APOE ε4 allele and homozygosity at polymorphic codon 129 in the PRNP gene are the major genetic risk factors for AD and human prion diseases, respectively. However, the roles of PRNP in AD, and APOE in CJD are controversial. In this work, we investigated for the first time, APOE and PRNP genotypes simultaneously in 474 AD and 175 sporadic CJD (sCJD) patients compared to a common control population of 335 subjects. Differences in genotype distribution between patients and control subjects were studied by logistic regression analysis using age and gender as covariates. The effect size of risk association and synergy factors were calculated using the logistic odds ratio estimates. Our data confirmed that the presence of APOE ε4 allele is associated with a higher risk of developing AD, while homozygosity at PRNP gene constitutes a risk for sCJD. Opposite, we found no association for PRNP with AD, nor for APOE with sCJD. Interestingly, when AD and sCJD patients were stratified according to their respective main risk genes , we found statistically significant associations for the other gene in those strata at higher previous risk. Synergy factor analysis showed a synergistic age-dependent interaction between APOE and PRNP in both AD , and sCJD . We propose that this statistical epistasis can partially explain divergent data from different association studies. Moreover, these results suggest that the genetic interaction between APOE and PRNP may have a biological correlate that is indicative of shared neurodegenerative pathways involved in AD and sCJD.Alzheimer's disease (AD) and Creutzfeldt-Jakob disease (CJD) represent two distinct clinical entities belonging to a wider group, generically named as conformational disorders that share common pathophysiologic mechanisms. It is well-established that the  APOE ε4 allele is a major risk factor for AD PRNP gene is a well-established susceptibility marker for human prion diseases APOE in CJD and PRNP in AD is matter of discussion.Both Alzheimer's disease (AD) and Creutzfeldt-Jakob disease (CJD) are brain amyloidoses associated with old age. Although, they represent two distinct clinical entities, AD and CJD are now considered as part of a wider group generically named as conformational disorders, and more specifically brain amyloidosis. These two disorders share common pathophysiologic mechanisms involving protein deposits in the brain due to the conversion of a soluble, normal protein into an insoluble, aggregated form leading to fatal degeneration PRNP codon 129 in AD, or the APOE ε4 allele in CJD yielded conflicting results. Some studies found an association between the PRNP codon 129 and AD APOE ε4 allele and CJD Previous studies on the distribution of APOE and PRNP genes has been rarely studied for CJD or AD. Furthermore, to our knowledge, an analysis of the potential epistasis of these genes in both disorders by using the same control population and statistical approach has never been materialized. In this study, we investigated a possible genetic interaction between APOE ε4 allele and the polymorphic codon 129 of the PRNP gene in both AD and sCJD populations.Since these disorders share several features, a genetic interaction between these genes might determine susceptibility to both diseases or modulate the clinical phenotypes. A potential genetic interaction between In this study, two different patient (AD and sCJD) and one control populations were analyzed. The control group consisted of 335 subjects; 198 (59.1%) women and 137 (40.9%) men. Age at study inclusion for control individual followed a non-normal distribution with a mean of 73.2 years and standard deviation of 11.0 years. The AD group consisted of 474 patients diagnosed with probable AD according to the NINCDS-ADRDA criteria; 323 were women (68.1%) and 151 were men (31.9%). Age at onset for control individuals followed a non-normal distribution with a mean of 74.5±8.2 years. The sCJD group (n = 175) included 140 (80.0%) definite, neuropathologically verified patients, and 35 (20.0%) probable cases that were diagnosed with sCJD. The gender distribution was 87 (49.7%) women and 88 (50.3%) men. Age at onset for this group followed a non-normal distribution with a mean of 67.3±11 years. No statistically significant differences in gender distribution or age at onset were found between definite and probable sCJD cases.Analysis of gender distribution showed statistically significant differences between AD (p = 0.010) or sCJD (p = 0.049) patients and the control group. Analysis of age distribution yielded statistically significant differences for sCJD patients (p<0.001), and for AD patients (p = 0.006) compared to the control group. Mean age and gender distribution of the control population were intermediate between those of AD and sCJD cases, supporting its validity as a common control group. In order to correct for potential effects of the differential gender and age distributions, odds ratios and synergy factors were obtained from logistic regression algorithms controlling by age and gender.PRNP and APOE genotypes in control subjects, AD and sCJD patients are shown in APOE polymorphic variants at codon 112 and codon 158, as well as the allelic haplotypes ε2/ε3/ε4, were in Hardy-Weinberg equilibrium in the three populations studied. As expected, the polymorphic codon 129 of the PRNP gene was in Hardy-Weinberg equilibrium in the healthy control and AD populations, but in clear disequilibrium (p<0.0001) in the sCJD population.The distribution of the polymorphic codon 129 of APOE ε4 allele carriers (OR = 4.51). Interestingly, when this association was studied in different strata according to PRNP codon 129 genotypes, we observed an increase in the OR associated with the presence of M129 alleles. Thus, the highest OR (7.28) was observed in M129M homozygous, while the lowest OR in V129V homozygous (2.06). The Mantel-Haenszel test for homogeneity indicated that these two homozygous strata were statistically significant different (p = 0.026). The fact that the OR for each stratum is different and the global OR appears to be an average OR suggested an interaction between APOE and PRNP, rather than PRNP acting as a confounding factor.As shown in APOE ε2 allele carriers . Similar to the results with the APOE ε4 allele carriers, when this analysis was performed in the 3 different strata defined by PRNP codon 129, we found a tendency to higher protection associated with the presence of PRNP V129 alleles .Additionally, we also observed a decreased risk of AD in PRNP codon 129  for M129M homozygous only in APOE ε4 carriers. In this APOE ε4+ stratum, the analysis of dose effect indicated a two-fold increase in AD risk  for each PRNP M129 allele carried. A similar analysis for APOE ε2 carriers did not show any significant association in any strata.The analysis of AD risk related to the status of odon 129  indicatePRNP gene is a susceptibility genetic risk factor of AD only in APOE ε4 carriers and prompted us to further explore the potential genetic interaction between APOE and PRNP in the AD population. In order to study the combined gene effects, we analyzed the risk of developing AD for subjects carrying both traits (APOE ε4 carriers and PRNP M129M homozygous) compared to subjects without these traits  increased risk of developing AD compared to subjects V129V with no APOE ε4 alleles, which is higher than the expected combined effect of the individual contributions of APOE ε4 and PRNP M129M, suggesting gene-gene interaction or epistasis  indicated a SF of 1.86 (p = 0.018) for the interaction APOE ε4 and a M129 allele  analysis following reported methods. According to this analysis, we obtained a SF of 3.59, which is statistically significant different (p = 0.027) from 1; indicating a synergistic interaction between two risk factors, nd M129M , upper. 9 allele , bottom.PRNP gene. Interestingly, when these associations were studied in different strata according to the APOE ε4 status, we observed an important increase in the OR for APOE ε4 carriers compared to non-ε4 carriers (28.5 vs. 3.5 for M129M and 13.2 vs. 2.43 for V129V). Mantel-Haenszel tests for homogeneity indicated that the APOE ε4 carriers were statistically significant different from APOE ε4 non-carriers for the M129M comparison (p = 0.022). These data, as in the case of the AD population, suggested an interaction between APOE and PRNP in sCJD. The analysis of the APOE ε4 allele dose effect  showed similar results, although of lower magnitude to the ones observed for the ε4+ and no-ε4 allele comparison , suggesting that the effect of APOE ε4 allele was dose dependent.As shown in APOE ε4 allele status and sCJD for the total population  . We also found a similar trend, although not statistically significant, in the V129V homozygous stratum. Opposite, a non significant protector effect for the APOE ε4 allele was found in M129V heterozygous stratum. Mantel-Haenszel test indicated significant differences between the M129M homozygous and M129V heterozygous strata (p = 0.022) or homozygous (M129M and V129V) vs. M129V heterozygous (p = 0.027). We did not find association for the APOE ε2, neither in the unselected domain , nor in any of the subpopulations defined by PRNP codon 129 genotypes.We found no association between  = 0.13) . HoweverAPOE and PRNP in sCJD, we analyzed the risk of developing sCJD for subjects carrying both traits (APOE ε4 carriers and PRNP homozygous) compared to subjects without these traits  increased risk of developing sCJD than subjects M129V heterozygous with no APOE ε4 alleles. This increased risk appears to be higher than a multiplicative effect of each independent factor, suggesting gene-gene interaction or epistasis. Synergy factor analysis yielded a SF of 7.26, which is statistically significant different (p = 0.005) from 1; indicating a synergistic interaction between two risk factors, APOE ε4 and PRNP homozygous. A similar analysis by PRNP genotypes (M129M vs. M129V) indicated a SF of 8.48 (p = 0.003) for the interaction APOE ε4 and PRNP M129M genotype , in agreement with previous reports APOE ε2 carriers compared to non-ε2 carriers , as previously reported PRNP .We further explored the influence of these genetic factors on disease onset for both AD and sCJD populations. In AD, we observed that PRNP V129 allele . Regarding APOE, we observed a later onset for APOE ε2 carriers compared to non-ε2 carriers  in agreement to previous reports APOE ε4 allele , also in agreement with previous results For sCJD, we found a non-significant tendency for earlier onset associated with the PRNP M129V heterozygous compared to PRNP homozygous (p<0.001) . These