Physical aggression scores were higher in men who had experienced early traumatic life events and who carried the low MAOA activity allele (MAOA-L). We repeated the analysis in the subgroup of healthy volunteers (N = 145) to exclude that the observed G×E interactions were due to the inclusion of psychiatric patients in our sample and were not generalizable to the population at large. The results for the subgroup of healthy volunteers were identical to those for the entire sample. The cumulative variance in the physical aggression score explained by the ANOVA effects involving the MAOA polymorphism was 6.6% in the entire sample and 12.1% in the sub-sample of healthy volunteers. Our results support the hypothesis that, when combined with exposure to early traumatic life events, low MAOA activity is a significant risk factor for aggressive behavior during adulthood and suggest that the use of dimensional measures focusing on behavioral aspects of aggression may increase the likelihood of detecting significant gene-by-environment interactions in studies of MAOA-related aggression.Previous research has reported that a functional polymorphism in the monoamine oxidase A (MAOA) gene promoter can moderate the association between early life adversity and increased risk for violence and antisocial behavior. In this study of a combined population of psychiatric outpatients and healthy volunteers (N = 235), we tested the hypothesis that MAOA genotype moderates the association between early traumatic life events (ETLE) experienced during the first 15 years of life and the display of physical aggression during adulthood, as assessed by the Aggression Questionnaire. An ANOVA model including gender, exposure to early trauma, and MAOA genotype as between-subjects factors showed significant MAOA×ETLE (F Individual differences in aggressive behavior are at least partly heritable and presumably result from the interaction between genetic and environmental factors The clearest genetic link to aggressive behavior exists for the monoamine oxidase A (MAOA) gene which plays a key role in the catabolism of monoamines, including dopamine (DA), norepinephrine (NE), and serotonin (5-HT) Recently, a large longitudinal study of children followed for 26 years since birth showed that MAOA VNTR genotype moderates the association between childhood maltreatment and violent and antisocial behavior 2,170 = 0.74, P = 0.47; AQ-PA, see 2 = 2.51, df = 4, P = 0.64) Because MAOA is an X-linked gene, male subjects were straightforwardly assigned to one of two genotype groups: 1) subjects carrying one low activity allele , and 2) subjects carrying one high activity allele (3.5-or 4-repeat form). Females were assigned to three genotype groups: 1) homozygous subjects carrying two low activity alleles, 2) homozygous subjects carrying two high activity alleles, and 3) heterozygous subjects carrying one low and one high activity allele. Expression studies have demonstrated that in female skin fibroblasts the MAOA gene undergoes X-inactivation and shows mono-allelic expression Participants were divided into two groups according to self-reported exposure to early traumatic life events: 1) those reporting none, and 2) those reporting one or more traumatic events. Thirty-four percent of the participants experienced at least one traumatic life event between 0 and 15 years of age, with some participants reporting the occurrence of multiple traumatic events .2 = 0.92, df = 1, P = 0.34) and exposure to ETLE . Compared to healthy controls, exposure to ETLE between 0 and 15 years of age was more frequent among psychiatric patients . Importantly, the ETLE groups did not differ significantly in MAOA genotype distribution, arguing against the possibility that genotype influenced exposure to traumatic events . Finally, the distribution of MAOA genotype did not differ between the healthy and psychiatric patient groups .Before assessing the effect of ETLE exposure and MAOA genotype on risk for increased physical aggression, we first tested whether subgroups in our sample differed significantly for the distribution of psychiatric diagnosis, ETLE exposure, and MAOA genotype. Such deviations might confound the interpretation of subsequent findings and reveal the existence of gene-by-environment correlation effects. Men and women did not differ in the distribution of psychiatric diagnosis .Scores on the AQ-PA scale in our sample ranged from 9 to 41 (median: 14.0). The percentage of psychiatric patients included in the subgroup of participants who scored in the top 25% of the score distribution (AQ-PA ≥ 19) was higher than that included in the subgroup of participants who scored in the bottom 25% (AQ-PA≤11)  and ETLE , but no main effect of MAOA genotype . As expected, physical aggression scores were higher among males and participants reporting exposure to early traumatic life events. The importance of MAOA genotype in modulating the impact of early trauma on adult physical aggression emerged when interaction effects were examined. We found significant MAOA×ETLE  and gender×MAOA×ETLE  interaction effects. The cumulative variance in the physical aggression score explained by the ANOVA effects involving the MAOA allele was 6.6%. Physical aggression scores were higher in men who had experienced early traumatic life events and who carried low MAOA activity alleles  and ETLE , no main effect of MAOA genotype , and significant interaction effects for MAOA×ETLE  and for gender×MAOA×ETLE . The cumulative variance in the physical aggression score explained by the ANOVA effects involving the MAOA allele was 12.1%  to exclude that the G×E interactions we found were due to the inclusion of psychiatric patients in our sample and were not generalizable to the population at large. The results for the subgroup of healthy volunteers were identical to those for the entire sample. We found significant main effects of gender . When we repeated the analysis in the sub-sample of healthy volunteers, the results did not change. Our results are consistent with the majority of previous reports Based on the findings of several independent studies, the relationship between the MAOA polymorphism and aggression appears to be fairly consistent. Yet, the intervening neural and psychological mechanisms are still unclear. One promising line of research has investigated the possibility that individuals with the low expression allele might be more sensitive to negative social experiences, which might ultimately result in defensively aggressive behavior. A recent fMRI study showed that the MAOA genotype at risk for impulsivity and violent behavior is associated with reduced gray matter volumes in limbic regions such as the amygdala, dorsal anterior cingulated cortex (dACC), and subgenual ACC and greater amygdala and subgenual ACC responsivity to negative emotional faces Although recent research has demonstrated that the allele×environment strategy is promising for detecting individual vulnerability to environmental risk, reported associations between gene variants and complex behaviors are in general weak. This is likely to reflect the fact that a number of several alleles contribute in various small ways to the interplay between environmental risk factors and development of complex behaviors. In the case of G×E interactions involving MAOA alleles and predicting antisocial behavior and/or conduct disorder, effect sizes reported to date are small on average. In this regard, the present study is no exception. The cumulative variance in the physical aggression score explained by the ANOVA effects involving the MAOA allele was 6.6% in the entire sample and 12.1% in the sub-sample of healthy volunteers. However, the significant G×E interaction effects found in the present study are remarkable considering its relatively small sample (N = 235) and require a methodological comment.The questionnaire we used to measure the occurrence of early traumatic life events explored a variety of adverse experiences other than physical and sexual abuse. There is a large body of evidence in the clinical literature demonstrating that childhood attachment-related trauma Another important difference between the present study and previous ones is the measure used to assess the behavioral phenotype. As dependent variable, we used the physical aggression scale of the Aggression Questionnaire This study has several limitations, the first of which is the small sample size. Studies relating genetic polymorphisms to behavioral or self-report assessments typically use much larger samples. Thus, the present results should be interpreted with caution until these findings have been replicated in larger samples. However, the within-study replication in psychiatric patients and healthy volunteers justifies a fairly high level of confidence. Second, we used a retrospective measure to assess the occurrence of traumatic events during childhood. When the type of events assessed is not limited to objective trauma  but also includes perceived situations , retrospective data may be subject to faulty recall or systematic distortions, even though the construct validity of our measure of early trauma was supported by its strong association with later-life aggression, in accordance with most other studies related to this topic. Although prospective studies are clearly desirable in delineating the role of early environment in the development of later-life aggressive behavior, such investigations face daunting methodological difficulties. Until such methodological problems can be overcome, reliance will have to be placed on a variety of sources of information, including retrospective data, to study G×E. Third, we used a self-report measure of aggression, and the social desirability bias of some subjects might have affected their self-reporting of aggression. Subjects motivated by need for social approval may not report as much aggression as those to whom social approval is less important. Such a bias, however, probably reduced our ability to detect interaction effects and thus implies that the findings are conservative estimates of G×E. Fourth, we included women in our sample to assess the impact of gender on the MAOA-early trauma interaction. Few studies investigating MAOA-related aggression have included female subjects In conclusion, despite its limitations, this study supports and extends the findings of previous reports showing that the MAOA polymorphism in combination with early experience modulates individual proclivity to later-life physical aggression. Its major methodological contribution is that the use of continuous self-report measures of aggression ranging from normative levels to pathological extremes and a wider focus in assessing early trauma may increase the likelihood of detecting interactions between the MAOA gene and childhood environment.The population sample of this study consisted of 90 outpatients  consecutively admitted to the day hospital of the psychiatric clinic at the University of Rome Tor Vergata and 145 healthy volunteers . All participants were of Caucasian origin. Diagnostic assessment was made by experienced clinical psychiatrists using the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-CV) All data were obtained under informed consent and using procedures and protocols approved by the University of Rome Tor Vergata Intramural Ethics Committee and the EMBL Bioethics Internal Advisory Committee (BIAC).2, 10 pmoles of each primer, 0.33 mM dNTPs, and 1.5 U of native Taq . PCR products were assayed on a 3% agarose gel. The primers used yielded 290, 320, 335, 350 and 380 bp fragments corresponding to the 2-, 3-, 3.5-, 4-and 5-repeat alleles, respectively For all participants DNA was obtained from buccal samples On the same day when DNA was collected, each subject completed the Aggression Questionnaire (AQ) The occurrence of traumatic life events during childhood was assessed using a questionnaire developed by Bandelow et al. Functional discrimination analysis was used to detect differences in levels of physical aggression among genotype groups of female participants. Chi-square tests were used to test differences between categorical variables. Main and interaction effects of gender, ETLE, and MAOA genotype on the physical aggression score were calculated by using three-way analysis of variance (ANOVA). All statistical analyses were carried out with the help of Statistica  and SPSS  software.


