Kikki Bodman-Smith

Increased pathogenicity of European porcine reproductive and respiratory syndrome virus is associated with enhanced adaptive responses and viral clearance

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases of swine worldwide. Since its first emergence in 1987 the PRRS virus (PRRSV) has become particularly divergent with highly pathogenic strains appearing in both Europe and Asia. However, the underlying mechanisms of PRRSV pathogenesis are still unclear. This study sets out to determine the differences in pathogenesis between subtype 1 and 3 strains of European PRRSV (PRRSV-I), and compare the immune responses mounted against these strains. Piglets were infected with 3 strains of PRRSV-I: Lelystad virus, 215-06 a British field strain and SU1-bel from Belarus. Post-mortem examinations were performed at 3 and 7 days post-infection (dpi), and half of the remaining animals in each group were inoculated with an Aujeszkys disease (ADV) vaccine to investigate possible immune suppression resulting from PRRSV infection. The subtype 3 SU1-bel strain displayed greater clinical signs and lung gross pathology scores compared with the subtype 1 strains. This difference did not appear to be caused by higher virus replication, as viraemia and viral load in broncho-alveolar lavage fluid (BALF) were lower in the SU1-bel group. Infection with SU1-bel induced an enhanced adaptive immune response with greater interferon (IFN)-γ responses and an earlier PRRSV-specific antibody response. Infection with PRRSV did not affect the response to vaccination against ADV. Our results indicate that the increased clinical and pathological effect of the SU1-bel strain is more likely to be caused by an enhanced inflammatory immune response rather than higher levels of virus replication.

Anti-heat shock protein-27 (Hsp-27) antibody levels in patients with chest pain : Association with established cardiovascular risk factors

BACKGROUND Antibody titres to several heat shock proteins (Hsps) have been shown to be associated with risk factors for cardiovascular disease (CVD), but there are no data for Hsp-27. We developed an ELISA for total IgG antibody concentrations, applying this to individuals with and without acute coronary syndrome, and have assessed the relationship between antibody levels and individual coronary risk factors. METHODS Blood was collected from 63 healthy controls without a history of chest pain or CVD and 60 patients admitted to hospital with acute cardiac chest pain on admission and approximately 12 h after the acute event. RESULTS Patients with chest pain had significantly higher Hsp-27 antibody levels than controls [median 0.16 (range 0.01-0.51) vs. 0.10 (range 0.00-0.32); p<0.001]. Furthermore, Hsp-27 antibody concentrations showed strong associations with age and hypertension (Standardised beta coefficient=0.343, p<0.001 and = -0.235, p<0.016, respectively), but not with other established cardiovascular risk factors. Logistic regression analysis showed age and diabetes were significant predictors of risk of CVD with OR 1.29 (95% CI 1.16 to 1.42, p=0.001) and 25.9 (95% CI 2.14>312, p=0.01) respectively. CONCLUSIONS Raised antibody levels to Hsp-27 were associated only with age and hypertension.

Daucus carota pentane-based fractions arrest the cell cycle and increase apoptosis in MDA-MB-231 breast cancer cells

BackgroundDaucus carota L.ssp.carota (wild carrot), an herb used in folk medicine worldwide, was recently demonstrated to exhibit anticancer activity. In this study we examined the anticancer effect of Daucus carota oil extract (DCOE) fractions on the human breast adenocarcinoma cell lines MDA-MB-231 and MCF-7 and clarified the mechanism of action.Methods and resultsUsing the WST assay, the pentane fraction (F1) and 1:1 pentane:diethyl ether fraction (F2) were shown to possess the highest cytotoxicity against both cell lines. Flow cytometric analysis revealed that both fractions induced the accumulation of cells in the sub-G1 phase, increase in apoptotic cell death and chromatin condensation. The increase in apoptosis in response to treatment was also apparent in the increase in BAX and the decrease in Bcl-2 levels as well as the proteolytic cleavage of both caspase-3 and PARP as revealed by Western blot. Furthermore, treatment of MDA-MB-231 cells with either fraction significantly reduced the level of phosphorylated Erk but did not show any effect on phosphorylated Akt. The combined treatment with a potent PI3K inhibitor (wortmannin) and F1 or F2 fraction had a synergistic inhibitory effect on cell survival which shows that these two drugs work on different pathways.ConclusionsThese results suggest that the pentane-based fractions of DCOE possess potential anti-cancer activity that is mainly mediated through the Erk pathway.