results are in concordance with previous reports about the classification sCJD in several subtypes characterized by a combination of PRNP codon 129 status and the size of protease resistant PrPSc fragments and disease phenotype APOE ε2 allele or APOE ε4 allele carriers.Additionally, we also explored the influence of these genetic factors on disease duration in sCJD population. We observed a significant longer disease duration in APOE ε4 and PRNP M129 was age dependent. For this purpose, we stratified cases and controls into two groups according to the median age for patients and control population pooled together. Analysis of AD risk related to the status of PRNP of the age stratified data indicated that the association to the PRNP M129 allele previously found in the APOE ε4 carriers stratum . We found statistically significant associations for the other gene in those strata at higher previous risk , suggesting an underlying interaction between APOE and PRNP as risk factors for both disorders . Interestingly, we found that these interactions between PRNP and APOE were also age-dependent for both disorders, but in opposite directions . The coexistence of AD and CJD pathology in a subgroup of sCJD patients Sc and Aβ peptides in amyloid lesions both in sCJD and AD ScCentral to both neurodegenerative pathologies is an analogous proteolytic processing of a membrane protein  that generates unstable, aggregation prone, neurotoxic fragments that are pathognomonic for each disease  was determined by Real-Time PCR Total DNA was isolated from peripheral blood or cerebral tissue following standard procedures. Genotyping of Statistical analyses of nominal or categorical variables were performed by Fisher's exact text or Pearson's chi-square test. Quantitative variables  were analyzed by non-parametric statistical hypothesis contrast with Mann-Whitney U test. Additionally, logistic regression models controlled by age (as a linear variable) and gender were used to compare genotypic and allelic frequencies and to calculate association adjusted odds ratio (OR) and 95% confidence intervals (CIs). The Hardy-Weinberg test for genotype frequency distributions was performed on the observed genotype frequencies for population, with significance based on a standard observed-expected 2 with 1 df. The Mantel-Haenszel test was used to estimate the common odds ratio and to test whether the overall degree of association across different genotype strata was significant. Deviations from normality of quantitative variables were checked by the Kolmogorov-Smirnov statistic with Lilliefors' significance. The synergy factor (SF), confidence intervals and significance were calculated as previously described


Dasypus novemcinctus) is a conspicuous example of a successful invader, having colonized much of the southern United States in the last 200 years. We used 15 years (1992–2006) of capture-mark-recapture data from a population of armadillos in northern Florida in order to estimate, and examine relationships among, various demographic parameters that may have contributed to this ongoing range expansion. Modeling across a range of values for γ, the probability of juveniles surviving in the population until first capture, we found that population growth rates varied from 0.80 for γ = 0.1, to 1.03 for γ = 1.0. Growth rates approached 1.0 only when γ ≥0.80, a situation that might not occur commonly because of the high rate of disappearance of juveniles. Net reproductive rate increased linearly with γ, but life expectancy (estimated at 3 years) was independent of γ. We also found that growth rates were lower during a 3-year period of hardwood removal that removed preferred habitat than in the years preceding or following. Life-table response experiment (LTRE) analysis indicated the decrease in growth rate during logging was primarily due to changes in survival rates of adults. Likewise, elasticity analyses of both deterministic and stochastic population growth rates revealed that survival parameters were more influential on population growth than were those related to reproduction. Collectively, our results are consistent with recent theories regarding biological invasions which posit that populations no longer at the leading edge of range expansion do not exhibit strong positive growth rates, and that high reproductive output is less critical in predicting the likelihood of successful invasion than are life-history strategies that emphasize allocation of resources to future, as opposed to current, reproduction.Understanding why certain species can successfully colonize new areas while others do not is a central question in ecology. The nine-banded armadillo ( Understanding why some species are able to successfully colonize new areas while others do not is a key question in ecology and conservation biology In addition to the aforementioned, intrinsic features of animal populations must inevitably play some role in determining the success of any invasion. For example, for a range to expand it is only logical to assume that populations produce sufficient individuals such that some leave current areas to colonize new ones. This could be accomplished by high reproductive output, high survivorship, or some combination of the two. Consequently, models to estimate population growth rates, coupled with prospective and retrospective perturbation analyses to identify parameters that most influence these rates, can provide valuable insights into the factors that might promote range expansion in a particular species.Dasypus novemcinctus; hereafter referred to as “armadillo”) is a dramatic example of a successful invader. Although widely distributed across much of the Americas Among mammals, the nine-banded armadillo (In this paper we use 15 years of capture-mark-recapture (CMR) data from a population of armadillos in northern Florida in an attempt to explore various demographic parameters that might contribute to range expansion. Specifically, we build on a previous study that focused on estimating apparent annual survival rate and transition probabilities between reproductive and non-reproductive states to estimate population growth rates. We then performed prospective perturbation analyses to quantify the relative influence of various demographic parameters on these estimates. A potential concern with these analyses was how our estimates might have been impacted by a three-year program of hardwood removal that eliminated much preferred habitat for armadillos at our study site Permission to conduct fieldwork was provided by the Director of Research, Tall Timbers Research Station. All field procedures followed American Society of Mammalogists guidelines Details of the field site and sampling methods can be found in Body mass was used to assign captured animals to one of three age categories: juveniles (young of the year) were individuals weighing <2 kg, yearlings weighed 2–3 kg, and adults weighed >3 kg Colinus virginianus; see Although our fieldwork ended in 2003, some data were available from 2004–2006 because of the harvesting of armadillos at Tall Timbers in order to remove nest predators of northern bobwhite . Finally, reproductive adult females that survive the year become non-reproductive adults the following year with probability ψrn, and remain reproductive with probability (1 - ψrn). The stage-structured population projection matrix was of the form:nF and rF are fertility rates for non-reproductive and reproductive adults. Fertility rates were estimated using post-breeding census methods We considered 4 stages, based on age and reproductive status: <1 year old = juveniles; ≥1 and <2 year old = yearlings; and ≥2 years old = adults nF = 0.5 * LS *γ * nS * ψnr and rF = 0.5 * LS *γ * rS * ψrr, where LS is litter size and γ is a composite parameter that quantifies the probability of survival until trappable age.LS and γ were estimated using a multistate capture-mark-recapture (CMR) modeling framework All parameters except LS was 4. Next, we created a variable, γ, to represent the proportion of quadruplets that survive to trappable age. Trappable age begins at first emergence of juveniles from their natal burrows  and treatment . The term iπ.To further examine the impact of hardwood removal on population dynamics, we used a fixed effect LTRE analysis tr = log(n(t+1)/n(t)) is a one-step population growth rate, n(t) and n(t+1) are projected population sizes at time t and t+1, respectively, and T = 50,000 steps Deterministic demographic analyses assume that the environment is constant, and there is no variability in vital demographic parameters. In reality, however, the environment as well as vital rates can vary unpredictably. Stochastic demographic methods allow explicit consideration of variability in vital rates. We used a simulation-based approach  to estimate stochastic population growth rate and stochastic elasticities u(t) and v(t) are stochastic stage structure and reproductive value vectors at time t, λ(t) is 1-time step population growth rate, and the term v(t) and u(t). Following t; (2) elasticities of sλ to the mean of matrix elements sλ to the variance of the matrix entries sλ to lower-level vital rates were calculated using methods described by Caswell where All analyses were performed using programs written in MATLAB .Overall estimates of demographic variables for the entire study period are presented in the Supplementary Materials . Across Matrix entry elasticities revealed that λ was proportionately most sensitive to changes in the probability of surviving and remaining in the reproductive adult stage, followed by the probability of surviving and remaining in the non-reproductive adult stage . As the rS, followed by nS and ψrr, as the most influential vital rates across all reasonable values of γ  were slightly lower than deterministic ones, but exhibited a similar relationship with γ Click here for additional data file.Figure S2Net reproductive rate as a function of γ. Net reproductive rate approaches 1.0 when γ ≈ 0.8.(TIFF)Click here for additional data file.Figure S3s).Elasticity of stochastic population growth rate (λ Elasticities are presented for (A) mean, and (B) standard deviation (SD) of vital demographic parameters for a range of values of γ. Symbols are: jS, yS, nS, and rS = survival of juveniles, yearlings, non-reproductive adults and reproductive adults, respectively; ψnr = probability of transitioning from non-reproductive to reproductive adult stage; ψrr = probability of reproductive adults remaining reproductive adults; LS = litter size; and γ = probability of surviving to trappable age.(TIFF)Click here for additional data file.


