39 research outputs found
Comparison of commercial kits to measure cytokine responses to Plasmodium falciparum by multiplex microsphere suspension array technology
<p>Abstract</p> <p>Background</p> <p>Multiplex cytokine profiling systems are useful tools for investigating correlates of protective immunity. Several Luminex and flow cytometry methods are commercially available but there is limited information on the relative performance of different kits. A series of comparison experiments were carried out to determine the most appropriate method for our subsequent studies.</p> <p>Methods</p> <p>Two Luminex methods were compared, the Bio-Rad human 17-plex panel and the Invitrogen (formerly BioSource) human cytokine 10-plex kit, and two flow cytometry methods, the Becton Dickinson Human Th1/Th2 Cytokine Kit (CBA) and the Bender MedSystems Human Th1/Th2 11plex FlowCytomix Multiplex Kit. All kits were tested for the measurement of cytokines in supernatants collected from human leukocytes stimulated with viable <it>Plasmodium falciparum </it>infected red blood cells (iRBC) or <it>P. falciparum </it>schizont lysates.</p> <p>Results</p> <p>Data indicated that the kits differed in sensitivity and reproducibility depending on the cytokine, and detected different quantities of some cytokines. The Bio-Rad 17-plex kit was able to detect more positive responses than the Invitrogen 10-plex kit. However, only when detecting IL-1, IL-6 or TNF did the two Luminex based methods correlate with one another. In this study, the flow cytometry based techniques were less variable and correlated better with one another. The two flow cytometry based kits showed significant correlation when detecting IFN-γ, IL-2, TNF, IL-10 and IL-6, but overall the BD kit detected more positive responses than the Bender MedSystems kit.</p> <p>Conclusions</p> <p>The microsphere suspension array technologies tested differed in reproducibility and the absolute quantity of cytokine detected. Sample volume, the number of cytokines measured, and the time and cost of the assays also differed. These data provide an accurate assessment of the four techniques, which will allow individual researchers to select the tool most suited for their study population.</p
A T Cell-inducing influenza vaccine for the elderly: safety and immunogenicity of MVA-NP+M1 in adults aged over 50 years
Current influenza vaccines have reduced immunogenicity and are of uncertain efficacy in older adults. We assessed the safety and immunogenicity of MVA-NP+M1, a viral-vectored influenza vaccine designed to boost memory T cell responses, in a group of older adults.Thirty volunteers (aged 50-85) received a single intramuscular injection of MVA-NP+M1 at a dose of 1·5×10(8) plaque forming units (pfu). Safety and immunogenicity were assessed over a period of one year. The frequency of T cells specific for nucleoprotein (NP) and matrix protein 1 (M1) was determined by interferon-gamma (IFN-γ) ELISpot, and their phenotypic and functional properties were characterized by polychromatic flow cytometry. In a subset of M1-specific CD8(+) T cells, T cell receptor (TCR) gene expression was evaluated using an unbiased molecular approach.Vaccination with MVA-NP+M1 was well tolerated. ELISpot responses were boosted significantly above baseline following vaccination. Increases were detected in both CD4(+) and CD8(+) T cell subsets. Clonality studies indicated that MVA-NP+M1 expanded pre-existing memory CD8(+) T cells, which displayed a predominant CD27(+)CD45RO(+)CD57(-)CCR7(-) phenotype both before and after vaccination.MVA-NP+M1 is safe and immunogenic in older adults. Unlike seasonal influenza vaccination, the immune responses generated by MVA-NP+M1 are similar between younger and older individuals. A T cell-inducing vaccine such as MVA-NP+M1 may therefore provide a way to circumvent the immunosenescence that impairs routine influenza vaccination.ClinicalTrials.gov NCT00942071
Preliminary Assessment of the Efficacy of a T-Cell–Based Influenza Vaccine, MVA-NP+M1, in Humans
A single vaccination with MVA-NP+M1 boosts T-cell responses to conserved influenza antigens in humans. Protection against influenza disease and virus shedding was demonstrated in an influenza virus challenge study
MIG and the Regulatory Cytokines IL-10 and TGF-β1 Correlate with Malaria Vaccine Immunogenicity and Efficacy
Malaria remains one of the world's greatest killers and a vaccine is urgently required. There are no established correlates of protection against malaria either for natural immunity to the disease or for immunity conferred by candidate malaria vaccines. The RTS,S/AS02A vaccine offers significant partial efficacy against malaria
High production of pro-inflammatory cytokines by maternal blood mononuclear cells is associated with reduced maternal malaria but increased cord blood infection
BACKGROUND: Increased susceptibility to malaria during pregnancy
is not completely understood. Cellular immune responses mediate
both pathology and immunity but the effector responses involved
in these processes have not been fully characterized. Maternal
and fetal cytokine and chemokine responses to malaria at
delivery, and their association with pregnancy and childhood
outcomes, were investigated in 174 samples from a mother and
child cohort from Mozambique. Peripheral and cord mononuclear
cells were stimulated with Plasmodium falciparum lysate and
secretion of IL-12p70, IFN-gamma, IL-2, IL-10, IL-8, IL-6, IL-4,
IL-5, IL-1beta, TNF, TNF-beta was quantified in culture
supernatants by multiplex flow cytometry while cellular mRNA
expression of IFN-gamma, TNF, IL-2, IL-4, IL-6, IL-10 and IL-13
was measured by quantitative PCR. RESULTS: Higher concentrations
of IL-6 and IL-1beta were associated with a reduced risk of P.
falciparum infection in pregnant women (p < 0.049).