This study was performed to compare levels of serum homocysteine (Hcy), vitamin B12 and folic acid in patients with primary open-angle glaucoma (POAG), pseudoexfoliative glaucoma (PEXG), normotensive glaucoma (NTG) and healthy controls.Twentyfive patients with POAG, 24 with PEXG, and 18 with NTG, along with 19 control healthy subjects were included this prospective study. Levels of serum Hcy were measured using immunoassay, and those of serum vitamin B12 and folic acid were measured using competitive chemiluminescent enzyme immunoassay.The mean Hcy concentration in the PEXG group was significantly higher (P < 0.001) as compared to the other groups. There were no significant differences with respect to the mean Hcy concentrations among other groups (P > 0.05). There were no statistical differences in serum vitamin B12 levels among POAG, PEXG, NTG and control subjects (P > 0.05).The mean serum folic acid level was significantly lower in the subjects with PEXG (P < 0.009). However, the mean folic acid concentrations among the other groups did not differ significantly (P > 0.05).Elevated levels of Hcy in PEXG may explain the role of endothelial dysfunction among patients with PEXG. Intraocular pressure (IOP) is assumed to be the most significant risk factor in glaucoma, but recent evidences indicate that vascular risk factors may also play a role. Impaired microcirculation and abnormal perfusion may cause glaucomatous damage in the optic nerve head. Anatomical or functional abnormalities of the vessels of the optic nerve head such as arteriosclerosis or vascular dysregulation might be the causative factor ,2.The metabolism of methionine via homocysteine (Hcy) to cysteine is a complex pathway involving an enzyme depending on vitamin B12, B6, and folic acid. Genetically inherited defects are the most important determinants of elevated levels of Hcy. Some studies showed that elevated Hcy may increase the risk of retinal vascular diseases, such as retinal artery and vein occlusion and non-artheritic ischemic optic neuropathy -7. Hcy-iAccording to one study, serum Hcy concentrations in patients with vascular disease are on average 30% higher than in normal subjects . In addiRecently, higher levels of plasma Hcy in primary open-angel glaucoma (POAG) and pseudoexfoliative glaucoma (PEXG) patients were reported -18. On tThe aim of this study is to evaluate the role of serum Hcy, vitamin B12 and folic acid levels in patients with POAG, PEXG, and NTG, and compare them to sex and age-matched group of control healthy subjects.Total of 25 patients with POAG, 24 with PEXG, and 18 with NTG, along with 19 control healthy subjects undergoing ocular surgery at the Gaziosmanpaşa University, Department of Ophthalmology between October 2004 and May 2005 were enrolled in this case control study. The study was conducted according to the tenets of the Declaration of Helsinki, and patients gave informed consent after the nature and intent of the study had been fully explained to them.All patients underwent a complete ophthalmic examination, including visual acuity, slit-lamp examination, gonioscopy, tonometry, fundoscopy, visual field examination, and systemic examination.Inclusion criteria were as follows:1-POAG was defined by the presence of an open angle on gonioscopy, IOP ≥ 22 mmHg measured with a Goldman Applanation Tonometer, typical glaucomatous cupping and visual field defect in at least one eye on standard automated perimetry (full-threshold or Swedish Interactive Threshold Algorithm (SITA) strategy, program 24-2, Humphrey Field Analyzer).2-Diagnosis of PEXG was based on the presence of typical exfoliation material on the anterior lens capsule in one or both eyes with typical glaucomatous cupping and visual field defect in at least one eye. IOP was ≥22 mmHg with an open angle in gonioscopic examination.3-NTG patients were characterized by the presence of glaucomatous optic neuropathy and visual field defect in at least one eye and IOP was <22 mmHg.4-Control subjects had no history of elevated IOP higher than 22 mmHg, no exfoliative material on the anterior lens capsule, normal visual fields and optic discs.Exclusion criteria included non-genetic factors associated with hyperhomocysteinemia, a history of diabetes mellitus, systemic hypertension, peripheral or coronary artery disease, cerebrovascular disease, renal dysfunction, ocular inflammation, retinal occlusive disease. In addition, we excluded those taking vitamin and other medications known to affect Hcy measurements, such as phenytoin, methotrexate, vitamins B6, B12 and folic acid.A single 5-ml venous blood sample for serum Hcy, vitamin B12 and folic acid detection was collected in the fasting state. The samples were centrifuged within 1 h after collection and stored at -40°C. The levels of serum Hcy were measured using an immunoassay procedure . This assay gives normal values of the serum Hcy as 12–15 μmol/l according to the manufacturer. The levels of serum vitamin B12 and folic acid were measured using competitive chemiluminescent enzyme immunoassay . For these assays, the normal serum values of vitamin B12 and folic acid are, respectively, 193–982 pg/ml and 3–7 ng/ml, also according to manufacturer's instruction.Analysis of variance (one-way ANOVA) was used to detect differences in continuous variables  among the 4 study groups. For categorical variables (gender), chi-square tests was used to assess differences between groups. P value <0.05 was considered statistically significant. Statistical analyses were performed using software SPSS version 10.00.There were no significant differences in gender and age between control group and the other groups Table .Levene's variance homogeneity analysis showed that the variances of groups were homogenous in serum Hcy, vitamin B12, folic acid, age and gender (P > 0.05).The parametric one-way ANOVA test indicated that the mean Hcy concentration in the PEXG group was significantly higher P < 0.001) than in the other groups. Mean ± standard deviation (SD) of Hcy levels in PEXG was 14.88 ± 3.26 μmol/l. The mean Hcy concentrations among the other groups were not significantly different (P > 0.05). Mean ± SD of Hcy levels in POAG, NTG and control subjects were 9.22 ± 3.70, 10.39 ± 2.89, 8.40 ± 2.77 μmol/l, respectively . Mean ± SD of vitamin B12 levels in POAG, PEXG, NTG and control subjects were 209.37 ± 104.44, 232.84 ± 67.55, 262.33 ± 85.94, 261.84 ± 126.22 pg/ml, respectively . Mean ± SD of folic acid levels in PEXG was 4.26 ± 1.69 ng/ml. The mean folic acid concentrations among the other groups were not significantly different (P > 0.05). Mean ± SD of folic acid levels in POAG, NTG and control subjects were 6.24 ± 2.88, 5.97 ± 1.85, 5.93 ± 1.70 ng/ml, respectively , and the other tissues.In our study, among various glaucoma types, only in PEXG type serum levels of Hcy was found to be increased and serum levels of folic acid was found to be decreased.Hcy: homocysteinePOAG: primary open-angle glaucomaPEXG: pseudoexfoliative glaucomaIOP: intraocular pressureSITA: Swedish Interactive Treshold AlgorithmADMA: asimmetric dimethylarginineThe author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


Cortical reorganization in poststroke aphasia is not well understood. Few studies have investigated neural mechanisms underlying language recovery in severe aphasia patients, who are typically viewed as having a poor prognosis for language recovery. Although test-retest reliability is routinely demonstrated during collection of language data in single-subject aphasia research, this is rarely examined in fMRI studies investigating the underlying neural mechanisms in aphasia recovery.The purpose of this study was to acquire fMRI test-retest data examining semantic decisions both within and between two aphasia patients. Functional MRI was utilized to image individuals with chronic, moderate-severe nonfluent aphasia during nonverbal, yes/no button-box semantic judgments of iconic sentences presented in the Computer-assisted Visual Communication (C-ViC) program. We investigated the critical issue of intra-subject reliability by exploring similarities and differences in regions of activation during participants' performance of identical tasks twice on the same day. Each participant demonstrated high intra-subject reliability, with response decrements typical of task familiarity. Differences between participants included greater left hemisphere perilesional activation in the individual with better response to C-ViC training. This study provides fMRI reliability in chronic nonfluent aphasia, and adds to evidence supporting differences in individual cortical reorganization in aphasia recovery.