There have been suggestions from previous studies that patients with Charcot–Marie–Tooth disease (CMT) have weaker dominant hand muscles. Since all studies to date have included a heterogeneous group of CMT patients we decided to analyse hand strength in 43 patients with CMT1X. We recorded handedness and the MRC scores for the first dorsal interosseous and abductor pollicis brevis muscles, median and ulnar nerve compound motor action potentials and conduction velocities in dominant and non-dominant hands. Twenty-two CMT1X patients (51%) had a weaker dominant hand; none had a stronger dominant hand. Mean MRC scores were significantly higher for first dorsal interosseous and abductor pollicis brevis in non-dominant hands compared to dominant hands. Median nerve compound motor action potentials were significantly reduced in dominant compared to non-dominant hands. We conclude that the dominant hand is weaker than the non-dominant hand in patients with CMT1X. Initially it was used to explain apparent activity-dependent weakness that developed in patients with post-polio syndrome Gap Junction Beta 1 (GJB1) gene that cause an X-linked form of the disease (CMT1X), the second commonest form of CMT http://www.molgen.ua.ac.be/CMTMutations). Mutations in GJB1 cause neuropathy through a loss of normal gene function GJB1 codes for a protein called Connexin 32. It is expressed by both Schwann cells and oligodendrocytes and is thought to act as a gap junction protein, though how loss of normal function leads to axon loss is yet to be elucidated [9].Charcot–Marie–Tooth (CMT) disease is a genetically heterogeneous inherited neuropathy. Over 300 mutations have been described in the Clinically, patients with CMT1X have a progressive motor and sensory neuropathy with distal wasting. Some patients also develop a central nervous system phenotype, which can either present acutely or chronically Since previous studies investigating hand strength in CMT have included a genetically mixed group of CMT patients, we decided to look at hand muscle strength in a single group of CMT patients, CMT1X. The aim of this study was to determine whether there was any difference in the strength of the muscles in patients with CMT1X between dominant and non-dominant hands. We also aimed to determine whether there were differences in strength between hand muscles innervated by different nerves as has been described previously with abductor pollicis brevis (APB) being weaker than first dorsal interosseous (FDIO) 2We retrospectively reviewed the notes of 53 patients attending the National Hospital for Neurology and Neurosurgery with a genetically confirmed diagnosis of CMT1X.We recorded handedness, age, gender, mutation, most recent CMT neuropathy score (CMTNS) The FDIO score for the dominant hand was tabulated against the FDIO score for the non-dominant hand and distributions were examined. In order to examine whether there was a difference in FDIO strength between hands, various MRC scores were tested as a cut off. For example, an FDIO score cut-off of 4− meant, we categorised scores into ⩽4− versus >4−. These categories were compared between the dominant and non-dominant hands using a McNemar’s test to see whether the same proportion of scores were considered to be ⩽4− for both hands. Similar comparisons were made for APB MRC scores.t-tests were used for normally distributed differences. Multilevel models were performed to account for the non-independence of observations in the dominant and non-dominant hands, where clustering was assumed to have occurred within each patient p value <0.01 was considered significant.In order to make comparisons between the dominant and non-dominant hands in nerve conduction outcomes and in MRC strength scores, the distribution of the difference in outcome values between hands was assessed. Non-parametric Wilcoxon sign-rank tests (i.e. matched-pairs tests) were used to compare skewed differences and paired Stata Statistical Software: Release 11. College Station, TX: StataCorp LP).All statistical analyses were performed using Stata (StataCorp. 2009. 3GJB1 gene (p < 0.01). Males had a mean CMTNS of 15.2  and females a CMTNS of 12.6 . Seventy-three percent of patients were examined by a single author (MMR).Of the 53 patients, nine were excluded because MRC score was not adequately documented and a further one was discounted due to having an additional diagnosis of CIDP. This left 43 patients, 22 males and 21 females, with 29 different mutations in the JB1 gene . Males wIn CMT1X patients, FDIO strength was higher in the non-dominant hand for 15/43 (34.9%) individuals and APB strength was higher in the non-dominant hand for 14/43 (32.6%) individuals. There were 22/43 (51.2%) patients with a weaker dominant hand based on a combined MRC score of FDIO and APB. No patient was found to have a stronger dominant hand for either FDIO or APB MRC strength scores individually or for the combined score.p = 0.003) and eight (18.6%) patients who had a non-dominant APB score >4− and a dominant APB score ⩽4− (p = 0.005). Using other MRC grades as a cut off did not show statistical significance in our sample population.When we categorised individuals into MRC strength scores of >4− and ⩽4−, there was a significant difference in strength according to hand-dominance. We identified nine (20.9%) individuals who had a non-dominant FDIO score >4− and a dominant FDIO score ⩽4− . Furthermore, the associated nerve conduction studies demonstrated that median nerve CMAPs (APB) were significantly lower than the ulnar nerve CMAPs (ADM) in CMT1X patients (p < 0.0001; p = 0.0005; When comparing FDIO to APB strength in the dominant hand of CMT1X patients, we found that APB was significantly weaker (4We reviewed the notes of 43 patients with CMT1X and demonstrated that patients with CMT1X had a weaker dominant hand. Median nerve CMAPs were reduced in the dominant compared to the non-dominant hand. Furthermore, we showed that the APB muscle was weaker than FDIO, confirming previous suggestions that there is a greater impact on median nerve function compared to the ulnar nerve There are several limitations to note with this study. Firstly, we used the MRC scoring system to measure hand strength. A recent study demonstrated that while there is a significant correlation between MRC scoring for hand muscles and muscle strength measured by the Rotterdam intrinsic hand myometer the scale is not linear and there is considerable overlap between MRC grades 4 and 5 Reassuringly, our study has confirmed several previous observations reported in CMT1X patients. Firstly we have shown that APB is weaker in males than in females. Additionally, our male subgroup was significantly younger and had a higher mean CMTNS. These findings all fit with males being more severely affected with an earlier disease onset Although the results in this study suggest the dominant hand is weaker in patients with CMT1X, these results should be confirmed by a prospective blinded study, ideally using muscle myometry, MRC scoring and nerve conduction studies. However, this study does highlight the need to investigate hand strength in an aetiologically homogenous population since previous hand strength studies in CMT have used a mixed population of patients with all forms of CMT It has been suggested in previous studies that a difference in hand strength between dominant and non-dominant hands may suggest that overwork weakness causes increased muscle weakness as the dominant hand will be used more by the patient


Overall pandemic A (H1N1) influenza vaccination rates remain low across all nations, including Japan. To increase the rates, it is important to understand the motives and barriers for the acceptance of the vaccine. We conducted this study to determine potential predictors of the uptake of A (H1N1) influenza vaccine in a cohort of Japanese general population.p = 0.001), to perceive high susceptibility to influenza (p = 0.03), to have willingness to pay even if the vaccine costs ≥ US$44 (p = 0.04), to have received seasonal influenza vaccine during the preceding season (p<0.001), and to have willingness to accept A (H1N1) influenza vaccine at baseline (p<0.001) compared to those who had not been vaccinated.By using self-administered questionnaires, this population-based longitudinal study was conducted from October 2009 to April 2010 among 428 adults aged 18–65 years randomly selected from each household residing in four wards and one city in Tokyo. Multiple logistic regression analyses were performed. Of total, 38.1% of participants received seasonal influenza vaccine during the preceding season, 57.0% had willingness to accept A (H1N1) influenza vaccine at baseline, and 12.1% had received A (H1N1) influenza vaccine by the time of follow-up. After adjustment for potential confounding variables, people who had been vaccinated were significantly more likely to be living with an underlying disease (While studies have reported high rates of willingness to receive A (H1N1) influenza vaccine, these rates may not transpire in the actual practices. The uptake of the vaccine may be determined by several potential factors such as perceived susceptibility to influenza and sensitivity to vaccination cost in general population. A new swine-origin influenza A (H1N1) emerged in early 2009 in Mexico and the United States and has since spread worldwide Vaccination is a primary public health measure to curb the spread of A (H1N1) influenza pandemic due to the lack of innate immunity as a result of the strain's novelty Early vaccination against the virus is cost-effective and may avert the deaths Evidence has emerged regarding factors associated with willingness to get A (H1N1) influenza vaccination. However, only a few studies have examined the association of these factors with the actual uptake of the vaccine, and most of these studies have been conducted in key populations such as healthcare workers, school teachers, people living with underlying diseases, elderly people, and pregnant women. The identified factors consistently reported in these studies include personal experience of getting vaccinated against seasonal influenza These findings are important in response to the need for revising or constructing a preparedness plan in early stage of the fight against the pandemic A (H1N1) influenza. However, findings from these studies are limited by the cross-sectional nature of the data. To the best of our knowledge, one longitudinal study has been conducted to explore the influencing factors related to the uptake of A (H1N1) influenza vaccine among school teachers in the United States We sent a postal mail in which an information sheet and a questionnaire were included to each selected person. In the information sheet, we fully explained the nature and possible consequences of the study and requested them to note if they were willing to participate in the study. For those who did not want to participate, we requested them to indicate on the sheet and return it to us. The study protocol was reviewed and approved by the Ethics Committee of the National Center for Global Health and Medicine, Tokyo, Japan.Baseline study was conducted in October 2009 and data for follow-up was collected in April 2010. We used multi-stage sampling strategy to select participants for this study. We first randomly selected four wards out of 23 wards and two cities out of 26 cities comprising metropolitan Tokyo. Of these two selected cities, one city refused participation. Number of towns in each ward and city ranges from 20 to 155, and approximately 10% of the towns were randomly selected. Probability proportional to size sampling was used to select adults aged 18–65 years randomly from the residential registries obtained from the municipal offices.Self-administered, anonymous questionnaires were mailed to participants in both baseline and follow-up survey by using the same study protocol. The survey questionnaires were developed based on measures adapted from previous studies and guidelines recommended by the Japanese government and non-government agencies. In baseline survey, we collected information regarding socio-demographic characteristics, history of living with underlying diseases, history of living with people in high risk groups, history of receiving seasonal influenza vaccine in 2008–2009, history of seasonal influenza infection during the current season (2008–2009), knowledge on A (H1N1) influenza and its vaccine, perception