Assessment of the Phenotype and Functionality of Porcine CD8 T Cell Responses following Vaccination with Live Attenuated Classical Swine Fever Virus (CSFV) and Virulent CSFV Challenge

ABSTRACT Vaccination with live attenuated classical swine fever virus (CSFV) induces solid protection after only 5 days, which has been associated with virus-specific T cell gamma interferon (IFN-γ) responses. In this study, we employed flow cytometry to characterize T cell responses following vaccination and subsequent challenge infections with virulent CSFV. The CD3+ CD4− CD8hi T cell population was the first and major source of CSFV-specific IFN-γ. A proportion of these cells showed evidence for cytotoxicity, as evidenced by CD107a mobilization, and coexpressed tumor necrosis factor alpha (TNF-α). To assess the durability and recall of these responses, a second experiment was conducted where vaccinated animals were challenged with virulent CSFV after 5 days and again after a further 28 days. While virus-specific CD4 T cell (CD3+ CD4+ CD8α+) responses were detected, the dominant response was again from the CD8 T cell population, with the highest numbers of these cells being detected 14 and 7 days after the primary and secondary challenges, respectively. These CD8 T cells were further characterized as CD44hi CD62L− and expressed variable levels of CD25 and CD27, indicative of a mixed effector and effector memory phenotype. The majority of virus-specific IFN-γ+ CD8 T cells isolated at the peaks of the response after each challenge displayed CD107a on their surface, and subpopulations that coexpressed TNF-α and interleukin 2 (IL-2) were identified. While it is hoped that these data will aid the rational design and/or evaluation of next-generation marker CSFV vaccines, the novel flow cytometric panels developed should also be of value in the study of porcine T cell responses to other pathogens/vaccines.

Interleukin-10 reduces hyperalgesia and the level of interleukin-1β in BALB/c mice infected with Leishmania major with no major effect on the level of interleukin-6

Infection with a high dose of Leishmania major has been shown to induce hyperalgesia in BALB/c mice accompanied by a sustained upregulation of Interleukin-1beta (IL-1beta) and an early upregulation of Interleukin-6 (IL-6). On the other hand, Interleukin 10 (IL-10) has been demonstrated to be hypoalgesic in other models such as rats exposed to UV rays. In this study, we injected BALB/c mice with a high dose of Leishmania major and treated them with IL-10 (15 ng/animal) for six consecutive days. Hyperalgesia was assessed using thermal pain tests and the levels of IL-1beta and IL-6 were also assessed at different post-infection days. Our results show that IL-10 can reduce the Leishmania major-induced hyperalgesia during the treatment period through a direct effect on the levels of IL-1beta which seems to play an important role in this hyperalgesia induction since its level was reduced during the period of IL-10 injection and was increased again when this treatment was stopped. On the contrary IL-10 has no direct effect on the levels IL-6 which seems to have no direct role in the induced hyperalgesia.

Daucus carota Pentane-Based Fractions Suppress Proliferation and Induce Apoptosis in Human Colon Adenocarcinoma HT-29 Cells by Inhibiting the MAPK and PI3K Pathways