Drs. Speiser, Cerottini, and Romero  correctlThe authors also point out that in their experience, they found that the majority of T cells generated with the heteroclitic Melan-A M26 peptide were tumor reactive, citing their studies in vitro , and in In our study , we anal


Nosocomial infection (NI), particularly with positive blood or cerebrospinal fluid bacterial cultures, is a major cause of morbidity in neonatal intensive care units (NICUs). Rates of NI appear to vary substantially between NICUs. The aim of this study was to determine risk factors for NI, as well as the risk-adjusted variations in NI rates among Canadian NICUs.From January 1996 to October 1997, data on demographics, intervention, illness severity and NI rates were submitted from 17 Canadian NICUs. Infants admitted at <4 days of age were included. NI was defined as a positive blood or cerebrospinal fluid culture after > 48 hrs in hospital.765 (23.5%) of 3253 infants <1500 g and 328 (2.5%) of 13228 infants ≥1500 g developed at least one episode of NI. Over 95% of episodes were due to nosocomial bacteremia. Major morbidity was more common amongst those with NI versus those without. Mortality was more strongly associated with NI versus those without for infants ≥1500 g, but not for infants <1500 g. Multiple logistic regression analysis showed that for infants <1500 g, risk factors for NI included gestation <29 weeks, outborn status, increased acuity on day 1, mechanical ventilation and parenteral nutrition. When NICUs were compared for babies <1500 g, the odds ratios for NI ranged from 0.2  to 8.6 (95% CI 4.1 to 18.2) when compared to a reference site. This trend persisted after adjustment for risk factors, and was also found in larger babies.Rates of nosocomial infection in Canadian NICUs vary considerably, even after adjustment for known risk factors. The implication is that this variation is due to differences in clinical practices and therefore may be amenable to interventions that alter practice. Hospital acquired  infection in neonatal intensive care units (NICUs) is a significant cause of mortality and morbidity, particularly in very low birth weight VLBW, <1500 g) babies. VLBW infants when affected by these infections are at increased risk for death, have a longer hospital stay, and utilize more resources than non-affected controls 500 g bab. SimilarRisk-adjustment has also been used to look at variations in rates of both neonatal mortality and morbidity . For exaThe aims of this study were to examine (a) risk factors for culture-proven hospital acquired infections for VLBW and HBW infants admitted to the NICU, (b) the risks associated with specific clinical interventions in the NICU and (c) the risk-adjusted variations in hospital acquired infection rates among Canadian NICUs.The study cohort included all infants admitted to 17 Canadian NICUs prior to 4 days of age. Neonates without a recorded birthweight were excluded. The study cohort was derived from a larger study of 9506 babies admitted to 17 tertiary NICUs in the Canadian Neonatal Network from January 1996 through to October 1997 . These uFor the purpose of this study, nosocomial infection (or NI) was defined as one or more positive single organism blood or CSF culture sampled after 48 h of admission in an infant with clinical suspicion of infection. To differentiate between nosocomial and primary  infections, the infant blood culture isolates were required to be different from maternal isolates or to occur at least 7 days after a treated positive blood culture obtained during the first 48 h of life. CSF samples were obtained when indicated according to local practices and policies. The time period of 2 days was chosen to exclude any neonates born with primary infection. Absence of NI included those infants who were never cultured and those with negative blood and/or CSF cultures. An infection episode was defined as a positive culture occurring at least 7 days after a previous treated culture or if the culture isolates were different from the previous culture . We defird percentile for weight . Major ld et al  using 6 ld et al . The subUnivariate and bivariate analyses using SPSS  were used to describe the study population and to explore the relationships between baseline population risks  and NI. Chi-square test was used to compare mortality and morbidity among infants with and without NI, for infants with birth weight less than 1500 g and 1500 g or more.We used a multivariable logistic regression model to develop a risk adjustment model for NI. The outcome variable was NI and independent variables were baseline population risks significantly correlated with NI on bivariate analysis. Correlation matrix was used to examine correlation between variables. The full model was created by adding population risk factors, then SNAP-II score, and finally therapy-related factors – variables were considered to be included if their p-value was <0.10. Backward and forward selections from the full model were applied to help determine the parsimonious model. Finally, centres were compared using adjusted odds ratios and 95% confidence intervals derived from the multivariable logistic regression analysis, using a reference centre . Sensitivity analyses were conducted by excluding outlier sites with large confidence intervals.This study was part of a larger study surveying morbidities and mortality in Canada, for which ethics approval for anonymous collation of data was obtained at each individual centre.16538 infants in the dataset met entry criteria (age less than 4 days on admission). 765 (23.5%) of 3253 VLBW infants, and 329 (2.5%) of 13244 HBW infants (41 were missing birth weight and were excluded), developed at least one episode of NI. Among VLBW infants who developed NI, 78.7% had only 1 episode, 16.2% had 2 episodes, and 5.1% had 3 or more episodes of NI, whereas among HBW infants, 87.9% had 1 episode, 9.3% had 2 episodes, and 2.8% had 3 or more episodes of NI. The median onset of the first infection from day of admission was at 19 days for VLBW infants and 15 days for HBW infants.Among infants with the first episode of NI, the incidence of CSF culture positive infections was 3.7% in VLBW infants, and 2.8% in HBW infants. The most common organism obtained from blood culture was coagulase negative Staphylococcus . The same was true of CSF isolates (68% and 41% respectively). Gram-negative organisms were the next most common pathogen in VLBW, whereas Gram positives, particularly group B Streptococcus, was the next most prevalent in HBW.Ninety percent of VLBW infants and 73.3% of HBW infants received at least one course of antibiotics in the NICU on either day 1 and / or day 3, as recorded by NTISSAmong VLBW infants, bivariate analysis showed that infants with NI were more likely to be outborn, delivered by vaginal birth, and have 5 minute Apgar score <7, but less likely to be SGA  indicates significant correlation beween NI and CVL and a contingency coefficient of 0.1 for either TPN present or absent. Therefore, there is no evidence that the presence of TPN or CVL in the models renders the other redundant.Mortality was more strongly associated with the NI group compared to the group without NI for HBW infants, but not for VLBW infants. Major morbidities  of retinopathy of prematurity) were more common in the NI group Table .In VLBW infants, factors associated with NI in the multivariable logistic model were: gestational age, use of assisted ventilation and IV fat and, and outborn status  with NI in the multivariable logistic model were: gestational age, use of central venous access, peripheral intravenous access, and use of IV fat or IV amino acid, assisted ventilation, outborn status, use of antibiotics and presence of congenital anomalies. Again, IV fat and IV amino acid could be used interchangeably in the model. Infants who received parenteral nutrition were more likely to have NI. Central venous access was associated with higher risk for NI, as was use of peripheral IV access.For VLBW infants, cross-tabulation of site and NI showed significant variation between sites . Crude rates of NI ranged from 6.7% to 74.5% of infants having at least one episode of NI. The crude incidences of NI for 8 hospitals were significantly (p < 0.05) different from the reference hospital (site O) . Crude rates of NI ranged from 0.1% to 17.0 %. When only site variables were in the regression model, 6 hospitals had significantly different (p < 0.05) NI rates from the reference hospital (F). Significant site differences largely corrected after adjustment for baseline population risks   Figure .Comparison of Figures The above results represent data from 17 tertiary NICUs, making up approximately three-quarters of level III neonatal cots in Canada. Canada has publicly-funded health care with a highly regionalized neonatal-perinatal care system in which each tertiary NICU is the referral centre for its geographic region. Consequently, our results approximate a population-based study for three quarters of the Canadian population.The bivariate analysis suggests that infants who experience at least one episode of nosocomial bacteremia or nosocomial bacterial meningitis are smaller, more preterm, and had higher illness severity on admission. They have lower 5-minute Apgar scores and are more likely to have been transported from another centre. VLBW infants with NI were more likely to have been delivered vaginally. Nosocomial infection rates will vary from institution to institution if these demographic factors vary from region to region. Adjusted data, to some extent, correct for these geographic variations.Our data show that significant variations in nosocomial infection do occur between units, with crude rates ranging 6.7% to 74.5% among VLBW infants, and 0.1% to 17.0% among HBW infants. These variations persist after adjustment for gestation, outborn status and admission illness severity. These findings are not unique to hospital acquired infections: using data from the same population of infants, other authors have identified between-site variations in the crude and adjusted rates of both mortality and a number of major neonatal morbidities, including chronic lung disease, retinopathy of prematurity, catheter-related sepsis and intraventricular lesions ,10,18. TMultivariate analysis suggests that, in addition to gestation and acuity, a number of therapeutic interventions are associated with nosocomial infection. These include mechanical ventilation, vascular access , and parenteral nutrition. These factors are predictive in cohorts of both VLBW and HBW babies – supporting their place as clinically significant risks factors for NI. The preponderance of NI among HBW babies with congenital anomalies may reflect surgical interventions, or the possibility of immunocompromise. We recognize that there are likely to be other, unrecorded demographic factors or interventions, that may account for risk, such as feeding practices or ethnicity – these remain unaccounted for in our study.There is little doubt from this study that nosocomial infection is associated with significant in-hospital morbidity, but the results on mortality are more subtle. Unadjusted mortality in VLBW infants was not increased by an episode of nosocomial sepsis. A possible explanation is that the effect size is small because most deaths among VLBW infants occur in the first few days of life from other causes, for example cardiorespiratory failure, whereas NI occurs more commonly at a later age. In addition, coagulase negative staphylococcal infections (the commonest isolate in NICU) are reported to have a relatively low mortality in VLBW infants (2% or less in three large cohort studies -21), makBetween-site analysis confirmed that inter-NICU variation in NI rates persisted after risk adjustment for birth weight, gestation and illness