of risk of getting infected with A (H1N1) influenza, perception of seriousness of pandemic A (H1N1) influenza, preventive behavior against A (H1N1) influenza, attitudes towards A (H1N1) influenza and its vaccine, and willingness to accept vaccination against A (H1N1) influenza. In follow-up survey, we additionally collected information regarding history of influenza infection during the current season (2009–2010) and history of the uptake of vaccine against seasonal influenza and A (H1N1) influenza during the current season (2009–2010).Willingness to accept vaccination against A (H1N1) influenza was assessed by asking: “Do you plan to get A (H1N1) influenza vaccine during this fall or winter if it becomes available?” with three response options including “yes,” “no,” and “not sure.” The uptake of influenza vaccine was assessed by asking the following yes/no questions: “Did you receive seasonal influenza vaccine during the last season (2008–2009)?”; “Did you receive seasonal influenza vaccine during this season (2009–2010)?”; and “Did you receive new swine A (H1N1) influenza vaccine during this season (2009–2010)?” To assess risk perception, we asked: “Do you think you are constitutionally susceptible to influenza infection?” using a 4-point scale that included “very high,” “high,” “not so high,” and “not high at all.” Regarding attitude towards vaccination, participants were asked whether they feel anxious about the side effects of swine A (H1N1) influenza vaccine with “a lot,” “some,” “not much,” and “not at all” as response choices. To assess willingness to pay for the vaccine, we asked: “Up to how much would you pay for receiving swine A (H1N1) influenza vaccine?” with four response options including “free of charge,” “US$ 1 to <22,” “US$ 22 to <44,” and “≥ US$ 44.”Regarding knowledge on A (H1N1) influenza and its vaccine, we evaluated the correct responses to four yes/no questions regarding modes of transmission, the fact that there have been healthy people who had died from A (H1N1) influenza, people at risk of A (H1N1) influenza, effectiveness of antiviral medicines such as Tamiflu and Relenza against A (H1N1) influenza, and side effects of vaccine against A (H1N1) influenza. To assess participants' preventive behavior, we asked whether participants washed their hands after returning home during the preceding week (yes/no).For socio-demographic characteristics, participants reported their age (continuous), gender , education attainment , marital status (unmarried or married), employment status (employed or unemployed), annual household income , and whether the participant was living with children who go to school (yes/no) and with people in high risk groups such as people with an underlying disease, pregnant women, children aged five years or younger, and elderly people aged 65 years or older (yes/no). In this study, underlying diseases included chronic respiratory diseases, chronic metabolic diseases, chronic heart diseases, kidney diseases, and immunodeficiency diseases.2 test or Fisher's Exact test for categorical variables and student t-test for continuous variables. Using cross tabulations, we assessed association between the uptake of A (H1N1) influenza vaccine with several characteristics of participants. We tested statistical significance of the association by using Pearson's χ2 test or Fisher's exact test.We compared characteristics of participants at baseline and follow-up and characteristics of people who successfully completed the study to those of people who participated in baseline but refused to remain in the cohort or did not return the questionnaires at follow-up by using χp≤0.10 in bivariate analyses. Two-sided p-values of <0.05 were regarded as statistically significant. We used SPSS version 17.0  for all the statistical analyses.A multiple logistic regression model was then constructed to determine potential predictors of the uptake of A (H1N1) influenza vaccine. Socio-demographic characteristics and variables on vaccination history, knowledge, perception, and behavior were included in the model if they were associated with the uptake of A (H1N1) influenza vaccine at a level of n = 428) were significantly more likely to have attained higher education (p = 0.03) and to live with people in high-risk groups (p = 0.009) compared to those who were lost to follow-up (n = 90).Characteristics of the participants at baseline and follow-up are shown in p = 0.001), to perceive high personal susceptibility to influenza (p = 0.03), to have willingness to pay for the vaccine even if it costs ≥ US$ 44 (p = 0.04), to have received seasonal influenza vaccination during the preceding season (2008–2009) (p<0.001), and to have willingness to accept A (H1N1) influenza vaccine at baseline (p<0.001) compared to those who had not been vaccinated.As shown in This study represents a few attempts to identify influencing factors associated with the actual uptake of A (H1N1) influenza vaccine using a longitudinal design in general population. Several potential predictors have been explored including willingness to accept A (H1N1) influenza vaccine at baseline, receiving seasonal influenza vaccine during the preceding season (2008–2009), willingness to pay for the vaccine even if it costs ≥ US$ 44, perceived high susceptibility to influenza, and living with an underlying disease.People who had willingness to accept A (H1N1) influenza vaccine at baseline were more than four times more likely to get vaccinated compared to those who had no willingness. However, among people who were willing to get a shot of the vaccine at baseline, only 17.6% had actually received it by the time of the follow-up. This finding may raise concerns in respect to other potential factors that might have diminished people's final decision making on whether to take the vaccination at different stages of the pandemic. Because baseline survey of this study was conducted when A (H1N1) influenza was at its peak in Japan, we can expect that people's willingness to get vaccinated was strongly anchored and well-defined in general population. The willingness might, however, be over-estimated, and people may change their mind when the pandemic situation become less severe and clinical evidence regarding efficacy and safety of the vaccine remained unavailable when vaccination campaigns were launched. In Japan, A (H1N1) influenza vaccine was not available for general population until January 2010 when the number of A (H1N1) influenza cases was going down In line with previous studies Among reasons for the low A (H1N1) influenza vaccine coverage rates, there may have been a lack of concern about the individual risk, which may translate into a lack of willingness or urgency to get vaccinated, particularly if there is mistrust of information provided by public health or government authorities People may perform a sort of trade-off between perceived risk and perceived benefit regarding the vaccine uptake. In this study, the majority (77.8%) of participants perceived their susceptibility to A (H1N1) influenza as “not so high” or “not high at all,” and those in this category were almost three times less likely to get vaccinated compared to those who perceived their susceptibility as “high or very high.” Meanwhile, more than two thirds of them were anxious about adverse effects of the vaccine. This suggests that perceived risks may exceed perceived benefit. In addition, the acceptability of A (H1N1) influenza vaccine was sensitive to cost, although Japan is a fairly affluent country with a high degree of vigilance for influenza. People who were willing to pay for the vaccine even if it costs ≥ US$ 44 were almost seven times more likely to get vaccinated compared to those who were willing to get vaccinated only if it is available for free. Such a cost sensitivity may also be true for the case of seasonal influenza vaccine for which, in Japan, people have to pay up to approximately US$ 25 to US$ 60 It is worth noting that some important factors, such as anxiety about adverse effects of A (H1N1) influenza vaccine and living with people in high risk groups, did not retain their significant association with the uptake of A (H1N1) influenza vaccine in this study. Concerns about vaccine safety and side effects have been consistently reported as a potential determinant of willingness to get vaccinated and/or the uptake of the vaccine The major strengths of this study include the longitudinal design with the ability to document not only people's willingness to accept A (H1N1) vaccine but also their actual practice of receiving the vaccine in six-month follow-up. Moreover, we were able to control for the effects of several potential confounding factors.Several limitations of this study should also be noticed. First, our findings were limited by the validity of self-reporting measures, which may lead to either under-reporting or over-reporting due to social desirability or conformity. Second, the relatively low response rate (54.8%) may have resulted in a biased sample and become potential threats to the generalizability of the findings to the whole population. This response rate, however, is comparable to or even higher than that of many mailing or telephone surveys in other countries Despite these limitations, this study contributes to the literature in several ways. While studies have reported high rates of willingness to receive A (H1N1) influenza vaccine, these rates may not transpire in the actual practices among general population. The uptake of the pandemic A (H1N1) influenza vaccine may be determined by several potential factors such as perceived susceptibility to influenza and sensitivity to vaccination cost. Although cultural differences could affect the acceptance of vaccines in general


As β2m can promote nerve regeneration, we investigated its potential role in amyotrophic lateral sclerosis (ALS) by investigating its expression level as well as the effect of genetically removing β2m on the disease process in mutant superoxide dismutase 1 (SOD1G93A) mice, a model of ALS. We observed a strong upregulation of β2m in motor neurons during the disease process and ubiquitous removal of β2m dramatically shortens the disease duration indicating that β2m plays an essential and positive role during the disease process. We hypothesize that β2m contributes to plasticity that is essential for muscle reinnervation. Absence of this plasticity will lead to faster muscle denervation and counteracting this process could be a relevant therapeutic target.Beta-2 microglobulin ( Within the central nervous system β2m has a predominantly motor neuronal expression pattern  class I proteins were originally discovered based on their critical role in the immune system, however immune-independent functions in the nervous system have recently been identified . Based on these mutations, ALS rodent models have been generated that predictably mimic the patient disease process  or human SOD1G93A and β2m knockout mice were purchased from The Jackson Laboratories  and maintained on a C57BL/6 background. The SOD1G93A and β2m knockout were interbred allowing for approx. 