Daucus carota L. ssp. carota (Apiacea, wild carrot, Queen Annes lace) has been used in folk medicine throughout the world and recently was shown to possess anticancer and antioxidant activities. This study aims to determine the anticancer activity of the pentane fraction (F1) and the 1:1 pentane:diethyl ether fraction (F2) of the Daucus Carota oil extract (DCOE) against human colon adenocarcinoma cell lines (HT-29 and Caco-2). Treatment of cells with various concentrations of F1 or F2 fractions produced a dose-dependent inhibition of cell proliferation. Flow cytometric analysis indicated that both fractions induced sub-G1 phase accumulation and increased apoptotic cell death. Western blot revealed the activation of caspase-3, PARP cleavage, and a considerable increase in Bax and p53 levels, and a decrease in Bcl-2 level. Treatment of HT-29 cells with either fraction markedly decreased the levels of both phosphorylated Erk and Akt. Furthermore, the combined treatment of F1 or F2 with wortmannin showed no added inhibition of cell survival suggesting an effect of F1 or F2 through the phosphatidyl inositol 3-kinase (PI3K) pathway. This study proposes that DCOE fractions (F1 and F2) inhibit cell proliferation by inducing cell cycle arrest and apoptosis in HT-29 cells through the suppression of mitogen-activated protein kinase (MAPK)/Erk and PI3K/Akt pathways.

Proteome-wide screening reveals immunodominance in the CD8 T cell response against classical swine fever virus with antigen-specificity dependent on MHC class I haplotype expression.

Vaccination with live attenuated classical swine fever virus (CSFV) vaccines induces a rapid onset of protection which has been associated with virus-specific CD8 T cell IFN-γ responses. In this study, we assessed the specificity of this response, by screening a peptide library spanning the CSFV C-strain vaccine polyprotein to identify and characterise CD8 T cell epitopes. Synthetic peptides were pooled to represent each of the 12 CSFV proteins and used to stimulate PBMC from four pigs rendered immune to CSFV by C-strain vaccination and subsequently challenged with the virulent Brescia strain. Significant IFN-γ expression by CD8 T cells, assessed by flow cytometry, was induced by peptide pools representing the core, E2, NS2, NS3 and NS5A proteins. Dissection of these antigenic peptide pools indicated that, in each instance, a single discrete antigenic peptide or pair of overlapping peptides was responsible for the IFN-γ induction. Screening and titration of antigenic peptides or truncated derivatives identified the following antigenic regions: core241–255 PESRKKLEKALLAWA and NS31902–1912 VEYSFIFLDEY, or minimal length antigenic peptides: E2996–1003 YEPRDSYF, NS21223–1230 STVTGIFL and NS5A3070–3078 RVDNALLKF. The epitopes are highly conserved across CSFV strains and variable sequence divergence was observed with related pestiviruses. Characterisation of epitope-specific CD8 T cells revealed evidence of cytotoxicity, as determined by CD107a mobilisation, and a significant proportion expressed TNF-α in addition to IFN-γ. Finally, the variability in the antigen-specificity of these immunodominant CD8 T cell responses was confirmed to be associated with expression of distinct MHC class I haplotypes. Moreover, recognition of NS21223–1230 STVTGIFL and NS31902–1912 VEYSFIFLDEY by a larger group of C-strain vaccinated animals showed that these peptides could be restricted by additional haplotypes. Thus the antigenic regions and epitopes identified represent attractive targets for evaluation of their vaccine potential against CSFV.

In Leishmania major-induced inflammation, interleukin-13 reduces hyperalgesia, down-regulates IL-1β and up-regulates IL-6 in an IL-4 independent mechanism.

Infection with high dose Leishmania major induces a sustained hyperalgesia in BALB/c mice while low dose induces a short lived hyperalgesia both accompanied with the upregulation of IL-1β and IL-6. Although IL-13 was shown to reduce the high dose L. major hyperalgesia during the treatment period, this effect was accompanied by a significant decrease in the levels of IL-1β and a significant increase in the levels of IL-6 in the paws of mice even beyond this period. Those results suggest that IL-13 exerts those effects via the induction of another mediator, IL-4 being a potential candidate due to its known hypoalgesic effects in other models and to its close functional closeness to IL-13 especially at the level of receptors. In this study we correlated the pain thresholds and the levels of IL-1β, IL-6 and IL-4 with the period of IL-13 treatment and beyond it in mice infected with high and low dose of L. major. The results of both models show that IL-1β plays no direct role in provoking the observed hyperalgesia after stopping the treatment with IL-13 which is in contrary to IL-6 which might be a key player after the treatment period. Furthermore we demonstrate that there is no correlation between the levels of IL-4, hyperalgesia, the decreased IL-1β levels and the increased levels of IL-6 in the paws of IL-13 treated and L. major (high and low dose) infected BALB/c mice.