severity in VLBW infants. The 3 sites with lower than median incidence of NI for VLBW infants, also had a lower incidence of NI for HBW infants. Three out of 4 sites with higher than median incidence of NI for HBW infants also had higher incidence of NI for VLBW infants. The impression that overall site performance is consistent for infants at different birth weight groups supports the contention that factors other that those recorded and adjusted for in this study, probably related to interventions or the NICU environment, contribute to the risk of nosocomial infection – especially in VLBW infants, with whom most of the burden of NI lies.We have seen that nosocomial infection rates vary across Canada and are significantly affected by clinical interventions. However, a number of important factors have not been adjusted for, particularly those related to organism virulence or infectivity, and population susceptibility. One could hypothesize that organism virulence or infectivity varies across the country, as does the susceptibility of population groups. There is some evidence to support variation in genetic susceptibility: Ahrens et al  reportedA limitation of our study is that it is possible that some positive blood or CSF cultures are in fact false positives. Struthers et al  estimateRates of nosocomial infection in Canadian NICUs vary considerably, even after correction for known risk factors. The data suggest that this variation is due, in part, to differences in clinical practices, and therefore may be amenable to interventions that alter practice. More study, and alternative study designs, may be required to evaluate contributing factors and effect practice change.Shoo K. Lee ; Wayne Andrews ; Ranjit Baboolal ; Jill Boulton ; David Brabyn ; David S.C. Lee ; Derek Matthew ; Douglas D. McMillan ; Christine Newman ; Arne Ohlsson ; Abraham Peliowski ; Margaret Pendray ; Koravangattu Sankaran ; Barbara Schmidt ; Mary Seshia ; Anne Synnes ; Paul Thiessen ; Robin Walker ; Robin Whyte .Holly Bavinton; Stella Karuri; Zhenguo Qiu; Sarka Lisonkova, Shawn Stewart.Nosocomial infection (NI)Neonatal Intensive Care Unit (NICU)Score for Neonatal Acute Physiology, Version II (SNAP-II)Neonatal Therapeutic Intensity Scoring System (NTISS)Very low birth weight (VLBW)Higher birth weight (HBW)Cerebrospinal fluid (CSF)Standard deviation (SD)Confidence interval (CI)The author(s) declare that they have no competing interests.KA interpreted data and drafted the manuscript. DDM, WA and MP were site investigators. ZQ performed statistical analysis and interpretation. SKL was the principal investigator and drafted the manuscript. All these individuals read and approved the final manuscript. The CNN represents all site investigators, and was responsible for organization and administration of the SNAP study, and subsequent data flow.The pre-publication history for this paper can be accessed here:


We review three approaches to the genetic analysis of the biology and pathobiology of human aging. The first and so far the best-developed is the search for the biochemical genetic basis of varying susceptibilities to major geriatric disorders. These include a range of progeroid syndromes. Collectively, they tell us much about the genetics of health span. Given that the major risk factor for virtually all geriatric disorders is biological aging, they may also serve as markers for the study of intrinsic biological aging. The second approach seeks to identify allelic contributions to exceptionally long life spans. While linkage to a locus on Chromosome 4 has not been confirmed, association studies have revealed a number of significant polymorphisms that impact upon late-life diseases and life span. The third approach remains theoretical. It would require longitudinal studies of large numbers of middle-aged sib-pairs who are extremely discordant or concordant for their rates of decline in various physiological functions. We can conclude that there are great opportunities for research on the genetics of human aging, particularly given the huge fund of information on human biology and pathobiology, and the rapidly developing knowledge of the human genome. PLoS Genetics and elsewhere have documented remarkable progress in genetic aspects of aging in model organisms. These studies have revealed what can be regarded as the first “public” mechanism of aging, that is to say, a biochemical genetic pathway, modulation of which can alter the life spans of diverse species , the product of which is a glycoprotein that probably functions as a secreted growth factor [Other articles in  species –3. We ap species . These m species . Human b species . It is tgenetics . Moreovegenetics . Third,  humans) ,7 . We shaHuman geneticists have only recently begun to use the tools of linkage analysis and association studies to identify alleles contributing to exceptionally long life spans. Interesting candidates have emerged from these studies. There is virtually no information, however, on the genetic basis of differential rates of decline in well-defined physiological functions among human populations. We shall argue that such studies, particularly those that are initiated in middle age, before the onset of complicating comorbidities, should have a high priority for research, as they would have the potential to discover genes that impact on rates of aging within various organ systems. Such investigations, moreover, would serve as tests of the null hypothesis that there is significant asynchrony in rates of physiological aging among major body systems in human subjects. Selective pressures can be expected to maintain a degree of synchrony in the rates at which various organ functions decline ,12, but The classical evolutionary biological theory of aging tells us that senescence occurs in age-structured populations because of the decline in the force of natural selection with age –13. ThatIt is not generally appreciated that the evolutionary theory of why we age provides clues to how we age –21. Clashttp://www.ncbi.nlm.nih.gov/sites/entrez?db=OMIM). These have the potential to reveal pathogenetic mechanisms. They can be divided into two major categories. One subset consists of mutations that impact upon a number of such phenotypes; they have been referred to as “segmental progeroid syndromes” [(WRN) of the RecQ family of helicases [WRN locus [ndromes” . A seconndromes” . We shoundromes” . Strikinndromes” ,28; thesndromes” . Patientelicases , exhibitelicases . While delicases , markerselicases . There aelicases  and the elicases . These aelicases ,34. Somaelicases . The disRN locus ,37 is coRN locus . WS has RN locus . PatientRN locus , also apRN locus . This maRN locus .APOE locus , numeroCentenarians have outlived any exceptionally long-lived invertebrate model by ∼100 years and any comparable rodent model by ∼30-fold. Human subjects in general and centenarians in particular outlive their nearest relatives, the common chimpanzee, by many decades . This haHomo sapiens. The life expectancies of centenarians at birth are nearly double that of most members of their birth cohort and, on average, have surpassed current life expectancy by 22 years. Environmental and stochastic contributions to human life span likely play important roles in the determination of such exceptionally long survivals, as inferred from the twin studies discussed below. Familial aggregates of exceptional longevity do not rule out major environmental factors that are the result of cultural inheritance ; such factors could explain, in part, why the progeny of long-lived members of the Framingham study exhibit advantageous cardiovascular risk profiles in middle age [Our task here, however, is to review progress towards the elucidation of genetic factors that contribute to exceptional longevities of individual members of ddle age . Neverthddle age . Siblingddle age . Such daPON1 [IGF-1 [PAPR-1 [During the last decade, centenarian populations ; (2) the apolipoprotein C-3 (APOC-3) gene codon A (−641) C variant (APOC-3 CC); and (3) a deletion at +2019 in the adiponectin (ADIPOQ) gene. The enrichment of the CETP genotype is supported by evidence from two independent populations [Studies performed at the Albert Einstein College of Medicine were based upon populations of Ashkenazi Jews and the following considerations . First, ulations ,86.While a significant overrepresentation of a single genotype among nonagenarians and centenarians operationally defines a candidate gene, several other criteria should be fulfilled before considering it to be an important longevity assurance gene ., appropriate alterations in plasma levels have been demonstrated [The first step by which the functionality of the genotype can be studied is by determining the serum and plasma levels of the coded protein, if it is secreted and circulating. For example, for each of the genotypes in nstrated ,85–89. Dnstrated . They inCETP and APOC3 differentially modulate lipoprotein characteristics. These effects may vary with age and should therefore be examined in cohorts of varying ages. The functional value of a genotype can also be assessed directly by functional studies of the mutant in a cellular system in vitro. Such studies may underestimate or overestimate the real physiological importance of the relevant gene action in vivo, however. If the gene encodes a protease, then studies of its specificity, tissue distribution, and regulation are called for. If the gene encodes a cell-surface receptor, then studies of the biochemistry of the receptor should be done. For example, the Einstein group recently identified novel mutations in the IGF-1 receptor of three centenarians. AKT phosphorylation was assessed in lymphoblastoid cell lines from these subjects and controls, both basal and induced levels (stimulating with IGF-1). A marked decrease in phosphorylation was observed in cells from the centenarians with the mutant IGF-1 receptors [A second step in establishing functionality is the identification of an intermediate phenotype. For example, alleles at eceptors ,90.CETP VV, the protection provided by certain genotypes can extend beyond a known role in a disease entity associated with its ascertainment . The CETP VV genotype is also associated with enhanced insulin sensitivity and lower risk for hypertension, the metabolic syndrome, and diabetes [CETP VV genotype protects against age-related cognitive decline and Alzheimer's disease [APOE locus [As noted in the Introduction, the enhancement of life spans in model organisms via single gene mutations raises the question of whether allelic variations at this pathway in human subjects might impact intrinsic biological aging within all tissues and thus lead to substantial increases in life span. Among the many billions of human beings who have lived since the time of recorded history, it is unlikely that a spontaneous mutant of this type, say leading to a doubling of the usual human life span, would have been missed. One could not rule out gene actions of this type, however, that contribute to the generation of rare, relatively healthy centenarians. This possibility is supported by at least three lines of evidence. First, as in the case of diabetes . Moreove disease , althougOE locus .A second example is a 2.5-fold increase, among centenarians, in the prevalence of an apoC III promoter variant. This variant is associated with significant declines in plasma levels of apoC III and a phenotype of large lipoprotein particles. There is also significantly less hypertension among subjects homozygous for this variant. The most striking data, however, were obtained from a retrospective study of a cohort of subjects bearing this variant. They live significantly longer; in fact, subjects <95 years old with this genotype lived on average over four years longer than those who were not homozygous for