Effects of antimicrobial peptides on membrane dynamics: A comparison of fluorescence and NMR experiments

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight resistant infections. They are commonly thought to kill bacteria by perturbing the permeability of their cell membranes. However, bacterial killing requires a high coverage of the cell surface by bound peptides, at least in the case of cationic and amphipathic AMPs. Therefore, it is conceivable that peptide accumulation on the bacterial membranes might interfere with vital cellular functions also by perturbing bilayer dynamics, a hypothesis that has been termed "sand in the gearbox". Here we performed a systematic study of such possible effects, for two representative peptides (the cationic cathelicidin PMAP-23 and the peptaibol alamethicin), employing fluorescence and NMR spectroscopies. These approaches are commonly applied to characterize lipid order and dynamics, but sample different time-scales and could thus report on different membrane properties. In our case, fluorescence anisotropy measurements on liposomes labelled with probes localized at different depths in the bilayer showed that both peptides perturb membrane fluidity and order. Pyrene excimer-formation experiments showed a peptideinduced reduction in lipid lateral mobility. Finally, laurdan fluorescence indicated that peptide binding reduces water penetration below the headgroups region. Comparable effects were observed also in fluorescence experiments performed directly on live bacterial cells. By contrast, the fatty acyl chain order parameters detected by deuterium NMR spectroscopy remained virtually unaffected by addition of the peptides. The apparent discrepancy between the two techniques confirms previous sporadic observations and is discussed in terms of the different characteristic times of the two approaches. The perturbation of membrane dynamics in the ns timescale, indicated by the multiple fluorescence approaches reported here, could contribute to the antimicrobial activity of AMPs, by affecting the function of membrane proteins, which is strongly dependent on the physicochemical properties of the bilayer

A large ongoing outbreak of hepatitis A predominantly affecting young males in Lazio, Italy; August 2016 - March 2017

The hepatitis A virus (HAV) is mainly transmitted through the faecal-oral route. In industrialized countries HAV infection generally occurs as either sporadic cases in travelers from endemic areas, local outbreak within closed/semi-closed population and as foodborne community outbreak. Recently, an increasing number of HAV infection clusters have been reported among young men-who-have-sex-with-men (MSM). The Lazio Regional Service for the epidemiology and control for infectious diseases (SeRESMI) has noticed an increase of acute hepatitis A (AHA) since September 2016. Temporal analysis carried out with a discrete Poisson model using surveillance data between January 2016 and March 2017 evidenced an ongoing outbreak of AHA that started at the end of August. Molecular investigation carried out on 130 out of 513 cases AHA reported until March 2017 suggests that this outbreak is mainly supported by an HAV variant which is currently spreading within MSM communities across Europe (VRD_521_2016). The report confirms that AHA is an emerging issue among MSM. In addition through the integration of standard (case based) surveillance with molecular investigation we could discriminate, temporally concomitant but epidemiologically unrelated, clusters due to different HAV variants. As suggested by the WHO, in countries with low HAV circulation, vaccination programmes should be tailored on the local epidemiological patterns to prevent outbreaks among high risk groups and eventual spillover of the infection in the general population

A large ongoing outbreak of hepatitis A predominantly affecting young males in Lazio, Italy; August 2016 - March 2017

The hepatitis A virus (HAV) is mainly transmitted through the faecal-oral route. In industrialized countries HAV infection generally occurs as either sporadic cases in travelers from endemic areas, local outbreak within closed/semi-closed population and as foodborne community outbreak. Recently, an increasing number of HAV infection clusters have been reported among young men-who-have-sex-with-men (MSM). The Lazio Regional Service for the epidemiology and control for infectious diseases (SeRESMI) has noticed an increase of acute hepatitis A (AHA) since September 2016. Temporal analysis carried out with a discrete Poisson model using surveillance data between January 2016 and March 2017 evidenced an ongoing outbreak of AHA that started at the end of August. Molecular investigation carried out on 130 out of 513 cases AHA reported until March 2017 suggests that this outbreak is mainly supported by an HAV variant which is currently spreading within MSM communities across Europe (VRD_521_2016). The report confirms that AHA is an emerging issue among MSM. In addition through the integration of standard (case based) surveillance with molecular investigation we could discriminate, temporally concomitant but epidemiologically unrelated, clusters due to different HAV variants. As suggested by the WHO, in countries with low HAV circulation, vaccination programmes should be tailored on the local epidemiological patterns to prevent outbreaks among high risk groups and eventual spillover of the infection in the general population

