Wild carrot pentane-based fractions suppress proliferation of human HaCaT keratinocytes and protect against chemically-induced skin cancer.

BACKGROUND: Previous studies in our laboratory showed that the Lebanese Daucus carota ssp. carota (wild carrot) oil extract possesses in vitro and in vivo anticancer activities. The present study aims to examine the cytotoxic effect of Daucus carota oil fractions on human epidermal keratinocytes and evaluate the chemopreventive activity of the pentane diethyl ether fraction on DMBA/TPA induced skin carcinogenesis in mice. METHODS: Wild carrot oil extract was chromatographed to yield four fractions (F1, 100% pentane; F2, 50:50 pentane:diethyl ether; F3, 100% diethyl ether; F4 93:7 chloroform:methanol). The cytotoxic effect of fractions (10, 25, 50 and 100 µg/mL) was tested on human epidermal keratinocytes (non-tumorigenic HaCaT cells and tumorigenic HaCaT-ras variants) using WST a ssay. Cell cycle phase distribution of tumorigenic HaCaT-ras variants was determined by flow cytometry post-treatment with F2 fraction. Apoptosis related proteins were also assessed using western blot. The antitumor activity of F2 fraction was also evaluated using a DMBA/TPA induced skin carcinoma in Balb/c mice. RESULTS: All fractions exhibited significant cytotoxicity, with HaCaT cells being 2.4-3 times less sensitive than HaCaT-ras A5 (benign tumorigenic), and HaCaT-ras II4 (malignant) cells. GC-MS analysis revealed the presence of a major compound (around 60%) in the pentane/diethylether fraction (F2), identified as 2-himachalen-6-ol. Treatment of HaCaT-ras A5 and HaCaT-ras II4 cells with F2 fraction resulted in the accumulation of cells in the sub-G1 apoptotic phase and decreased the population of cells in the S and G2/M phases. Additionally, F2 fraction treatment caused an up-regulation of the expression of pro-apoptotic (Bax) and down-regulation of the expression of anti-apoptotic (Bcl2) proteins. A decrease in the phosphorylation of AKT and ERK was also observed. Intraperitoneal treatment with F2 fraction (50 or 200 mg/kg) in the DMBA/TPA skin carcinogenesis mouse model showed a significant inhibition of papilloma incidence (mice with papilloma), yield (number of papilloma/mouse) and volume (tumor relative size) at weeks 15, 18 and 21. CONCLUSION: The present data reveal that F2 fraction has a remarkable antitumor activity against DMBA/TPA-induced skin carcinogenesis, an effect that may be mediated through inhibition of the MAPK/ERK and PI3K/AKT pathways.

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

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.

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

BACKGROUND: Daucus 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 RESULTS: Using 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. CONCLUSIONS: These results suggest that the pentane-based fractions of DCOE possess potential anti-cancer activity that is mainly mediated through the Erk pathway.

A Ru(II)-Strained Complex with 2,9-Diphenyl-1,10-phenanthroline Ligand Induces Selective Photoactivatable Chemotherapeutic Activity on Human Alveolar Carcinoma Cells via Apoptosis.

[Ru(bipy)2(dpphen)]Cl2 (where bipy = 2,2'-bipyridine and dpphen = 2,9-diphenyl-1,10-phenanthroline) (complex 1) is a sterically strained compound that exhibits promising in vitro photocytotoxicity on an array of cell lines. Since lung adenocarcinoma cancer remains the most common lung cancer and the leading cause of cancer deaths, the current study aims to evaluate the plausible effect and uptake of complex 1 on human alveolar carcinoma cells (A549) and mesenchymal stem cells (MSC), and assess its cytotoxicity in vitro while considering its effect on cell morphology, membrane integrity and DNA damage. MSC and A549 cells showed similar rates of complex 1 uptake with a plateau at 12 h. Upon photoactivation, complex 1 exhibited selective, potent anticancer activity against A549 cells with phototoxicity index (PI) values of 16, 25 and 39 at 24, 48 and 72 h, respectively. This effect was accompanied by a significant increase in A549-cell rounding and detachment, loss of membrane integrity and DNA damage. Flow cytometry experiments confirmed that A549 cells undergo apoptosis when treated with complex 1 followed by photoactivation. In conclusion, this present study suggests that complex 1 might be a promising candidate for photochemotherapy with photoproducts that possess selective anticancer effects in vitro. These results are encouraging to probe the potential activity of this complex in vivo.