the variant. This is indeed a very large impact upon life span when one considers the conclusions of demographers, who have noted that the elimination of ischemic heart disease, a disorder that was responsible for 25.73% of all deaths in 1985, would increase life expectancy at birth by only 3.0 years for females and 3.55 years for males .FOXO-1, a transcription factor homologous to the DAF16 gene of Caenorhabditis elegans. DAF16 is a key regulator of a downstream suite of genes that are thought to protect the organism from macromolecular damage and thus enhance life span [CETP locus upon age-related diseases and longevity may be a special feature of the biology of humans, there is also evidence that the fruits of research on the genetic modulation of the life spans of worms, flies, and mice may in fact be applicable to our species.A third argument suggesting that some longevity genes are not merely disease specific is the marked conservation of some of these loci. For example, apoC III is under the control of ife span . Some ceife span . Thus, wSome favorable “longevity genotypes” may act to buffer the deleterious effects of genes that lead to age-related diseases. As a result, the frequencies of deleterious genotypes may, paradoxically, be increased among individuals with extreme life spans. This may explain why the cholesteryl ester transfer protein (CETP-VV) genotype appears to exhibit an additional advantageous effect—the neutralization of the deleterious effects of the lipoprotein(a) (Lp(a)) gene . Such buC. elegans grown in liquid cultures with axenic medium [C. elegans [A study of uncensored pairs of Danish human twins has indicated that only about one quarter of the heritability of life span can be attributed to the constitutional genotype . There ac medium . One can elegans . There i elegans ,105. The elegans ; moreove elegans . A secon elegans  and mito elegans  genomes. elegans . In cont elegans , such re elegans . Whateve elegans , but we http://www.perlegen.com/index.htm?whatweoffer/why_whole_genome.html) and statistical methodologies [Human geneticists, most of whom are practicing medical geneticists, suffer from a biased ascertainment of their subjects. With the notable exception of studies of nonogenarians and centenarians discussed above, their subjects present themselves because of dysfunctions and never because of remarkably robust function. Centenarians, however, often have a variety of comorbidities. Moreover, given their extreme old age, it is not feasible to carry out longitudinal studies of the rates of change of specific physiological functions. An argument has been developed for a different approach to the discovery of allelic variants that are associated with unusual degrees of maintenance of structure and function during aging . The sugdologies  should g


Anopheles darlingi was studied. Habitat profile was characterized by collecting environmental data. Remote sensing and GIS techniques were applied to extract additional spatial layers of land use, distance maps, and relief characteristics for spatial model building.Hydropower plants provide more than 78 % of Brazil's electricity generation, but the country's reservoirs are potential new habitats for main vectors of malaria. In a case study in the surroundings of the Manso hydropower plant in Mato Grosso state, Central Brazil, habitat suitability of An. darlingi. Probabilities of presence strongly vary as a function of land cover and distance from the lake shoreline. Vector presence was associated with spatial proximity to reservoir and semi-deciduous forests followed by Cerrado woodland. Vector absence was associated with open vegetation formations such as grasslands and agricultural areas. We suppose that non-significant differences of vector incidences between rainy and dry seasons are associated with the availability of anthropogenic breeding habitat of the reservoir throughout the year.Logistic regression analysis and ROC curves indicate significant relationships between the environment and presence of A. darlingi habitats in the studied hydropower reservoir area. Vector presence is significantly increased in forested areas near reservoirs in bays protected from wind and wave action. Construction of new reservoirs under the tropical, sub-humid climatic conditions should therefore be accompanied by entomologic studies to predict the risk of malaria epidemics.Satellite image classification and multitemporal shoreline simulations through DEM-based GIS-analyses consist in a valuable tool for spatial modeling of  Anopheles darlingi Root and four other dipters species of the Anophelines genus: Anopheles aquasalis, Anopheles albitarsis, Anopheles cruzi and Anopheles bellator. An. darlingi is considered the most anthropophilic and endophilic species among the Amazonian anophelines  = a + BX + e   (1)where:p: probability that the event Y occursln [p/(1-p)]: log odds ratio, or "logit"a: coefficient on the constant term,B: coefficient(s) on the independent variable(s),X: independent variable(s), ande: error term.The discrimination capacity of logistic regression models can be measured by cross-classifying observations and predictions in a two-by-two table, and calculating indices of classification performance  such as An. darlingi is widely described as an endophilic or endophagic [An. darlingi specimens had been captured in the interior of houses. As shown in Fig. dophagic . In campAn. darlingi flight ranges of rural settlement housing or tourist infrastructure .Following the argumentation of Charlwood , we suppVarious studies have identified the relation between climatic conditions such as precipitation and temperature and human-biting rates of malaria vectors ,36. FiguExploratory analysis indicates slightly higher HBR during the wet season Fig. , which cLand cover classification was initially stratified in forest, savannah, cattle farming, crop farming/open soil and water. Only one small crop farming area was observed during field work. As this test site showed spectral signatures similar to recently reformed pastures, and as this land use is supposed to occupy less than 1 % of the reservoir influence area, pasture and crop farming areas were joined in one class in the final thematic layer. Proximity of sample points to waterbodies is already represented by the reservoir distance layer Fig. . To miniTable Overall accuracy for the three land cover classes used as predictors in LR model building was determined to be 84.1. Producer's and User's accuracies are similarly high for Pasture and Savannah formations, whereas forest classification is less accurate. Denser savannah woodlands ("Cerradão"), build ecotones with the semi-deciduous forests, causing higher rates of misclassifications between both classes. Thematic map Fig.  shows SaFigures From the 73 extra-domicile sampling campaigns, 31 were carried out at a distance between 0 a 200 m of reservoir shoreline, 12 at a distance between 200 and 500 m, 25 between 500 and 1,000 m and 5 at a distance greater than 1 km Fig. .Figure An. darlingi, followed by savannah.Table An. darlingi is the most important malaria vector in Brazil, knowledge about its habitats is limited, particularly on a local scale and in the influence area of hydropower plant reservoirs.Although Anopheline incidence did not increase during the summer rainy season. Contrary to our findings, An. darlangi incidences in the surroundings of the Serra da Mesa power plant in the Cerrado savannah region of Goias state was found to be higher during the rainy season [Various studies have reported the importance of climatic factors on vector incidence and malaria prevalence in semi-humid tropical climates; increased rain and temperatures, for example, can have positive effects on vector breeding and development rates . Our resy season .Multispectral Landsat ETM imagery classification and DEM-based GIS methods were applied to local scale prediction of vector presence probabilities. As climatic factors showed no significant influence on vector presence, only a unique logistic regression model was developed.An. darlingi for locations covered with denser savannah formations . None of their sampling sites, however, was covered by semi-deciduous forest, which showed the highest incidences in our study. It is important to mention that vegetation units in the hilly terrain of the Manso power plant region are highly fragmented, reflecting variability in geoecological conditions as well as anthropic transformations. Forest formations in the study area are spatially limited to corridors of straight river valleys along the outlets of inflowing streams. Semi-deciduous forests as well as Cerrado woodlands and savannahs are reminiscent of patches interlaced by managed pastures. None of the sampling points was located in a continuous singular vegetation unit whose area would exceed the flight ranges of An. darlingi. Therefore, we see no contradiction to studies that have reported an increase of An. darlingi incidence in Amazon regions suffering recent deforestation [First, higher incidences are associated with spatial proximity to reservoir and semi-deciduous forests followed by Cerrado woodland. Vector absence was associated with open vegetation formations such as natural grasslands, pastures and agricultural areas. As in our study, Guimaraes et al.  noted hiestation ,26. As rAn. darlingi is estimated to vary between 2 km and 7 km [An. darlingi may follow edges of the forest margin following road systems. As the implantation of resettlements and tourist infrastructure has opened potential flight paths, we believe that mosquitoes may reach areas in a distance of at least 2.5 km from the supposed breeding habitats in the hydropower reservoir. This thesis seems to be confirmed by some HBR above 10 in a distance more than 1.6 km from the shoreline. In reservoirs such as that of the Manso power plant, which experience extensive water level variations as a function of operation schemas for power generation and seasonal hydrological stream regimes, distance estimates based on DEM analysis should be done for date specific water levels. Maximum water level amplitude of about 10 m can result in horizontal alterations of shoreline distances of more than 200 m. Vector presence and reservoir proximity had significant positive correlation in an univariate as well as in the multivariate LR model (p < 0.05). Since preferred breeding habitats of An. darlingi are large pools of stagnant water and slow-moving streams [Maximum flight range of and 7 km , An. dar streams , we deveAn. darlingi in the surroundings of the Manso hydropower plant in the Central Brazilian Cerrado region is highest near the reservoir and inside semi-deciduous forest. Savannah scrub- and woodlands are more suitable habitats than pastures or cropland.According to the presented results, we conclude in the framework of formulated hypothesis: the incidence of Only slight seasonal differences among HBR were observed, indicating that the increase of mosquito presence in the wet season as reported for other sub-humid tropical regions is balanced by the presence of an artificial reproduction habitat.An. darlingi habitats.Remote Sensing and GIS techniques such as digital land cover classification and DEM based buffering were found to contribute to habitat characterization and mapping. GIS-based morphographic classification, still little explored for habitat suitability simulation, was found to be a promising technique that should be included in future studies of PZ and ESS conceived the study design, performed statistical and spatial analysis and drafted the manuscript. AR and RM carried out the entomologic survey and participated in data analysis. MS participated in study design and was responsible for project coordination. All authors participated in the elaboration of final manuscript.