Precision measurement of the η→π+π−π0\eta\to\pi^+\pi^-\pi^0 Dalitz plot distribution with the KLOE detector

Using $1.6$ fb$^{-1}$ of $e^+ e^-\to\phi\to\eta\gamma$ data collected with the KLOE detector at DA$\Phi$NE, the Dalitz plot distribution for the $\eta \to \pi^+ \pi^- \pi^0$ decay is studied with the world's largest sample of $\sim 4.7 \cdot 10^6$ events. The Dalitz plot density is parametrized as a polynomial expansion up to cubic terms in the normalized dimensionless variables $X$ and $Y$. The experiment is sensitive to all charge conjugation conserving terms of the expansion, including a $gX^2Y$ term. The statistical uncertainty of all parameters is improved by a factor two with respect to earlier measurements.Comment: 11 pages, 9 figures, supplement: an ascii tabl

Transduction of SIV-Specific TCR Genes into Rhesus Macaque CD8+ T Cells Conveys the Ability to Suppress SIV Replication

The SIV/rhesus macaque model for HIV/AIDS is a powerful system for examining the contribution of T cells in the control of AIDS viruses. To better our understanding of CD8(+) T-cell control of SIV replication in CD4(+) T cells, we asked whether TCRs isolated from rhesus macaque CD8(+) T-cell clones that exhibited varying abilities to suppress SIV replication could convey their suppressive properties to CD8(+) T cells obtained from an uninfected/unvaccinated animal.We transferred SIV-specific TCR genes isolated from rhesus macaque CD8(+) T-cell clones with varying abilities to suppress SIV replication in vitro into CD8(+) T cells obtained from an uninfected animal by retroviral transduction. After sorting and expansion, transduced CD8(+) T-cell lines were obtained that specifically bound their cognate SIV tetramer. These cell lines displayed appropriate effector function and specificity, expressing intracellular IFNγ upon peptide stimulation. Importantly, the SIV suppression properties of the transduced cell lines mirrored those of the original TCR donor clones: cell lines expressing TCRs transferred from highly suppressive clones effectively reduced wild-type SIV replication, while expression of a non-suppressing TCR failed to reduce the spread of virus. However, all TCRs were able to suppress the replication of an SIV mutant that did not downregulate MHC-I, recapitulating the properties of their donor clones.Our results show that antigen-specific SIV suppression can be transferred between allogenic T cells simply by TCR gene transfer. This advance provides a platform for examining the contributions of TCRs versus the intrinsic effector characteristics of T-cell clones in virus suppression. Additionally, this approach can be applied to develop non-human primate models to evaluate adoptive T-cell transfer therapy for AIDS and other diseases

GagCM9-Specific CD8+ T Cells Expressing Limited Public TCR Clonotypes Do Not Suppress SIV Replication In Vivo

Several lines of evidence suggest that HIV/SIV-specific CD8+ T cells play a critical role in the control of viral replication. Recently we observed high levels of viremia in Indian rhesus macaques vaccinated with a segment of SIVmac239 Gag (Gag45–269) that were subsequently infected with SIVsmE660. These seven Mamu-A*01+ animals developed CD8+ T cell responses against an immunodominant epitope in Gag, GagCM9, yet failed to control virus replication. We carried out a series of immunological and virological assays to understand why these Gag-specific CD8+ T cells could not control virus replication in vivo. GagCM9-specific CD8+ T cells from all of the animals were multifunctional and were found in the colonic mucosa. Additionally, GagCM9-specific CD8+ T cells accessed B cell follicles, the primary residence of SIV-infected cells in lymph nodes, with effector to target ratios between 20–250 GagCM9-specific CD8+ T cells per SIV-producing cell. Interestingly, vaccinated animals had few public TCR clonotypes within the GagCM9-specific CD8+ T cell population pre- and post-infection. The number of public TCR clonotypes expressed by GagCM9-specific CD8+ T cells post-infection significantly inversely correlated with chronic phase viral load. It is possible that these seven animals failed to control viral replication because of the narrow TCR repertoire expressed by the GagCM9-specific CD8+ T cell population elicited by vaccination and infection