Antioxidant and hepatoprotective activities of the oil fractions from wild carrot (Daucus carota ssp. carota)

Abstract Context: Wild carrot, Daucus carota L. ssp. carota (Apiacae), is widely distributed throughout the world and has various uses in traditional medicine in Lebanon. Objective: The present study aimed to fractionate and analyze the chemical composition of the Daucus carota oil extract (DCOE) fractions and to evaluate their antioxidant and hepatoprotective properties in vitro and in vivo. Materials and methods: DCOE was chromatographed on silica gel column to produce four fractions: pentane (F1), 50:50 pentane:diethyl ether (F2), diethyl ether (F3), and 93:7 chloroform: methanol (F4). Qualitative and quantitative analyses of oil fractions were performed by GC-MS and HPLC techniques. The in vitro antioxidant properties were assessed using DPPH, FIC, and ferric-reducing antioxidant power (FRAP) assays. The hepatoprotective property was determined by examining the levels of serum markers (alanine transaminase (ALT) and aspartate transaminase (AST)) and hepatic antioxidant (superoxide dismutase (SOD), catalase (CAT), and glutathione-S-transferase (GST)) enzymes in CCl4-intoxicated mice pretreated with intraperitoenal 50, 100, or 200 mg/kg b.w. of the oil fractions for 5 d. Results: GCMS analysis of F2 revealed the presence of 2-himachalen-6-ol (61.4%) which is reported for the first time in Daucus carota species. F3 and F4 were rich in phenolics and flavonoids and demonstrated significant DPPH activity (IC50 = 0.29 and 0.38 mg/ml, respectively) and high FRAP values (225.11 and 437.59 µmol FeSO4/g, respectively). The sesquiterpene-rich fraction F1 had the highest FIC ability (IC50 = 0.28 mg/ml). Pretreatment with F1 and F4 reversed the CCl4-induced decrease in SOD, CAT, and GST levels and reduced significantly hepatic damage. Discussion and conclusion: The current results suggested that wild carrot oil fractions exhibited a unique chemical composition and possessed significant antioxidant activities as well as hepatoprotective effects against CCl4-induced hepatotoxicity.

Partial Activation of Natural Killer and γδ T Cells by Classical Swine Fever Viruses Is Associated with Type I Interferon Elicited from Plasmacytoid Dendritic Cells

ABSTRACT Vaccination with live attenuated classical swine fever virus (CSFV) vaccines can rapidly confer protection in the absence of neutralizing antibodies. With an aim of providing information on the cellular mechanisms that may mediate this protection, we explored the interaction of porcine natural killer (NK) cells and γδ T cells with CSFV. Both NK and γδ T cells were refractory to infection with attenuated or virulent CSFV, and no stimulatory effects, as assessed by the expression of major histocompatibility complex (MHC) class II (MHC-II), perforin, and gamma interferon (IFN-γ), were observed when the cells were cultured in the presence of CSFV. Coculture with CSFV and myeloid dendritic cells (mDCs) or plasmacytoid dendritic cells (pDCs) showed that pDCs led to a partial activation of both NK and γδ T cells, with upregulation of MHC-II being observed. An analysis of cytokine expression by infected DC subsets suggested that this effect was due to IFN-α secreted by infected pDCs. These results were supported by ex vivo analyses of NK and γδ T cells in the tonsils and retropharyngeal lymph nodes from pigs that had been vaccinated with live attenuated CSFV and/or virulent CSFV. At 5 days postchallenge, there was evidence of significant upregulation of MHC-II but not perforin on NK and γδ T cells, which was observed only following a challenge of the unvaccinated pigs and correlated with increased CSFV replication and IFN-α expression in both the tonsils and serum. Together, these data suggest that it is unlikely that NK or γδ T cells contribute to the cellular effector mechanisms induced by live attenuated CSFV.