EPC number and functionality are assumed to reflect the endogenous vascular repair capacity with the EPC pool declining in higher ages and being exhausted by unfavorable life-style and risk factors. This intriguing and clinically highly relevant concept, however, has so far been derived from small case-control studies and patient series.In the population-based Bruneck Study EPC number and EPC-colony forming units (EPC-CFU) were assessed as part of the fourth follow-up evaluation (2005) in 571 and 542 subjects, respectively. EPC number declined with age (p = 0.013), was significantly lower in women (p = 0.006) and higher in subjects on statin, hormone replacement or ACE inhibitor/angiotensin-receptor blockers, and correlated positively with moderate alcohol consumption. Unexpectedly, a positive relation between EPC number and several vascular risk factors emerged. In a step forward multivariate linear regression analysis EPC number was independently related with SDF1α, MMP-9, triglycerides, alcohol consumption, and Hba1c. EPC-CFU in turn was related to SDF1α and diastolic blood pressure. Moreover, EPC number showed a significant positive association with the Framingham risk score (P = 0.001). Finally, there was an inverse association between EPC number and common carotid artery intima-media thickness (p = 0.02) and the carotid artery atherosclerosis score (p = 0.059).Our population-based data confirm the decline of EPC number with advancing age and lend first epidemiological support to a role of SDF-1α and MMP9 in EPC differentiation, mobilization and homing, but are conflict with the view that EPC number is unfavorably affected by cardiovascular risk factors. EPC number increases with the cardiovascular risk estimated by the Framingham risk score (FRS), which in the absence of similar changes for EPC-CFU. Finally, we demonstrate a significant inverse association between EPC number and extent of carotid atherosclerosis even though this association was only of moderate strength and not entirely consistent in other vascular territories. Asahara and colleagues first isolated circulating angioblasts from human peripheral blood, which had the potential to differentiate in vitro into endothelial cells and to contribute to neoangiogenesis after tissue ischemia in vivo, and defined this cell population as endothelial progenitor cells (EPC)Functionally, it is believed that EPC play an important role in regeneration of ischemic and damaged tissues via angiogenesis and repairing denuded endothelium in the injured vesselsPopulation recruitment was performed as part of the Bruneck StudyPBMNC isolation from Venous blood and EPC identification were performed as previously reportedThe EPC-CFU assay performed as described previouslyTo assess reproducibility, the cEPC and EPC-CFU numbers were determined twice in two separate blood samples obtained at least one week apart from 10 subjects in our preliminary experiment. The samples were analyzed independently by two observers who were blinded to subjects' clinical profiles. The interobserver correlations were 0.965 and 0.975 for cEPC and EPC-CFU assay, whereas the intraclass correlations obtained from a single observer who analyzed two blood samples collected at least one week apart from 10 subjects, were 0.939 and 0.961, respectively.Plasma granulocyte-colony stimulating factor (G-CSF), stromal cell-derived factor-1α (SDF1-α), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) levels were determined using commercially available kits . All ELISA tests were carried out at room temperature on freshly thawed plasma samples. The concentration of all cytokines was determined by comparison with a standard curve, following manufacturer's instruction. Other laboratory parameters were all examined by standard methodsThe ultrasound protocol involves the scanning of the internal  and common carotid arteries  of either side with a 10-MHz imaging probee-transformed to satisfy the assumption of normality and constant variance of the residuals. The association between EPC number and EPC-CFU and IMT and the carotid atherosclerosis score was tested by means of linear regression analysis. The multivariate models included the variables age (years), sex , smoking (number of cigarettes smoked daily), alcohol consumption (g/day), diabetes , body mass index (kg/m2), HDL and LDL cholesterol (mg/dL), loge-transformed triglycerides (mg/dL), loge-transformed hs-CRP level (mg/L), loge-transformed Lp(a) level (mg/dL), loge-transformed urinary ACR (ratio), ferritin concentration (µg/L), systolic blood pressure (mmHg) and facultatively various types of medication. A two-sided p value <0.05 was considered significant.The data were analyzed using the SPSS 12.0 and BMDP software packages. Levels of variables according to tertile groups for EPC number and EPC-CFU were presented as mean values±SD or as medians with corresponding 25th and 75th percentiles (continuous variables), and as absolute numbers and percentages (dichotomous variables). Associations of EPC number and EPC-CFU tertile groups with vascular risk factors, life-style and demographic variables, IMT and severity of atherosclerosis were assessed using generalized linear models and logistic regression analysis. We performed tests for linear trend by treating the medians in each category of EPC number and EPC-CFU as a continuous variable. Levels of C-reactive protein, lipoprotein (a), D-dimer, triglycerides and urinary ACR were logfigure S1). The EPC number in circulation was investigated in all the 571 subjects, but only 542 subjects were subject to EPC colony analysis due to the limitation of blood sample and PBMNC number in 29 subjects. In this study, we have determined the EPC number and EPC-CFU number by adjusting to per 106 PBMC and per 1 ml of blood, to avoid any discrepancy caused by total circulating PBMNC variation happened in some situation. The later data (per 1 ml of blood) are summarized in the online supplementary data, which are very similar for most aspects to former data (per 106 PBMC) (Data not shown). 6 PBMC), which indicate that the EPC and EPC-CFU numbers from a majority of subjects (higher than 70%) fall into (0-1000 EPC) and (0-250 EPC-CFU) score groups, respectively. Figure S2B illustrates a moderate but significant decline of EPC number (P = 0.013) but not EPC-CFU number (per 106 PBMC) with age.Detailed Characterization of EPC in colony formation and quantification assay was carried out in present study (data not shown). and e-transformed EPC number was independently related with loge-transformed SDF-1α , loge-transformed MMP-9 (P = 0.005), loge-transformed triglycerides (P = 0.009), alcohol consumption (P = 0.002) and Hba1c (P = 0.052). Loge-transformed EPC-CFU number in turn was related to loge-transformed SDF-1α and diastolic blood pressure .Our study lends further support to the previous observations that EPC number declines with advancing age and is influenced by standard cardiovascular drugs, refutes the concept that the circulating EPC pool is exhausted and repair capacity impaired by vascular risk factors and an unfavorable life-style and reports important novel findings like the significant association between EPC number and cytokine levels as well as carotid artery IMT.Most important consideration in EPC experiment is the methods to choice. Various surface markers are expressed on EPCs and are used for EPC characterizationObviously, our findings are different compared to the other published clinical studies in cardiovascular disease. As mentioned above, such discrepancy may be due to different methods used to characterise and quantify putative EPCs, preventing straightforward comparisons between them and probably explaining the difference in the results. The methods to measure the circulating numbers of EPCs by flow cytometry using either antibodies against CD34 and KDR or CD133 were used in many previous studies. However, double positive cells for DiI-Ac-LDL and Lectin in EPC culture were counted as EPCs and used in the present study to analyze the EPC numbers in circulation. The advantage for our method is that the number of cultured EPCs not only reflect the levels of circulating EPCs in humans, but also the proliferative potential of EPC which is responsible for EPC function in angiogenesis and vasculogenesis.Another interesting observation in the current study is the number of cultured EPCs, but not EPC-CFUs, was associated with some cardiovascular risk factors, FRS and common carotid artery atherosclerosis. Since the number of cultured EPCs include total EPCs with lower or higher proliferative potential, whereas EPC-CFU assay assesses a proportion of EPCs with higher proliferative ability and clonogenic capacity. One possible explanation for such difference between the number of cultured EPCs and EPC-CFU assay is that they represent different functional subpopulation of circulating EPCs. As controversy still exists over the exact definition of circulating EPCs, comparison between studies using different technique to assess EPC must be considered.Past studies were highly consistent in reporting inverse associations between EPC number and ageSeveral case-control studies and evaluations in patient series indicated that the EPC number is inversely associated with cardiovascular risk factorsVascular repair is crucial to atherogenesis especially in its advanced stages in which plaque fissuring and endothelial denudation are common phenomena. Circulating EPCs have been suggested to crucially contribute to vascular repair. Rauscher and colleagues have explored the potential effects of EPC on vessel pathology in ApoE deficient miceThere is solid evidence from experimental studies that progenitor-related cytokines, such as VEGFCounter expectation, in our study EPC number did not significantly differ between subjects with and without a history of previous cardiovascular disease. On interpreting these finding, however, it has to considered that most events had occurred already years before conduction of these study and that patients had been subject to extensive life-style