50% of the mice to be littermate controlled in this study. Chow and water were provided ad libitum and mice were housed in the specific pathogen free animal facility of the KU Leuven under standard conditions according to the guidelines of the KU Leuven. End stage was defined as the age at which mice could no longer right themselves within 30 s when placed on their back. End stage is used as a measurement of survival and is the condition at which mice are euthanized to prevent further suffering. Disease onset was defined as the age at which mouse weight dropped below 90% of the average day 90–105 weight. The animal caretakers and scientists were blinded to the genotypes of the mice when assessing “end stage”. All animal experiments were performed with the approval of the Animal Ethical Committee of KU Leuven (020/2010).Mice overexpressing human wild-type 2, were collected. At least 1,500 motor neurons were dissected for each animal.Murine spinal cords were snap-frozen in Tissue-Tec  to make cryostat sections of 20-μm thickness. Then, cresyl violet–stained motor neurons, located in the ventral horn of the lumbar spinal cord, were collected on membrane slides 1.0 PEN , using dissection by a laser-dissection microscope (Carl Zeiss AG) and capturing in Adhesive Cap 500 opaque (Carl Zeiss AG). Only motor neurons in which the nucleus was visible and with soma area > 250 μmgapdh (Mm.PT.39a.1), β2m (Mm00437762_m1) and cd8b1 (Mm.PT49a.10182911). For this analysis, presymptomatic tissue was collected at 90 days of age and symptomatic at 120 days of age. The scientist performing the qPCR was blinded to the genotypes of the samples.Isolation of mRNA was performed using the TriPure  method and the RNeasy kit . Reverse transcriptase polymerase chain reaction (PCR) used random hexamers  and Moloney Murine Leukemia Virus Reverse Transcriptase . Quantitative PCR (qPCR) was performed with the StepOnePlus (Life Technologies) and TaqMan Universal PCR Master Mix (Life Technologies). Gene expression assays were purchased from Life Technologies and IDT DNA : ® . Images were collected by Zeiss Axio Imager M1 microscope (Carl Zeiss AG) with AxioCam Mrc5 camera (Carl Zeiss AG). The number of (motor) neurons was quantified by measurement of the soma area as visualized by cresyl violet staining in ImageJ  on multiple 40 μm thick sections in the ventral horn of the lumbar spinal cord. Characterisation of motor neurons occurred as previously  and subsequently with 4% formaldehyde. Spinal cords were post-fixed with 4% formaldehyde overnight at 4°C and transferred to 30% sucrose for an additional night. After snap freezing, tissue was sectioned by cryostat at 40 μm thickness and stained with a polyclonal antibody directed against ubiquitin . Images were collected by Zeiss Axio Imager M1 microscope (Carl Zeiss AG) with AxioCam Mrc5 camera (Carl Zeiss AG). Ubiquitin immunopositive aggregates were counted per ventral horn using ImageJ of 2–6 ventral horns of the lumbar spinal cord of 2–4 mice per group and presented as the average of the number of aggregates per ventral horn. The scientist performing the immunohistochemistry and aggregate quantification was blinded to the genotypes of the samples.t-test. Differences between more than two groups were analyzed by ANOVA with Bonferroni correction for multiple testing. Significance was assumed at p < 0.05. Error bars represent the standard deviation.Analysis was performed with the statistical software package Prism Origin . Survival was analyzed by Log-Rank testing. Differences between two groups were analyzed using a Student’s β2m to have a functional role in ALS pathogenesis, we assessed gene expression in the spinal cords of non-transgenic, WTSOD1 and SOD1G93A mice. We observed a strong increase in β2m gene expression during disease progression in SOD1G93A mice  was identified by qPCR on neurons from SOD1G93A mice compared to neurons from SOD1WT mice isolated by laser dissection microscopy . The complete genetic ablation of β2m did not affect onset of disease (data not shown), but significantly decreased average survival of SOD1G93A mice by 8.9 days  plasticity by β2m knockout mouse we cannot exclude that the decrease of survival of ALS mice lacking β2m may be due to the effect of removing β2m in the immune system. β2m is necessary for the differentiation of CD8+ T cells and natural killer T (NKT) cells  (VanGuilder Starkey et al., β2m is localized at synapses and in synaptosomes (Shatz, β2m and PirB are associated with decreased plasticity and recovery in other neurodegenerative conditions such as stroke (Adelson et al., β2m or PirB in these models may be due to the role of the immune system. This notion is supported by work by Linker et al. that show that reconstitution of CD8+ cells in β2m knockout mice delays the effect of EAE compared to β2m knockout littermates (Linker et al., The mechanism to which s Shatz, . Alternaβ2m knockout in ALS and identifies β2m signaling as a potential new direction for the development of therapeutic strategies counteracting ALS.In conclusion, this work shows the detrimental effect of Kim A. Staats and Susann Schönefeldt performed the murine behavioral analyses. Kim A. Staats and Marike Van Rillaer conducted the staining experiments. Kim A. Staats and Annelies Van Hoecke analyzed gene expression of motor neurons excised by laser dissection microscopy. Philip Van Damme, Wim Robberecht, Adrian Liston and Ludo Van Den Bosch supervised and designed the experiments. Kim A. Staats, Adrian Liston and Ludo Van Den Bosch wrote the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


E) to the inner membrane of Escherichia coli when envelope stress is low. Elevated envelope stress triggers RseA cleavage by the sequential action of two membrane proteases, DegS and RseP, releasing σE to activate an envelope stress reducing pathway. Revertants of a ΔdegP ΔbamB strain, which fails to grow at 37°C due to high envelope stress, harbored mutations in the rseA and rpoE genes. Null and missense rseA mutations constitutively hyper-activated the σE regulon and significantly reduced the major outer membrane protein (OMP) levels. In contrast, a novel rpoE allele, rpoE3, resulting from the partial duplication of the rpoE gene, increased σE levels greater than that seen in the rseA mutant background but did not reduce OMP levels. A σE-dependent RybB::LacZ construct showed only a weak activation of the σE pathway by rpoE3. Despite this, rpoE3 fully reversed the growth and envelope vesiculation phenotypes of ΔdegP. Interestingly, rpoE3 also brought down the modestly activated Cpx envelope stress pathway in the ΔdegP strain to the wild type level, showing the complementary nature of the σE and Cpx pathways. Through employing a labile mutant periplasmic protein, AcrAL222Q, it was determined that the rpoE3 mutation overcomes the ΔdegP phenotypes, in part, by activating a σE-dependent proteolytic pathway. Our data suggest that a reduction in the OMP levels is not intrinsic to the σE-mediated mechanism of lowering envelope stress. They also suggest that under extreme envelope stress, a tight homeostasis loop between RseA and σE may partly be responsible for cell death, and this loop can be broken by mutations that either lower RseA activity or increase σE levels.RseA sequesters RpoE (σ Escherichia coliDegP is the major periplasmic protease in et al. degP requires DegP's protease activity degP is under the control of the σE and Cpx regulons degP expression to overcome the potentially lethal envelope stress caused by aberrant OMP assembly Spiess sohA (suppressor of htrA) sohA reversed the growth phenotype of ΔdegP could not be determined. Prior to this study, Kiino and Silhavy prlF and hypothesized that it was involved in some aspect of protein localization. The prlF gene, which turned out to be the site of the sohA mutation, together with yhaV, was recently determined to constitute a toxin-antitoxin module sohA/prlF alleles overcome the growth defects of ΔdegP remains unknown.Compensatory mutations have been isolated that overcome the temperature sensitive growth phenotype of cells lacking DegP. The first of this was isolated by Baird and Georgopoulos degP suppressor mutation was obtained somewhat serendipitously and mapped to the cyaA gene cyaA ΔdegP background overcame the temperature sensitive growth phenotype by inducing the σE and Cpx stress regulons. While it remains to be determined exactly how reduced cAMP levels leads to up-regulation of the envelope stress response systems, another alarmone, ppGpp, was shown to up-regulate σE activity in a RseA-independent manner E activity both directly, by influencing σE-dependent transcription, and indirectly, via altering competition for the core polymerase in favor of σEThe second ΔdegP strain also lacking bamB, a nonessential component of the BAM complex degP and bamB confers a severe growth phenotype even at 30°C envZ was identified as one of the genes in which suppressor alterations improved the growth phenotype of the ΔdegP ΔbamB strain In this study, we sought revertants of a ΔdegP ΔbamB strain. Mutations in rseA constitutively activate the σE regulon and reduce envelope stress in part by lowering the major OMP levels. In contrast, an rpoE mutation, which causes a novel duplication/truncation of the rpoE gene, does not reverse the growth defect by lowering the major OMP levels; instead, it appears to activate a proteolytic pathway that partly compensates for the loss of DegP.Here we report two new sites of suppressor mutations that overcome the growth defects of a ΔdegP and bamB displays a synthetic and conditional lethal phenotype at or above 37°C and grows poorly even at 30°C One of the ways to understand the consequences of a conditional lethal mutation is by isolating and characterizing revertants that have acquired so-called suppressor mutations and overcome the harmful effects of the original mutation. We have previously shown that a mutant simultaneously lacking degP ΔbamB double mutant were grown in LB at 30°C, diluted, spread onto LB agar plates, and incubated at 37°C until colonies appeared (between 24 and 36 hours). Temperature resistant colonies, which arose at a frequency of around 10−7, were then purified at 30°C and suppressor mutants categorized based on growth robustness and OMP levels. Using these criteria, the suppressors were classified into three groups  a W33R substitution, which was previously shown to directly affect the RseA-σE binding pocket rseA, and (c) an IS1 insertion element at nucleotide 454 of rseA. In each case, the mutations presumably abrogated RseA's ability to sequester σE at the inner membrane, thereby elevating the σE-mediated envelope stress response. Thus, rseA mutations suppressed the ΔdegP ΔbamB-mediated conditional lethal phenotype, in part, by lowering the load of OMPs in the envelope, and additionally by increasing the synthesis of periplasmic chaperones.The second group of revertants were distinct from those carrying missense mutations in jor OMPs , lane 2,degP bamB+ strain. Because of these two properties, we chose to investigate this suppressor further in the ΔdegP bamB+ strain and not in the original ΔdegP ΔbamB strain, which grows very poorly and prone to accumulating suppressors.The last group was represented by one suppressor, which was noted to have the unusual ability to suppress lethality without significantly altering steady-state OMP levels ; lane 3.degP cells, growth was measured by culturing cells in LB for five hours at 39°C, which is the sub-lethal growth temperature for the ΔdegP mutant, with cell density measured every thirty minutes. In contrast to ΔdegP-only cells, ΔdegP cells containing the suppressor mutation grew at a rate indistinguishable from the degP+ strain  insertion library into a ΔdegP strain harboring the suppressor mutation and selected for tetracycline resistant (TetR) transductants. Over a thousand TetR transductants were screened for the reversion to temperature sensitivity  by replica plating at 40°C and 30°C. One colony became temperature sensitive, indicating that either the insertion of Tn10 in a gene confers the growth defect or the Tn10 brought in the flanking wild type DNA and replaced the suppressor mutation. To distinguish the two possibilities, we determined the linkage of Tn10 to the temperature sensitivity phenotype by transducing the TetR marker into the ΔdegP strain containing the suppressor mutation. Approximately 50% of the time the resulting TetR transductants became temperature sensitive, indicating that the Tn10 is closely linked to the suppressor mutation and not inserted in a gene that confers a temperature sensitive growth phenotype.Before determining the mechanism of Δ10, arbitrarily primed polymerase chain reaction (AP-PCR) was utilized. PCR products obtained after the second, high-stringency reaction were analyzed on a 4% w/v agarose gel, excised and sequenced using the appropriate Tn10 specific primers used in the reaction. Using this method, the Tn10 was found to disrupt yfiF located at 58.5′ on the chromosome, at nucleotide 883. A known Kanr insertion in glrK (57.9′ on the E. coli chromosome) was utilized to determine which side of yfiF::Tn10 the suppressor mutation was located. ΔglrK::Kanr was introduced into ΔdegP suppressor-yfiF::Tn10 cells by P1 transduction, and Kanr transductants were screened for Tets and temperature sensitive phenotypes. Using this method, the gene order was determined to be glrK-suppressor-yfiF.To conclusively determine the chromosomal location of the TnrpoE-nadB region, we noted the presence of a PCR product from the suppressor strain that was approximately 1 kb larger than that amplified from the wild-type strain (data not shown). Sequence analysis indicated the presence of duplicated DNA in the suppressor strain.Several