Pro-inflammatory cytokines IL-6, IL-1beta and TNF strongly
correlated among themselves (rho > 0.5, p < 0.001). Higher
production of IL-1beta was significantly associated with
congenital malaria (p < 0.046) and excessive TNF was
associated with peripheral infection and placental lesions (p
< 0.044). CONCLUSIONS: Complex network of immuno-pathological
cytokine mechanisms in the placental and utero environments
showed a potential trade-off between positive and negative
effects on mother and newborn susceptibility to infection
IL-23 signaling in Th17 cells is inhibited by HIV infection and is not restored by HAART: Implications for persistent immune activation.
HIV infection causes a profound depletion of gut derived Th17 cells, contributing to loss of mucosal barrier function and an increase in microbial translocation, thus driving systemic immune activation. Despite normalization of circulating CD4+ T cell counts with highly active antiretroviral therapy (HAART), Th17 frequency and function often remain impaired. Given the importance of interleukin (IL)-23 in the generation and stabilization of Th17 cells we hypothesized that impaired IL-23 signaling causes persistent Th17 dysfunction in HIV infection.The effects of in vitro HIV infection on responses to IL-23 in Th17 cells were examined. These included the production of IL-17, phosphorylated STAT3 (pSTAT3) and the transcription of retinoic acid orphan receptor C (RORC) gene. Blood derived Th17 cells from untreated and HAART-treated HIV-infected individuals were also examined for the IL-23 induced production of phosphorylated STAT3 (pSTAT3) and the expression of the IL-23 receptors.In vitro HIV infection significantly inhibited IL-17 production and IL-23 induced pSTAT3 while expression of RORC RNA was unaffected. Th17 cells isolated from untreated and HAART-treated HIV-infected individuals showed complete loss of IL-23 induced pSTAT3 without a decrease in the expression of the IL-23 receptors.This study is the first to demonstrate an effect of HIV on the IL-23 signaling pathway in Th17 cells. We show that in vitro and in vivo HIV infection results in impaired IL-23 signaling which is not reversed by HAART nor is it a result of reduced receptor expression, suggesting that HIV interferes with IL-23-activated signaling pathways. These findings may explain the inability of HAART to restore Th17 frequency and function and the resulting persistent chronic immune activation observed in HIV infected individuals
Clinical characteristics of HIV-infected study subjects.
<p>Clinical characteristics of HIV-infected study subjects.</p
HIV infection does not downregulate IL-23R expression.
<p>Circulating Th17 cells were isolated from HIV-seronegative or untreated HIV-infected individuals and expression of IL-23R was assessed by flow cytometry. <b>(A)</b> Representative histograms demonstrating IL-23R expression on circulating Th17 cells isolated from HIV-seronegative controls, HIV-infected untreated, HIV-infected HAART treated individuals and the matched IL-23R isotype control. <b>(B)</b> Summary of % cells expressing IL-23R on circulating Th17 cells from HIV seronegative and untreated HIV-infected patients. <b>(C)</b> Western blot demonstrating expression of IL-12Rβ1 on blood Th17 cell lysates from HIV-seronegative and HIV infected, untreated donors. Figure is representative of 3 out of 6 donors tested.</p
HIV reduces IL-17 secretion and intracellular expression in blood-derived and <i>in vitro</i>-generated Th17 cells.
<p>Blood-derived and <i>in vitro</i> generated Th17 cells were infected with HIV for 24 hours. <b>(A)</b> Th17 cells were cultured for 3 days with anti-CD3 and anti-CD28 mAbs. Supernatants were harvested and assayed for secreted IL-17 by ELISA; * p < 0.001, n = 9; ** p = 0.045, n = 9. Data shown are mean ± SEM. <b>(B)</b> Representative histograms showing intracellular expression of IL-17 in Th17 cells stimulated for 6 hours with PMA and onomycin in the presence of brefeldin A. (<b>C)</b> Summary of the proportion of blood-derived and <i>in vitro</i> generated Th17 cells expressing IL-17 following PMA and ionomycin stimulation is shown with mean frequency indicated; + p = 0.026, n = 9, ++ p = 0.012, n = 5.</p