modifications and drug therapy. Furthermore, many patients had again entered a clinically inactive stage of vessel disease. In contrast, the recent intriguing studies revealing lower levels of EPC in subjects with cardiovascular disease had a focus on patients with symptomatic coronary artery disease and acute coronary syndromes. In these patients differences in EPC number and functionality may be more pronounced because of an amplified consumption or inadequate mobilization of circulating EPC.In summary, our data implicated that the changes of the number of EPCs are loosely associated with certain risk factors for the cardiovascular disease and may not directly associated with the disease development. In additional, one of our major finding in the current study is unfavourable to the traditional view that the EPC number is negatively affected by cardiovascular risk factors, indicating that the role of EPC is more complex than assumed previously. Our knowledge about the role of EPCs is far from complete and will require further revisions. Compared to previously data from case-control studies, our data also implicated that the capacity to mobilze EPCs in acute disease is more important than EPC basline levels.Table S1(0.06 MB DOC)Click here for additional data file.Text S1(0.07 MB DOC)Click here for additional data file.Figure S1Characterisation of EPC-CFU and EPC(2.88 MB TIF)Click here for additional data file.Figure S2Distribution of EPC and EPC-CFU, and the decline of EPC and EPC-CFU numbers with age.(1.17 MB TIF)Click here for additional data file.


Dysregulation of the immune system has been shown to occur during spaceflight, although the detailed nature of the phenomenon and the clinical risks for exploration class missions have yet to be established. Also, the growing clinical significance of immune system evaluation combined with epidemic infectious disease rates in third world countries provides a strong rationale for the development of field-compatible clinical immunology techniques and equipment. In July 2002 NASA performed a comprehensive immune assessment on field team members participating in the Haughton-Mars Project (HMP) on Devon Island in the high Canadian Arctic. The purpose of the study was to evaluate the effect of mission-associated stressors on the human immune system. To perform the study, the development of techniques for processing immune samples in remote field locations was required. Ten HMP-2002 participants volunteered for the study. A field protocol was developed at NASA-JSC for performing sample collection, blood staining/processing for immunophenotype analysis, whole-blood mitogenic culture for functional assessments and cell-sample preservation on-location at Devon Island. Specific assays included peripheral leukocyte distribution; constitutively activated T cells, intracellular cytokine profiles, plasma cortisol and EBV viral antibody levels. Study timepoints were 30 days prior to mission start, mid-mission and 60 days after mission completion.The protocol developed for immune sample processing in remote field locations functioned properly. Samples were processed on Devon Island, and stabilized for subsequent analysis at the Johnson Space Center in Houston. The data indicated that some phenotype, immune function and stress hormone changes occurred in the HMP field participants that were largely distinct from pre-mission baseline and post-mission recovery data. These immune changes appear similar to those observed in astronauts following spaceflight.The immune system changes described during the HMP field deployment validate the use of the HMP as a ground-based spaceflight/planetary exploration analog for some aspects of human physiology. The sample processing protocol developed for this study may have applications for immune studies in remote terrestrial field locations. Elements of this protocol could possibly be adapted for future in-flight immunology studies conducted during space missions. The growing diagnostic significance of clinical immunology combined with epidemic microbial disease rates in third-world countries make it clear that improved field-laboratory capabilities may be needed in the future to supportimmune testing or research. Whereas technological advances have already made it possible to provide field-testing for other laboratory disciplines , field immunology research has remained a challenge. This is due in part to the labile nature of cytokines and chemokines, the requirement for high-quality cellular samples with surface antigen integrity intact, and in some cases the need for live cell cultures. The size, weight and power requirements of most of the required instrumentation  also largely preclude their routine use in the field.Dysregulation of the immune system has been documented to occur during spaceflight. There have been several excellent reviews published regarding this phenomenon -4. SpeciTo evaluate the effects of mission-associated factors on human physiology, ground-based 'spaceflight analogs' may be used . A variepolar desert  is an international field research project centered on the scientific study of the Haughton meteorite impact structure and surrounding terrain on Devon Island, Nunavut Territory, Canadian High Arctic. The HMP is viewed as an analog for planetary exploration, in particular for exploration of the Moon and Mars figure  42]. Ha. Ha42]. • Long travel to and from Devon Island • Relatively harsh polar desert environment• Disrupted circadian rhythms (24 hours of daylight during the Summer field season)• Relative isolation from the outside world (with limited exception)• Limited local infrastructure • Moon and Mars-relevant  exploration/field/EVA activities• Reliance on remote telemedicine and communications equipment.These factors make the HMP potentially a good analog for spaceflight-associated immune dysfunction studies. The objectives of the present study were to (1) develop and field-evaluate methods for processing biological samples to support immune function testing in remote locations; and (2) utilize the protocol to assess mission-associated immune changes during a HMP mission. These methods could be adapted for monitoring the immune system of astronauts during exploration-class spaceflight or for immunology research studies in third-world/field locations.For this study, a deployment to the HMP Research Station was carried out in July 2002, during which a mobile biological sample processing facility was established in the station's laboratory tent facility. A panel of assays was developed that would comprehensively assess for each participant: (1) the presence or absence of specific immune responses; (2) the functional responsiveness immune system; 3) the presence or absence of latent EBV reactivation; (4) physiological stress. Ten subjects deployed to Devon Island during the 2002 field season participated in the study . The remaining 4 individuals showed no change in IgG titer  to develop and validate a protocol for collecting, processing and stabilizing biological samples in remote field locations for immune assessment testing; and (2) to assess immune status in subjects participating in a simulated planetary exploration (spaceflight) analog. The data generated from the samples processed on-location demonstrate that a variety of field immunology laboratory techniques may be performed in remote settings without access to sophisticated equipment, provided transport to a laboratory for analysis may be arranged within approximately two weeks of sample processing. All whole blood cultures, immunophenotype, and plasma assays were successfully performed using the field-collected samples figure . ImmunopGiven the epidemic disease rates in third-world countries, and the increasing sophistication of clinical laboratory equipment (limiting field use), we anticipate that protocols such as those validated here may have important future field research applications. In addition, protocols such as these could be modified for immune function testing on-orbit, to facilitate space physiology studies required prior to exploration-class space missions. Indeed, some of the microgravity-compatible hardware required to implement such investigations has already been developed. This study utilized the BCPSA for preservation of leukocytes figure , and ourDetermining any clinical risk related to the effects of space flight on the human immune system presents several daunting challenges. First among these is a lack of in-flight data. The vast majority of flight immune studies have been pre-, and post-flight assessments. Unfortunately, post-flight assessments may be skewed by the effects of landing and readaptation to the 1 × G environment. Because of this, it is difficult to extrapolate post-flight data to the in-flight condition. Until comprehensive studies are initiated to assess in-flight immunity, it is useful to perform ground-based analog studies. The HMP represents a potentially good ground-based spaceflight analogs with regards to flight-associated immune dysfunction. Several likely causes of this phenomenon are shared between flight and HMP: long difficult travel, remoteness and isolation, extreme environment, disrupted circadian rhythms, and mission-associated stress. HMP participants keep rigorous schedules with respect to actual  exploration activities, hardware evaluation, and science studies, to maximize the benefit from their time on Devon Island. The only potential causes of flight immune dysregulation NOT shared with the HMP to some degree are high-energy radiation and microgravity. Interestingly, HMP participants are exposed to elevated levels of ultraviolet radiation due to the thinned atmospheric ozone concentrations in the Arctic , althougThe 10 HMP field participants and their environment had several important features in common with astronauts serving onboard the International Space Station. Both groups represent otherwise healthy and fit individuals performing mission activities in a remote and stressful environment. We believe that the data generated during this study accurately