A photoactivatable Ru (II) complex bearing 2,9-diphenyl-1,10-phenanthroline: A potent chemotherapeutic drug inducing apoptosis in triple negative human breast adenocarcinoma cells.

The photoactivatable Ru (II) complex 1 [Ru(bipy)2(dpphen)]Cl2 (where bipy = 2,2'-bipyridine and dpphen = 2,9-diphenyl-1,10-phenanthroline) has been shown to possess promising anticancer activity against triple negative adenocarcinoma MDA-MB-231 cells. The present study aims to elucidate the plausible mechanism of action of the photoactivatable complex 1 against MDA-MB-231 cells. Upon photoactivation, complex 1 exhibited time-dependent cytotoxic activity with a phototoxicity index (P Index) of >100 after 72 h. A significant increase in cell rounding and detachment, loss of membrane integrity, ROS accumulation and DNA damage was observed. Flow cytometry and a fluorescent apoptosis/necrosis assay showed an induction of cell apoptosis. Western blot analysis revealed the induction of intrinsic and extrinsic pathways and inhibition of the MAPK and PI3K pathways. The photoproduct of complex 1 showed similar effects on key apoptotic protein expression confirming that it is behind the observed cell death. In conclusion, the present study revealed that complex 1 is a potent multi-mechanistic photoactivatable chemotherapeutic drug that may serve as a potential lead molecule for targeted cancer chemotherapy.

Rapid quantification of ruthenium(ii) polypyridyl anti-cancer drugs using a selective ligand dissociation LC-MS/MS method.

Research on Ru anti-cancer drugs is on the rise with many complexes in clinical trials. Inductively coupled plasma-mass spectrometry (ICP-MS) has been the standard technique for bioanalytical studies on Ru and Pt complexes in biological media. Tedious ICP-MS methods rely on detecting and quantifying the element while lacking important structural information of the original complexes. Despite being equally sensitive, more accessible, and highly selective to the target species, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has not been validated for the analysis of Ru drugs. Using USFDA guidelines, we report here the optimization and validation of a facile LC-MS/MS method for the detection and quantification of three Ru(ii) polypyridyl complexes in cells, plasma, and urine matrices. Importantly, a fast (10 min), single-step procedure was efficient for both extraction and sample purification, and analytes were rapidly eluted over a 3 min simple isocratic run. Specific parent ions were differentially fragmented by tandem MS, thus forming a unique and rational ligand dissociation chemistry that exhibits high selectivity to the target species with no measurable interferences or matrix effects. The developed LC-MS/MS method was advantageous vis-à-vis the prototypical ICP-MS based techniques both in vitro and in vivo, paving the way for its utilization in elaborate cellular uptake, pharmacokinetics, and pharmacodynamics studies.

Enhanced cellular uptake and photochemotherapeutic potential of a lipophilic strained Ru(ii) polypyridyl complex.

The use of ruthenium complexes as chemotherapeutic agents has been recently explored as one of the alternatives to conventional treatments. In the present study, two Ru(ii) polypyridyl complexes were synthesized and characterized: a strained [Ru(bipy)2(BC)]Cl2 (complex 1) where [bipy = 2,2'-bipyridine and BC = bathocuproine] along with the unstrained control [Ru(bipy)2(phen)]Cl2 (complex 2) where [phen = 1,10-phenanthroline]. The photophysical and photochemical analyses proved that unlike the photostable complex 2, complex 1 ejected both bipy and BC ligands at a ratio of 3 : 1 respectively. Results showed that the activity of complex 1 was significantly enhanced upon photoactivation. The response was however particularly significant in B16-F10 melanoma cells where phototoxicity index (PI = IC50 dark/IC50 light) was >900. When compared to cisplatin, the photoproducts were more potent against all tested cell lines, implying that the complex acquired significant chemotherapeutic potential upon irradiation. Cellular uptake of complex 1 and the free BC ligand were found to be significantly facilitated as evidenced by 400-600 fold increase in concentration of the compounds inside the cells relative to the extracellular culture medium. Complex 2 exhibited 35 times lower cellular concentration relative to complex 1. Flow cytometry and plasmid DNA gel electrophoresis measurements showed that complex 1 interacts with DNA inducing apoptosis in the dark and either late-apoptosis or necrosis upon irradiation. These findings corroborate the importance of lipophilic ligands such as BC to enhance uptake and subsequently improve the photochemotherapy potential of Ru(ii) polypyridyl complexes.