sets of primers were designed to amplify 1 to 3 kb long DNA from the 57.93 to 58.53 minute region of the chromosome from wild-type and suppressor-containing strains and the products were sequenced. When amplifying the rpoE and reading into rpoE (primer 1) and one primer landing upstream of nadB and reading into nadB (primer 2)  and 124 bp downstream from the nadB ATG (primer 4) produced an expected 291-bp long PCR fragment from wild type strain  . Becauseimer set . Anothere strain . Howevere strain , confirme strain .rpoE . In addition to coding for five non-native amino acids, the region immediately after F122 was identical to the DNA sequence from 21 bases upstream of the nadB translation start to the translation start site of the native, full-length copy of rpoE was reached  to six-fold (in ΔdegP background) higher than in their rpoE+ counterparts (rpoE expression resulting from the rpoE3-mediated promoter duplication (in degP+ background) and increased envelope stress (in ΔdegP background). Despite these increases in σE levels, the steady-state levels of OMPs were not reduced in the rpoE3 mutant . This increase correlates well with a significant reduction in the OMP levels (rpoE3), 20% (ΔdegP), and 70% (rpoE3 ΔdegP)  . The addE pathways are important in reducing envelope stress caused by aberrant β-barrel OMP assembly degP is critical for this reduction in envelope stress cpxP, a prototypical member of the Cpx regulon, is modestly upregulated in ΔdegP cells grown at 39°C . In order to test for proteolysis in this background, degP was also removed by a degP::Kanr insertion.For this, we employed AcrAdegP::Kanr or rpoE3 did not significantly affect AcrAL222Q levels  responsible for AcrAL222Q degradation in the absence of DegP in the ropE3 and ΔrseA strains.Since OMP levels are not reduced by degPsohB nor degQ is known to be regulated by σE and there is no experimental data showing SohB is actually a protease. yfgC, a gene regulated by σE, is predicted to encode a periplasmic metalloprotease L222Q stability or the ΔdegP phenotypes (data not shown). It should be noted that activation of a proteolytic pathway likely represents only a component of the broader defensive response triggered by σE. Therefore, we believe that the collective action of increased proteolysis and other defensive measures most likely account for the rpoE3-mediated reversal of the ΔdegP phenotypes.Overexpression of SohB and DegQ from plasmids has been reported to rescue the temperature sensitive growth phenotype of ΔE levels and rescue the ΔdegP phenotypes are either in rseA, a gene encoding for the negative regulator of σE, or the rpoE gene itself suggests that even under high envelope stress conditions the amount of free σE becomes limiting. If growth at high temperatures without DegP indeed leads to the accumulation of misfolded OMPs, then these accumulated OMPs should trigger the DegS/RseP-mediated proteolytic pathway to degrade RseA and elevate free σE levels degP cells begin to die at 39°C suggests that the defensive response system may have maxed out, perhaps because newly released and activated σE allows for a greater synthesis of RseA, which in turn re-captures σE. In other words, under extreme envelope stress, a tight homeostasis loop between RseA and σE may partly be responsible for cell death. This loop can be broken by mutations that either lower RseA activity or increase σE levels. An alternative means to overcoming the growth phenotype of ΔdegP without directly influencing the RseA-σE loop would be through mutations that influence the cAMP-CRP prlF/sohA mutations likely provide yet another mechanism to overcome the temperature sensitive growth phenotype of ΔdegP.The fact that mutations required to increase σEscherichia coli K-12 bacterial strains used in this study were constructed in an MC4100 background −1 ampicillin, 25 µg ml−1 kanamycin, 12.5 µg ml−1 chloramphenicol, 10 µg ml−1 tetracycline and 0.2% L-(+) arabinose (Sigma). All other chemicals were of analytical grade. Bacterial growth curves were performed by diluting overnight cultures 1∶100 into 25 ml fresh pre-warmed media supplemented with appropriate antibiotics. Cultures were vigorously shaken at 39°C in baffle-bottom flasks in a water incubator with cell density checked every 30 min.egP, followed by the removal of the antibiotic resistance cassette was carried out using the λ-red mediated gene deletion method as previously described Standard bacterial genetic methods were carried out as previously described 10 marker was identified by the arbitrary-primed PCR (AP-PCR) method 10 marker (see below). That round of PCR used both ARB1 and ARB6 primers 10 left or Tn10 right primers to amplify fragments on either side of the Tn10. First round products were analyzed on an 0.8% agarose gel, and fragments unique to the Tn10 strain were excised using a QIAquick gel extraction kit (QIAGEN) and subjected to a second, high-stringency round of PCR using the appropriate Tn10 primer and ARB2 10 primer or ARB2. Sequences were analyzed to obtain non-Tn10 sequence, and unassigned sequences were analyzed using BLASTn to determine the location of the Tn10.The unknown Tn600 of 0.025. Cell pellets, collected from freshly grown cells, were resuspended in a lysis buffer containing 20 mM Tris-HCl pH 7.5, 2 mM phenylmethylsulfonylfluoride (PMSF), 10 mM MgCl2 and 25 µg ml−1 DNase I. Samples were passed twice through a French pressure cell, followed by low-speed centrifugation of lysates to remove unlysed cells. Crude lysates were the centrifuged at 100,000 g for 1 h at 4°C to pellet membranes. Periplasm was extracted using the gentle osmotic shock method −1 lysozyme) and incubated on ice for 30 min. Samples were centrifuged at 100,000 g for 15 min and periplasm drawn off as soluble fraction. Spheroplast pellets were then washed with cold 10 mM Tris-HCl pH 7.5 and subjected to French press lysis as described above. After high speed spin, cytoplasm was harvested from the soluble fraction. Membranes pellets were routinely washed in 10 mM Tris-HCl pH 7.5 and resuspended according to cell density in the same buffer.Fresh cultures were grown by sub-culturing overnight cultures into fresh media with appropriate antibiotics at a starting OD−1 bromophenol blue, 5% v/v β-mercaptoethanol and 2% w/v sodium dodecyl sulfate [SDS]). Samples were boiled for 5 min and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). When required, 4 M urea was added in order to resolve OmpC and OmpF. Membrane, periplasmic and cytoplasmic fractions were prepared in the same buffer as whole-cell samples. After electrophoresis, proteins were transferred to Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore) using a Bio-Rad mini-transblot. Membranes were blotted with antibodies raised against OmpF (1∶16 667), LamB (1∶10 000), AcrA (1∶10 000), TolC (1∶10 000), MalE (1∶10 000) RpoE (1∶5 000) or GroEL (1∶25 000). Blots were developed as described previously For Western blot analysis from whole cells, aliquots of cultures, normalized to cell density, were pelleted in a microcentrifuge. Pellets were resuspended in sample buffer  normalized to cell density (OD562) in each well. All assays were performed in quadruplicate.β-galactosidase activity was determined by an established method Figure S1Effects of ΔdegP and rpoE3 mutations on RybB::LacZ activity. Two independent cultures were grown at 37°C to a mid-log phase and used for β-galactosidase assays. LacZ activities are relative to the wild-type strain. Relevant genotypes are shown at the bottom.(TIF)Click here for additional data file.Figure S2L222Q (A) and wild type Levels of AcrAacrA (B) in different genetic backgrounds. AcrA levels were determined by Western blot analysis of whole cell lysates prepared from overnight grown cultures at 37°C. GroEL, LamB and AcrA were detected using specific antibodies. GroEL served as a gel loading control. Each lane contains protein samples from equal number of cells, based on OD600. Relevant genotypes are shown at the top.(TIF)Click here for additional data file.Table S1A list of Escherichia coli K-12 strains used in this study.(DOC)Click here for additional data file.


Xenopus oocytes at room temperature  were compared to those recorded at either physiological temperature (37°C) or at lower temperature (4°C). Alterations in temperature had dramatically differing effects on the amplitude of whole-cell responses detected with these three nAChR subtypes. Compared to responses at RT, the amplitude of agonist-evoked responses with α4β2 nAChRs was increased at high temperature  and reduced at low temperature , whereas the amplitude of α7 responses was reduced at high temperature  and increased at low temperatures . In contrast to the effects of temperature on α4β2 and wild type α7 nAChRs, the amplitude of α7 nAChRs containing the L247T mutation was unaffected by changes in temperature. In addition, changes in temperature had little or no effect on current amplitude when α7 nAChRs were activated by the largely non-desensitizing allosteric agonist 4BP-TQS. Despite these differing effects of temperature on the amplitude of agonist-evoked responses in different nAChRs, changes in temperature had a consistent effect on the rate of receptor desensitization on all subtypes examined. In all cases, higher temperature resulted in increased rates of desensitization. Thus, it appears that the differing effects of temperature on the amplitudes of whole-cell responses cannot be explained by temperature-induced changes in receptor desensitization rates.We have examined the effect of temperature on the electrophysiological properties of three neuronal nicotinic acetylcholine receptor (nAChR) subtypes: the rapidly desensitizing homomeric α7 nAChR, the more slowly desensitizing heteromeric α4β2 nAChR and on α7 nAChRs containing a transmembrane mutation (L247T) that results in dramatically reduced desensitization. In all cases, the functional properties of receptors expressed in  Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop family of ligand-gated ion channels which also includes receptors for 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA) and glycine Seventeen nAChRs subunits  have been identified in vertebrate species and can co-assemble into a large number of nAChR subtypes with considerable diversity in subunit composition A variety of experimental approaches have confirmed that conventional orthosteric agonists such as acetylcholine bind at an extracellular site located at the interface of two subunits Xenopus oocytes, we have examined heteromeric α4β2 and homomeric α7 nAChRs. In addition, we have examined the effect of changes in temperature on responses evoked by both orthosteric and allosteric agonists, as well as on α7 nAChRs containing a mutation that slows the rate of desensitization caused by orthosteric agonists such as acetylcholine. Changes in temperature resulted in changes in the magnitude of agonist-evoked responses. However, opposing effects were observed on different nAChR subtypes. Changes in temperature were also associated with changes in rates of receptor desensitization, but this does not appear to explain the differences observed in current amplitudes at different temperatures.A particular advantage of studies conducted with recombinant nAChRs in artificial expression systems is the ability to examine the properties of receptors with defined subunit composition, as well as the ability to examine the effects of alterations in amino acid composition by means of techniques such as site-directed mutagenesis Xenopus oocytes was examined by two-electrode voltage-clamp recording. When acetylcholine-evoked responses were recorded at room temperature , rapidly desensitizing currents were observed  saline. A calcium-free saline is typically used with α7 nAChRs to minimize the possibility of calcium-activated chloride channels . In order to examine whether differences in saline composition might explain differences in the effect of temperature on current amplitudes, experiments with α4β2 nAChRs were repeated in calcium-free saline. The effect of temperature on current amplitude with α4β2 nAChRs was consistent between the two saline solutions , Table 1EC50 concentration (0.4 µM) of acetylcholine or was used (A further series of experiments was performed with the human α7 nAChR containing a single point mutation (L247T) in the second transmembrane domain. As has been described previously was used , Table 1P = 0.002), reduction