reflect what would be anticipated from such individuals. No consistent changes were observed in the distribution of any major peripheral leukocyte subsets. The leukocyte differential, lymphocyte subsets and T cell subsets were essentially unchanged between the pre-, mid-, and post-mission timepoints figure . In addiWe believe that the immune subset data is consistent with otherwise healthy individuals in a stressful environment, and that these changes do not relate to the presence or absence of immune functional changes. Indeed, when a legitimate indicator of immune function was assessed, the cytokine profiles of the T cell subsets, sharp mission-associated decreases were observed. The ability of the T cell subsets (CD4+ and CD8+) to be stimulated to produce Interleukin-2 was significantly decreased mid-mission, as was the CD8+ population that could be stimulated to produce IFNg figure . All funOne consistent in-flight observation in astronauts is that the reactivation of latent herpes viruses occurs during spaceflight to high levels -22. It iIt is well established that periods of physiological stress correlate with dysregulated immune function . ClassicSpaceflight itself may be a physiological stressor, and certainly landing, with a high-G entry and readaptation to the 1 × G environment after lengthy microgravity deconditioning, is a tremendous physiological stress. Following spaceflight, alterations in stress hormones have been observed in astronauts. Stowe et al. found that following spaceflight of 8–15 days there was no change in plasma cortisol or adrenocorticotropic hormone levels. In contrast, urinary epinephrine, norepinephrine and cortisol were significantly elevated following flight . A more Taken together, these data support the establishment of the HMP as a unique ground-based spaceflight/planetary exploration analog that may have significant utility for terrestrial human physiology studies. The HMP may be particularly useful for assessments of human physiology such as immunology, that are particularly influenced by stress, isolation and disrupted circadian rhythms.This study protocol was designed to be analogous to spaceflight research: baseline testing was performed 30–60 days prior to the start of the mission to establish baseline values; mid-mission testing performed to assess mission-associated changes; and post-mission testing performed 30–60 days after return to confirm recovery to baseline values. Ten healthy male subjects scheduled to participate in the HMP-2002 field season were enrolled in this study. No female HMP 2002 participants were identified who could be solicited for this study, primarily for logistical reasons. The subjects were from a variety of locations and institutions located throughout the US and Canada figure . On DevoWhole blood samples consisted of 10.0 ml EDTA and 10.0 ml heparin whole blood collected per subject per timepoint using standard phlebotomy techniques during the morning hours. Because it was necessary to collect pre- and post-mission samples near the local residence of the subject, clinical laboratories from local hospitals were enlisted to perform the pre- and post-mission sample collections. Pre- and post-mission samples were shipped overnight to JSC for analysis. Previous in-house validation has shown that a 24-hour delay in sample processing did not affect the results for the parameters measured during this study (data not shown). For consistency of data, all mid-mission blood samples collected on Devon Island were held for 24 hours in the laboratory tent at ambient temperature  prior to processing to duplicate the pre- and post-mission sample shipping delays. Specific sample collection points for each subject are presented in figure A 5-color flow cytometry antibody matrix was created  that assessed most of the major leukocyte/lymphocyte subsets, as well as various memory/naïve and activated T cell subsets  according to the manufacturer's instructions. EBV IgM antibody was detected using the Diamedix EBV VCA IgM assay  according to the manufacturer's instructions on the MAGO automated microplate robotic system (Diamedix).EBV PCR was performed by real time PCR using primers/probe set first described by Kimura, H. et al. . 200 micThe measurement of plasma cortisol was performed by enzyme-linked immunosorbant assay  as has previously been described in detail ,51.No laboratory equipment was transported for real-time field analysis. Instead, techniques were developed allowing field culture, processing and preservation of samples for subsequent laboratory analysis after mission completion. In general, the field activities consisted of the following activities:• Collection of whole blood samples.• Setup of whole blood culture for T cell activation.• Preservation of whole blood for flow cytometry assays.• Preservation of activated leukocytes for intracellular cytokine detection.• Purification and aliquoting of plasma for stress hormone and viral assays.The sample processing performed on Devon Island rendered all sample types stable for storage and transport to JSC for subsequent analysis. During the deployment, processing was aided by the development of novel hardware for processing blood samples, the use of a newly developed commercial leukocyte preservatives and the development of field sample processing techniques. In most cases, processing rendered the samples stable for up to two weeks without cold storage allowing easy transport off Devon Island for subsequent analysis at Johnson Space Center. The individual modifications for field use per assay are as follows:On Devon Island, a phlebotomy station was set up in the main laboratory tent. Samples were collected from each subject by venipuncture using sterile techniques and butterfly type collection equipment. This does not represent a significant deviation from standard blood collection techniques.Whole blood was treated with a recently developed commercial preservative   that maiTo facilitate field sample processing, and to reduce the amount of biohazardous waste, whole blood preservation was performed on Devon Island using the Blood Collection/Preservation/Storage Apparatus (BCPSA) Figure . This de2. Since short term culture in buffered media does not require exogenous CO2, whole blood cultures were performed on Devon Island in 12 × 16 mm polystyrene tubes that were tightly sealed. A similar method for whole blood culture in portable devices was previously described [All cell culture reagents were transported to Devon Island chilled and stored chilled until use. Previous in-house testing has validated the stability of all the cell culture mitogens  without loss of activity for up to two weeks when stored in this manner (data not shown). Typical laboratory incubators supply two items to the incubating cells: 37 degree heat, and COescribed . A portaTo purify and aliquot EDTA plasma on-location at Devon Island for viral and cortisol assays, a small centrifuge was transported to the island. EDTA whole blood was centrifuged for 10 minutes, and plasma was removed and stored in labeled cryo-vials. Plasma samples were stored at 4–5°C when possible, and briefly at room temperature otherwise (during air travel), for transport to JSC for analysis. Salivary cortisol has been found to be extremely stable when stored at 5°C, with values unchanged following storage of up to 3 months . We havet test was used and p values of less than 0.05 were considered significant. Where appropriate, significant data is indicated by '*'.To determine mission-associated alterations, mid-mission data were compared to pre-mission baseline values. Statistical operations were performed by using Microsoft Excel 2003. The Student's paired The author(s) declare that they have no competing interests.BC, CS and PL participated in conception of this study. BC performed all field sample processing, all flow cytometry assessments, all data analysis and drafted the manuscript. PL was responsible for all HMP 2002 logistical concerns, and serves as principal investigator of HMP. RS performed all hormone assessments, contributed to the manuscript draft. JJ and RE are NASA flight surgeons and HMP veterans; they contributed to the conception of the study, aided clinical interpretation of the data and contributed to the manuscript draft. RW was responsible for all viral assays and provided inputs to the manuscript. CS is principal investigator of the NASA-JSC Immunology Laboratory, where majority of testing performed, and provided inputs to the manuscript. All authors have read and approved the final version of the manuscript.


First, [T]he results only pertain to the sample itself, and should not be used to make quantitative statements about any population – including the population from which the sample was selected.” In their article, According to p-values become meaningless.The weakness of all nonprobability sampling is its subjectivity that precludes the development of a theoretical framework for it. Finally, as former U.S. EPA scientists who pioneered agency exposure science, we are disappointed that this article was cleared for publication by the U.S. EPA because it is not in accordance with U.S. EPA (and other agency) requirements to follow the Office of Management and Budget’s (OMB) data collection policies  that reqProbability sampling must be used at each stage of respondent selection. You may encounter difficulties in clearing the survey through OMB if you do not insist that probability selection methods be used.Recent samples of high-risk subpopulations and their exposures to particles were undertaken by the U.S. EPA using doctor-identified subjects, and these were therefore not probability-based samples. The OMB allowed these studies but required that a statement be made in all resulting publications that the results could be applied only to the participants, even if chosen in this case by expert judgment, and must not be extrapolated to larger populations. We believe a similar statement should be made in all publications of studies using alternatives to probability-based sampling.In summary,