Trivalent Adenovirus Type 5 HIV Recombinant Vaccine Primes for Modest Cytotoxic Capacity That Is Greatest in Humans with Protective HLA Class I Alleles

If future HIV vaccine design strategies are to succeed, improved understanding of the mechanisms underlying protection from infection or immune control over HIV replication remains essential. Increased cytotoxic capacity of HIV-specific CD8+ T-cells associated with efficient elimination of HIV-infected CD4+ T-cell targets has been shown to distinguish long-term nonprogressors (LTNP), patients with durable control over HIV replication, from those experiencing progressive disease. Here, measurements of granzyme B target cell activity and HIV-1-infected CD4+ T-cell elimination were applied for the first time to identify antiviral activities in recipients of a replication incompetent adenovirus serotype 5 (Ad5) HIV-1 recombinant vaccine and were compared with HIV-negative individuals and chronically infected patients, including a group of LTNP. We observed readily detectable HIV-specific CD8+ T-cell recall cytotoxic responses in vaccinees at a median of 331 days following the last immunization. The magnitude of these responses was not related to the number of vaccinations, nor did it correlate with the percentages of cytokine-secreting T-cells determined by ICS assays. Although the recall cytotoxic capacity of the CD8+ T-cells of the vaccinee group was significantly less than that of LTNP and overlapped with that of progressors, we observed significantly higher cytotoxic responses in vaccine recipients carrying the HLA class I alleles B*27, B*57 or B*58, which have been associated with immune control over HIV replication in chronic infection. These findings suggest protective HLA class I alleles might lead to better outcomes in both chronic infection and following immunization due to more efficient priming of HIV-specific CD8+ T-cell cytotoxic responses

Long-Term Programming of Antigen-Specific Immunity from Gene Expression Signatures in the PBMC of Rhesus Macaques Immunized with an SIV DNA Vaccine

While HIV-1-specific cellular immunity is thought to be critical for the suppression of viral replication, the correlates of protection have not yet been determined. Rhesus macaques (RM) are an important animal model for the study and development of vaccines against HIV/AIDS. Our laboratory has helped to develop and study DNA-based vaccines in which recent technological advances, including genetic optimization and in vivo electroporation (EP), have helped to dramatically boost their immunogenicity. In this study, RMs were immunized with a DNA vaccine including individual plasmids encoding SIV gag, env, and pol alone, or in combination with a molecular adjuvant, plasmid DNA expressing the chemokine ligand 5 (RANTES), followed by EP. Along with standard immunological assays, flow-based activation analysis without ex vivo restimulation and high-throughput gene expression analysis was performed. Strong cellular immunity was induced by vaccination which was supported by all assays including PBMC microarray analysis that identified the up-regulation of 563 gene sequences including those involved in interferon signaling. Furthermore, 699 gene sequences were differentially regulated in these groups at peak viremia following SIVmac251 challenge. We observed that the RANTES-adjuvanted animals were significantly better at suppressing viral replication during chronic infection and exhibited a distinct pattern of gene expression which included immune cell-trafficking and cell cycle genes. Furthermore, a greater percentage of vaccine-induced central memory CD8+ T-cells capable of an activated phenotype were detected in these animals as measured by activation analysis. Thus, co-immunization with the RANTES molecular adjuvant followed by EP led to the generation of cellular immunity that was transcriptionally distinct and had a greater protective efficacy than its DNA alone counterpart. Furthermore, activation analysis and high-throughput gene expression data may provide better insight into mechanisms of viral control than may be observed using standard immunological assays

Variability of Bio-Clinical Parameters in Chinese-Origin Rhesus Macaques Infected with Simian Immunodeficiency Virus: A Nonhuman Primate AIDS Model