in current amplitude.Whereas activation of wild-type α7 nAChRs by acetylcholine results in rapidly desensitizing responses, activation of α7 nAChRs by the allosteric agonist 4BP-TQS has a much slower onset and causes much less desensitization One possible explanation for these results is that the relatively small effect of high temperature on current amplitude in α7 nAChRs activated by 4BP-TQS might be due to instability of the compound at 37°C. To examine this possibility, control experiments were performed with solutions of 4BP-TQS that had been stored at 37°C for three hours and then cooled to room temperature. No differences in agonist responses were observed, when compared with freshly prepared solutions (data not shown).L247T nAChRs and for wild-type α7 nAChRs activated by 4BP-TQS, despite changes in temperature having little or no effect on current amplitude .It has been known for several decades that changes in temperature can influence the properties of nAChRs. For example, a series of studies conducted in the early 1970s demonstrated that end-plate currents recorded at the frog neuromuscular junction are influenced by temperature 3 receptors 3 receptors has been reported to be independent of temperature, at least between 25°C and 40°C LymnaeaAn increase in current amplitude with increased temperature, similar to the effect we have observed with α4β2 nAChRs, has been reported recently for recombinant P2XL247T nAChRs. Changes in temperature had a greatly reduced effect on the amplitude of α7 responses in response to 4BP-TQS compared to responses to acetylcholine. Indeed, no significant effect was observed when responses at 4°C were compared to those at RT (We have found that introduction of a single point mutation (L247T) in the α7 nAChR completely abolishes the effect of temperature on current amplitude, whilst retaining an effect of temperature on the rate of desensitization of the macroscopic response. This observation adds to the many effects that have been reported for the L247T mutation in α7 nAChRs. These include an increase in agonist potency, reduction in receptor desensitization, increased spontaneous openings, the conversion of competitive antagonists into agonists and the conversion of allosteric potentiators into allosteric agonists se at RT . This isTorpedo, expressed in cultured mammalian cell lines It has been reported previously that exposure of some nAChRs to low temperature for several hours can facilitate more efficient protein folding and assembly. This was first demonstrated for the muscle-type nAChR from the marine ray It is unclear why changes in temperature should have opposing effects on current amplitude in two closely related ion channels (α4β2 and α7 nAChRs) or why these effects should be largely or completely abolished by either a single point mutation or by activation by an allosteric agonist, rather than by the conventional orthosteric agonist, acetylcholine. It appears, however, that these effects are not a consequence of changes in the rate of receptor desensitization observed during macroscopic oocyte responses, since changes in temperature were found to have a broadly consistent effect on this parameter. In addition, changes in temperature had a consistent effect on the rate of receptor deactivation after removal of agonist (faster at 37°C and slower at 4°C), presumably reflecting changes in the off-rate of agonists from their binding sites. It is possible that receptors can adopt multiple open or desensitized states, and that entry into these various states occurs at different rates and can be affected differently by phenomena such as changes in temperature, mutagenesis or by allosteric modulators. This idea is consistent with models that have been advanced to explain mechanisms of allosteric modulation of nAChRs LymnaeaIt is possible that the changes in current amplitude that we have observed with α4β2 and α7 nAChRs may be a consequence of temperature-induced changes in single-channel conductance. Indeed, it has been reported that the conductance of muscle-type nAChRs increases with increasing temperature BamHI and purified with QIAquick PCR purification kit (Qiagen). In vitro synthesis of cRNA was performed using the SP6 mMessage mMachine SP6 kit (Ambion).Plasmid pSP64GL constructs containing human α4, α7 and β2 cDNA have been described previously Xenopus laevis frogs were obtained from the European Xenopus Resource Centre (University of Portsmouth). Oocytes were isolated and defolliculated as described previously 2, 0.77 mM CaCl2, 2.4 mM NaHCO3, and 15 mM Tris, 50 mg/l tetracycline, 50 mg/l penicillin and 50 mg/l streptomycin (pH 7.5 with HCl). Experiments were performed on oocytes 3 to 5 days of injection. Oocytes were placed in a recording chamber , which was continuously perfused with a modified Ringer solution . Oocytes were impaled by two microelectrodes filled with 3 M KCl (0.5–1.5 MΩ) and voltage-clamped at −60 mV using a GeneClamp 500 amplifier (Axon Instruments). Drugs were applied by switching between control and drug-containing solution using a BPS-8 valve control system (ALA Scientific). Agonists were applied for either 5 seconds (acetylcholine) or up to 40 seconds (4BP-TQS) with 2 minute (acetylcholine) or 8 minute (4BP-TQS) intervals between applications. The experiments were performed at room temperature . To detect effects of changed temperature on kinetics of nAChRs the modified Ringer solution was warmed/cooled to 37°C or 4°C in water bath or on ice. Agonist solutions were prepared fresh and warmed or cooled for 10–15 min immediately prior to use. In control experiment, it was confirmed that warming agonist solutions, even for 3 hours did not alter agonist potency. To examine effects of temperature, oocytes were continuously perfused with warm/cold modified Ringer solution for 40 sec prior to agonist application. Peak current amplitudes evoked by warm/cold acetylcholine were normalized to the peak amplitudes at RT from the same oocyte. To examine reversibility of any temperature induced changes, oocytes were then perfused with RT Ringer for 5 mins, followed by application of acetylcholine at RT. Peak amplitudes, the rise time and time to decay to half of the maximum amplitude were measured using Clampfit 9.2 (Molecular Devices). Effects of temperature on current amplitude were determined by measuring peak responses, however, results were not significantly different when responses were measure as area above the curve/total charge transfer. Statistical significance was determined using with SigmaStat  using non-paired Student's t-test. The effects of raising or lowering temperature on agonist-evoked responses were normalized to responses recorded at RT on the same oocyte. All data are reported as mean ± S.E.M.Adult female


Burkholderia pseudomallei infection (melioidosis) is an important cause of community-acquired Gram-negative sepsis in Northeast Thailand, where it is associated with a ∼40% mortality rate despite antimicrobial chemotherapy. We showed in a previous cohort study that patients taking glyburide ( = glibenclamide) prior to admission have lower mortality and attenuated inflammatory responses compared to patients not taking glyburide. We sought to define the mechanism underlying this observation in a murine model of melioidosis.2 cfu B. pseudomallei intranasally, then treated with therapeutic ceftazidime  in order to mimic the clinical scenario. Glyburide (50 mg/kg) or vehicle was started 7 d before inoculation and continued until sacrifice. The minimum inhibitory concentration of glyburide for B. pseudomallei was determined by broth microdilution. We also examined the effect of glyburide on interleukin (IL) 1β by bone-marrow-derived macrophages (BMDM).Mice (C57BL/6) with streptozocin-induced diabetes were inoculated with ∼6×10B. pseudomallei growth in vitro or in vivo. Glyburide directly reduced the secretion of IL1β by BMDMs in a dose-dependent fashion.Diabetic mice had increased susceptibility to melioidosis, with increased bacterial dissemination but no effect was seen of diabetes on inflammation compared to non-diabetic controls. Glyburide treatment did not affect glucose levels but was associated with reduced pulmonary cellular influx, reduced bacterial dissemination to both liver and spleen and reduced IL1β production when compared to untreated controls. Other cytokines were not different in glyburide-treated animals. There was no direct effect of glyburide on Diabetes increases the susceptibility to melioidosis. We further show, for the first time in any model of sepsis, that glyburide acts as an anti-inflammatory agent by reducing IL1β secretion accompanied by diminished cellular influx and reduced bacterial dissemination to distant organs. We found no evidence for a direct effect of glyburide on the bacterium. Burkholderia pseudomallei infection  is a common cause of bacterial infection in Northeast Thailand, where the mortality rate is 43% despite appropriate antibiotic treatment. We showed previously that patients taking glyburide ( = glibenclamide) prior to admission have lower mortality rates and lower levels of inflammation in the blood. In this study, we used a mouse model to better understand the mechanism underlying this observation. In this study, we used a mouse model of diabetes and infected the mice with B. pseudomallei. Half the mice were treated with glyburide and half were not. We also performed in vitro experiments to find the minimum concentration of glyburide that would inhibit the growth of B. pseudomallei. We found that glyburide treatment was associated with reduced inflammation (as measured by the flow of cells into the lungs and by interleukin-1β production) and reduced spread of the bacterium to liver and spleen when compared to untreated controls. There was no direct effect of glyburide on B. pseudomallei growth in vitro or in vivo. Because the effect of glyburide is on the host and not on the bacterium, it is possible that this effect will be seen in other causes of sepsis, not just melioidosis. Burkholderia pseudomallei, an environmental soil saprophyte B. pseudomallei has a myriad of clinical presentations ranging from acute pneumonia to chronic skin abscess B. pseudomallei has recently been classed a tier 1 bioterror threat agent Melioidosis is a severe community-acquired sepsis in Southeast Asia, caused by infection with B. pseudomallei: specifically, it impairs cytokine responses B. pseudomalleiDiabetes is the most commonly identified risk factor for sepsis in general Paradoxically, melioidosis patients with a pre-admission diagnosis of diabetes have a lower risk of mortality than patients without diabetes, which appears to contradict our current knowledge of the effect of diabetes on the host. We have shown previously, in a cohort study of 1,160 patients, that this reduction in risk is associated with glyburide use, and does not appear to be a characteristic of diabetes itself ATP-channel blocker and broad-spectrum ATP-binding cassette (ABC) transporter inhibitor used to treat type 2 diabetes. It costs less than USD 0.10 per dose and appears on the World Health Organization (WHO) model list of essential medicines Glyburide USAN ( = glibenclamide rINN) is a KATP-channels in pancreatic β-cells, which leads to the stimulation of insulin secretion. There is, however, evidence from experimental models that glyburide also has a wide range of anti-inflammatory effects The relevant pharmacological mechanism of glyburide in diabetes is the inhibition of KIn this study, we used an experimental model of melioidosis to provide insights into mechanisms underlying the effect of glyburide on inflammation, the best described of which is an inhibitory effect on the host inflammasome, an intracellular protein complex present in macrophages that activates caspase 1 and cleaves pro-IL1β and pro-IL18 to their active forms when presented with an appropriate inflammatory stimulus Mouse experiments were performed at the Academic Medical Center (AMC), University of Amsterdam and were approved by the animal experimentation committee of the AMC (number DIX101725). Mouse experiments were performed in accordance with Dutch law (Wet op de dierproven 1996) and European Council directive (86/609/EEC). Bone marrow-derived macrophage experiments were performed at the Friedrich Loeffler Institut für Medizinische Mikrobiologie, Universitätsmedizin Greifswald, in compliance with the relevant laws.Nnt, that appeared in the Jackson