion of the portal tracts without further hepatic pathology. For immunohistochemistry tissue samples were frozen in liquid nitrogen and stored at -80°C until use or fixed in buffered 4% formaldehyde and routinely embedded in paraffin. Immunohistochemical studies were performed with the previously described primary antibodies recognizing ET-1 , eNOS , and anti-VCAM-1 . In brief, after de-paraffinization, endogenous peroxidase activity was blocked with 3% hydrogen peroxide in absolute methanol for 10 min at room temperature. After incubation with the primary antibody for 12 hours at room temperature, sections were exposed to the secondary biotin-conjugated goat anti-rabbit or goat anti-mouse IgG. This was followed by amplification using system SAB  with 3,3'-diaminobenzidine hydrochloride. Sections were counterstained with Ehrlich hematoxylin. Immunohistochemical staining for endothelial nitric oxide synthetase (eNOS) and vascular endothelial adhesion molecule-1 (VCAM-1) showed extensive expression in the spleen but not the liver, while endothelin-1 (ET-1) staining was weak . VCAM-1 concentration was initially markedly elevated in serum , while ET-1 was normal. One year after surgery, VCAM-1 levels in serum decreased to near normal. Due to the excellent clinical course of the disease, no further measurements were performed.While some reports of IPH in childhood exist, those published so far were of patients with a much milder disease course . In our Accompanying disease was described in 9 patients Table . An accoIn clearer cases of IPH, splenectomy turned out to be curative  activation [On this basis, VCAM-1 overexpression would not be a prime mechanism propagating portal hypertension but rather a secondary event that may be related to chronic vascular stress, inflammation, and phagocyte migration leading to nuclear factor tivation -33. VCAMtivation . The immtivation . While ativation ,36.We report the first case of uncontrolled splenic hyperperfusion and enlargement with subsequent hypersplenism leading to life-threatening complications of IPH in infancy and show that disease remission after splenectomy in early childhood points to the spleen as the main organ underlying pathogenesis and progression of IPH. The success of surgical interventions targeting hypersplenism suggests that the primary defect in the regulation of splenic blood flow seems to be crucial for the development of IPH. This view of the pathophysiology is supported by the fact that i) liver function abnormalities do not occur in IPH, ii) abnormal eNOS and VCAM-1 expression was only seen in splenic but not hepatic tissue, iii) the signs of portal hypertension resolved in our patient after splenectomy, and iv) both the adult patients reported before and our young patient have not developed recurrent signs of portal hypertension, liver pathologies or complications after splenectomy. Thus, beside other treatment options  total  splenectomy needs to be considered as a prime therapeutic option for IPH.IPH: Idiopathic portal hypertension; eNOS: endothelial nitric oxide synthetase; VCAM-1: vascular endothelial adhesion molecule-1; ET-1: endothelin-1 (ET-1).The authors declare that they have no competing interestsJD designed and performed the research, analyzed the data and wrote the paper; JW designed and performed the research and analyzed the data; UM performed magnetic resonance imaging of the abdomen, scintigraphic imaging with In-111-oxinate tagged, abdominal and Doppler ultrasound; BK performed splenectomy and liver biopsies; GK carried out histology and immunohistochemistry; CD cared for the patient and helped with analyzing date; MCF cared for the patient and helped with analyzing date; DF cared for the patient, designed and analyzed the research and helped with writing the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/10/122/prepub