BACKGROUND: Although Chinese-origin Rhesus macaques (Ch RhMs) infected with simian immunodeficiency virus (SIV) have been used for many years to evaluate the efficacy of AIDS vaccines and therapeutics, the bio-clinical variability of such a nonhuman primate AIDS model was so far not established. METHODOLOGY/PRINCIPAL FINDINGS: By randomizing 150 (78 male and 72 female) Ch RhMs with diverse MHC class I alleles into 3 groups (50 animals per group) challenged with intrarectal (i.r.) SIVmac239, intravenous (i.v.) SIVmac239, or i.v. SIVmac251, we evaluated variability in bio-clinical endpoints for 118 weeks. All SIV-challenged Ch RhMs became seropositive for SIV during 1-2 weeks. Plasma viral load (VL) peaked at weeks 1-2 and then declined to set-point levels as from week 5. The set-point VL was 30 fold higher in SIVmac239 (i.r. or i.v.)-infected than in SIVmac251 (i.v.)-infected animals. This difference in plasma VL increased overtime (>100 fold as from week 68). The rates of progression to AIDS or death were more rapid in SIVmac239 (i.r. or i.v.)-infected than in SIVmac251 (i.v.)-infected animals. No significant difference in bio-clinical endpoints was observed in animals challenged with i.r. or i.v. SIVmac239. The variability (standard deviation) in peak/set-point VL was nearly one-half lower in animals infected with SIVmac239 (i.r. or i.v.) than in those infected with SIVmac251 (i.v.), allowing that the same treatment-related difference can be detected with one-half fewer animals using SIVmac239 than using SIVmac251. CONCLUSION/SIGNIFICANCE: These results provide solid estimates of variability in bio-clinical endpoints needed when designing studies using the Ch RhM SIV model and contribute to the improving quality and standardization of preclinical studies

Mathematical model insights into arsenic detoxification

<p>Abstract</p> <p>Background</p> <p>Arsenic in drinking water, a major health hazard to millions of people in South and East Asia and in other parts of the world, is ingested primarily as trivalent inorganic arsenic (iAs), which then undergoes hepatic methylation to methylarsonic acid (MMAs) and a second methylation to dimethylarsinic acid (DMAs). Although MMAs and DMAs are also known to be toxic, DMAs is more easily excreted in the urine and therefore methylation has generally been considered a detoxification pathway. A collaborative modeling project between epidemiologists, biologists, and mathematicians has the purpose of explaining existing data on methylation in human studies in Bangladesh and also testing, by mathematical modeling, effects of nutritional supplements that could increase As methylation.</p> <p>Methods</p> <p>We develop a whole body mathematical model of arsenic metabolism including arsenic absorption, storage, methylation, and excretion. The parameters for arsenic methylation in the liver were taken from the biochemical literature. The transport parameters between compartments are largely unknown, so we adjust them so that the model accurately predicts the urine excretion rates of time for the iAs, MMAs, and DMAs in single dose experiments on human subjects.</p> <p>Results</p> <p>We test the model by showing that, with no changes in parameters, it predicts accurately the time courses of urinary excretion in mutiple dose experiments conducted on human subjects. Our main purpose is to use the model to study and interpret the data on the effects of folate supplementation on arsenic methylation and excretion in clinical trials in Bangladesh. Folate supplementation of folate-deficient individuals resulted in a 14% decrease in arsenicals in the blood. This is confirmed by the model and the model predicts that arsenicals in the liver will decrease by 19% and arsenicals in other body stores by 26% in these same individuals. In addition, the model predicts that arsenic methyltransferase has been upregulated by a factor of two in this population. Finally, we also show that a modification of the model gives excellent fits to the data on arsenic metabolism in human cultured hepatocytes.</p> <p>Conclusions</p> <p>The analysis of the Bangladesh data using the model suggests that folate supplementation may be more effective at reducing whole body arsenic than previously expected. There is almost no data on the upregulation of arsenic methyltransferase in populations chronically exposed to arsenic. Our model predicts upregulation by a factor of two in the Bangladesh population studied. This prediction should be verified since it could have important public health consequences both for treatment strategies and for setting appropriate limits on arsenic in drinking water. Our model has compartments for the binding of arsenicals to proteins inside of cells and we show that these comparments are necessary to obtain good fits to data. Protein-binding of arsenicals should be explored in future biochemical studies.</p