Laboratories colony some time between 1951 and 1970 Nnt gene B. pseudomallei by BALB/c mice is deficient compared to C57Bl/6 mice et al. have reported that BALB/c macrophages express lower levels of the lysosomal enzyme, β-glucuronidase, in response to macrophage-activating lipopeptide-2 (a synthetic TLR2 ligand) and to E. coli LPS when compared to C57Bl/6 macrophages The C57Bl/6J substrain (Jackson Laboratories) is a model for diet-induced type 2 diabetes, because it carries a spontaneous 17.8 kb deletion in the nicotinamide nucleotide transhydrogenase gene, We used a streptozocin model of diabetes, because that model permits a diabetic phenotype to be induced on any background, at any time, and the cohort become diabetic synchronously B. pseudomallei 1026b is a clinical isolate collected in 1993 from a 29-year-old Thai female rice farmer with bacteremia, soft tissue, cutaneous lesions, septic arthritis and splenic abscesses 2B. pseudomallei 1026b intranasally 4–5 weeks after streptozocin treatment  Glyburide 50 mg/kg  or vehicle was administered intraperitoneally once daily starting 7 days before inoculation and continued until sacrifice. Glyburide solutions were prepared in 20% dimethylsulfoxide (DMSO); control mice were given 20% DMSO. This dose of glyburide was chosen because it is equivalent to the highest human dose  after taking into account differences in pharmacokinetics  and water, and were group-housed in polycarbonate cages with 12-hour light/dark cycles, temperature 18–22°C, humidity 40–65%. Eight animals from each group were sacrificed 48, 72 and 96 hours after inoculation. At sacrifice, mice were sedated with ketamine/medetomidine intraperitoneally (12.5 mg/20 µg per 100 g weight), then exsanguinated by cardiac puncture. Death was confirmed by cutting the diaphragm. Glucose measurements were obtained prior to sedation as medetomidine itself elevates glucose concentrations. The left lung was lavaged with three 300 µl aliquots of sterile 0.9% saline then collected for culture. Left lung, liver and spleen were homogenized in sterile saline in a ratio of 1 g organ to 4 ml of saline. Organ homogenates, blood and bronchoalveolar lavage fluid (BALF) were plated onto blood agar in serial dilutions to quantify bacterial burdens. The remaining lung homogenate was incubated for 30 minutes with Greenberger lysis buffer then centrifuged at 650× g for 10 minutes. The supernatant was passed through a 0.2 µm filter  to remove viable bacteria, then stored at −20°C for cytokine measurements. Heparinized blood was centrifuged at 700× g for 10 minutes; the plasma was filtered and stored at −20°C pending analysis.Animals were allowed Creatinine was measured with a commercial available kit  using a Hitachi analyzer  according to the manufacturers' instructions. In lung homogenates and BALF, interleukin (IL) 1β, IL6, IL10, CXCL5 (LIX) and tumor necrosis factor-α (TNFα) were measured by sandwich ELISA . Cytokine levels in plasma were measured by particle immunoassay  because of limited sample quantity. Protein concentrations were measured using the DC™ protein assay .B. pseudomallei final concentration 5×105 cfu/ml) were pipetted into each well of a flat-bottomed 96-well plate. The final concentration of DMSO in every well was 1%. Cultures were incubated at 37°C (5% carbon dioxide) for 18 hours. Growth was defined as a pellet ≥2 mm or turbidity on visual inspection. The MIC of glyburide was read as the lowest concentration of glyburide that inhibited the growth of B. pseudomallei.The minimum inhibitory concentration of glyburide was established by broth microdilution and the protocol was adapted from the CLSI guidelines for microdilution assays of non-fastidious organisms g for 10 minutes. Cells were resuspended in RPMI 1640 (Invitrogen) containing Panexin BMM 5% v/v , 2 ng/ml recombinant murine GM-CSF (PAN Biotech) and 50 µM 2-mercaptoethanol. Cells were cultured for 10 days at 37°C and 5% carbon dioxide. Cultures were washed with PBS to remove non-adherent cells. Adherent cells were then detached with Trypsin-EDTA  and by scraping. Viable cells were counted in a Neubauer hemocytometer by Trypan blue exclusion. Six-well plates were seeded with 650,000 cells per well, then incubated overnight to allow the cells to adhere.Bone marrow-derived macrophages were generated as previously described B. pseudomallei 1026b overnight on Columbia agar with 5% sheep blood. Colonies were suspended in PBS to OD595 0.25 (∼1.2×108 cfu/ml). This suspension was then diluted with RPMI 1640 to the required concentration. Inoculum size was verified by plating serial dilutions onto Columbia agar. Cells were infected with bacteria for 30 minutes, then washed twice in PBS to remove extracellular bacteria. RPMI 1640+Panexin 5%+kanamycin 120 µg/ml (∼4× MIC for this strain to kill extracellular bacteria) was added to each well. Cell culture supernatants were harvested 24 hours after infection, then stored at −20°C pending assay. Supernatants were treated with ultraviolet light for 10 minutes prior to assay in order to sterilize them.Cells were pre-treated with varying concentrations of glyburide for 60 min prior to infection . Controls were treated with vehicle (RPMI 1640+Panexin 5%+DMSO 0.1%). The inoculum was prepared by culturing p-value for each group, but also estimates size of difference and permits interaction testing. Models were fit by the method of variance-weighted least-squares, because (unlike ANOVA) the method does not assume homoscedasticity . Responses were not assumed to vary linearly with time. Separate p-values were reported for each time point only when justified by a test for interaction. Results from bone marrow-derived macrophage experiments were analysed by repeated measures ANOVA. Graphs were plot in GraphPad Prism 5.0c for Mac OS X .Statistical analyses were performed on Stata 11 . Bacterial loads and cytokine concentrations were log-normal. Linear regression was used in preference to ANOVA because it reports not just a www.uniprot.org.IL1β (Swiss-Prot identifier P01584 human and P10749 murine), IL18 (Swiss-Prot ID Q14116 human and P70380 murine); gamma interferon (Swiss-Prot ID P01579 human and P01580 murine); caspase 1 (Swiss-Prot ID P29466 human and P29452 murine); PYCARD (Swiss-Prot ID Q9ULZ3 human and Q9EPB4 murine); NLRP3 (Swiss-Prot ID Q96P20 human and Q8R4B8 murine); Streptozocin (PubChem CID 29327), Glyburide/glibenclamide (PubChem CID 3488). Swiss-Prot online database may be found at p = 0.05, Welch t-test).In our murine model of diabetes blood glucose levels began to rise one week after the fifth streptozocin dose was given and reached a plateau after a further week  left. Lo2B. pseudomallei intranasally. So as to mimic the clinical situation more closely, all animals received ceftazidime treatment starting 24 h after inoculation  to determine bacterial loads in lungs and BALF (the primary site of the infection), liver, spleen and blood  of glyburide, which we found to be >1000 µM .The host response is altered by changes in glucose concentration. The primary action of glyburide in diabetes is to reduce blood glucose concentration by stimulating insulin secretion by the β-islet cells of the pancreas. Streptozocin treatment destroys the β-cells of the pancreas, making animals hyperglycemic and unresponsive to the action of glyburide. This allowed us to dissect out the separate effects of glyburide on glucose and on the inflammatory response. Blood glucose measurements taken at sacrifice confirmed that in our model of diabetes, blood glucose concentrations are indeed unaltered by glyburide treatment compared to vehicle-treated controls . Inflammasome inhibition would be expected to inhibit the secretion of mature IL18, and in turn reduce IFNγ secretion p = 0.17, results not shown). We also found no differences in IFNγ concentrations in lung or in blood +/+Lepr(db)/J mice), and found a defect in intracellular killing by peritoneal macrophages taken from diabetic animals compared to non-diabetic animals We have developed a murine model of diabetes and shown that it is more susceptible to melioidosis than wild-type animals, as evidenced by higher bacterial loads. This adds to the growing body of evidence that diabetes in vitro and in vivo reports We next presented evidence for an anti-inflammatory effect of glyburide in a diabetic mouse model of melioidosis and this anti-inflammatory effect appears specific to IL1β. This was supported by data from stimulated BMDMs, in which glyburide reduced IL1β secretion in a dose dependent fashion. This is consistent with the results of previous We hypothesized that the effect of glyburide was independent of its effect on glucose control. This is because in our previously reported clinical study A limitation of clinical studies of infection has always been that the interplay between the burden of infection and the inflammatory response has been difficult to tease out. Cytokine levels may be higher because bacterial burdens are higher, or bacterial burdens may be higher because of an ineffective innate host response We found that glyburide limits the cellular influx into the lungs, with effects on both neutrophil and monocyte numbers. Neutrophils are a crucial part of the host response to melioidosis B. pseudomallei is critically dependent on cellular immunity B. pseudomallei is a facultative intracellular pathogen that is capable of parasitizing macrophages B. pseudomallei is not known, but it is possible to speculate that the reduced influx of leukocytes into the lungs reduces the number of cells parasitized by the bacterium and therefore impairs the ability of B. pseudomallei to disseminate The host response to We found no effect on cytokines unrelated to the inflammasome. Specifically, there was no effects seen on CXCL5, IL6, IL10 or TNFα secretion, which is again consistent with previous reports in the literature et al. demonstrated that glyburide is able to inhibit assembly of the NLRP3 inflammasome in response to stimulation with lipopolysaccharide (LPS) and adenosine triphosphate (ATP) Salmonella enterica serovar Typhimurium was used as a stimulus, glyburide was not able to prevent inflammasome assembly, as measured by caspase 1 cleavage  The mechanism by which glyburide is able to inhibit IL1β secretion is not known. Lamkanfi Our IL1β assay does not distinguish between immature pro-IL1β and mature cleaved IL1β. The concentrations of IL1β we report in lung tissue therefore represent a combination of newly synthesised immature IL1β and mature IL1β. However, only mature IL1β is secreted into BALF and it therefore a marker of inflammasome assembly. In our model, glyburide-treatment reduced IL1β secretion in BALF, but did not block it completely. This means that there must have been some inflammasome assembly despite glyburide treatment. Immature IL18 is also cleaved by the inflammasome, so our finding that inflammasome assembly is not completely blocked by glyburide in melioidosis is supported by the fact that we observed a slight but non-significant reduction in IL18.B. pseudomallei infection B. pseudomallei lung infection et al. recently demonstrated that the IL1β response appeared to be harmful to the host We know that inflammasome-activation is necessary for the host response to et al. have presented evidence that glyburide may block the secretion of mature IL1β by an inflammasome-independent mechanism Leishmania parasites Our study does not exclude other mechanisms for the action of glyburide. Aside from an effect on the inflammasome, Hamon In conclusion, we have replicated our clinical finding that glyburide treatment exerts beneficial effects in melioidosis Figure S1Glyburide does not influence production of non-inflammasome cytokines in pulmonary B. pseudomallei infection. Note. BALF = bronchoalveolar lavage fluid; IL = interleukin; TNFα = tumor necrosis factor-alpha. Mice were treated with glyburide or vehicle for 7 days prior to inoculation with ∼6×102 cfu B. pseudomallei. All mice were treated with ceftazidime starting 24 h after inoculation until sacrifice . Error bars indicate standard deviations. A single p-value is reported for each cytokine unless there is evidence from a test of interaction that effects at each time point are different. A horizontal interrupted line marks the limit of detection for the assay. Glyburide did not influence the production of IL6, IL10, CXCL5 and TNFα in the systemic or pulmonary compartment.(EPS)Click here for additional data file.