ß-2-Himachalen-6-ol inhibits 4T1 cells-induced metastatic triple negative breast carcinoma in murine model.

ß-2-himachalen-6-ol (HC), a major sesquiterpene isolated from the Lebanese wild carrot umbels, was shown to possess remarkable in vitro and in vivo anticancer activities. The present study investigates the anti-metastatic activity of HC post 4T1 breast cancer cells inoculation in a murine model. The effect of HC on 4T1 cell viability was assessed using WST-1 kit, while cell cycle analysis was performed using flow cytometry. Tumor development and metastasis were evaluated by injecting 4T1 cells in the mice mammary gland region followed by either HC or cisplatin treatment. The 6-thioguanine assay was used for the quantification of metastatic cells in the blood. HC treatment caused a dose-dependent decrease in cell viability with IC50 and IC90 values of 7 and 28 µg/mL respectively. Concomitant treatment with cisplatin significantly reduced cell viability when compared to cells treated with cisplatin or HC alone. Flow cytometry revealed a significant increase (p˂0.05) in cell count in the Sub-G1 phase at HC 10 µg/mL, and total DNA fragmentation (p˂0.001) at HC 25 µg/mL. Annexin/PI staining showed early and late apoptotic mode of cell death upon treatment with HC. Histopathological evaluation revealed less incidence of primary and metastatic tumor/inflammation in the HC and cisplatin treated groups. Tumor size and colony-forming units were significantly decreased in the HC treated group. HC treatment induced cell cycle arrest, promoted apoptosis and reduced the incidence of primary and metastatic lesions caused by 4T1 cells. The present findings suggest that HC has an anti-metastatic potential against aggressive types of cancer.

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.

The chemotherapeutic effect of ß-2-himachalen-6-ol in chemically induced skin tumorigenesis.

ß-2-himachalen-6-ol (HC), a novel sesquiterpene derived from Lebanese wild carrot, was shown to possess a remarkable anticancer activity. The present study investigates the in vitro anticancer activity of HC and its effect on papillomas induced using a DMBA/TPA skin carcinogenesis mouse model. HaCaT-ras II-4 epidermal squamous cell viability was assessed using WST-1 kit. Cell cycle was analyzed by flow cytometry, and pro/anti-apoptotic proteins were measured using western blot. Mice papillomas were induced by DMBA and promoted with TPA for 18 weeks. At week 12, animals were divided into four groups: HC topically treated (5%Top), HC intraperitoneally treated (25 mg/kg; HC25), Cisplatin treated (2.5 mg/kg), and control (DMSO treated). Papilloma yield, volume, histology, and mice weight and liver function were assessed. HC treatment decreased significantly cell survival (IC50 = 7 and IC90 = 40 µg/ml) and increased significantly cells undergoing late apoptosis and necrosis. It also significantly decreased the levels of pro-caspase-3, p53, Bcl-2, p-Erk/Erk and p-Akt/Akt and increased p21 and Bax proteins. Treatment with HC25, HC5%Top or Cisplatin showed a significant decrease in papilloma yield and volume. Only Cisplatin treatment caused a significant decrease in body weight and increase in serum ALT. In conclusion, ß-2-himachalen-6-ol induced significant tumor shrinkage, an effect partly mediated via promoting apoptosis through inhibition of the MAPK/ERK and PI3K/AKT pathways, with no significant toxicity to laboratory mice.

Interleukin-10 reduces hyperalgesia and the level of Interleukin-1beta 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.

ß-2-himachalen-6-ol protects against skin cancer development in vitro and in vivo.

BACKGROUND: Previous studies in our laboratory showed that Daucus carota oil extract (DCOE) possesses remarkable in-vitro anticancer activity and antitumour promoting effect against DMBA/TPA skin carcinogenesis in mice. Chemical analysis of DCOE led to the isolation of the ß-2-himachalen-6-ol (HC), major sesquiterpene with a potent anticancer activity against various colon, breast, brain and skin cancer cells. This study investigated the anticancer activity of HC against invasive epidermal squamous cell carcinoma cells and evaluated its effect in a DMBA/TPA skin carcinogenesis Balb/c murine model. METHODS: HaCaT-ras II-4 epidermal squamous cells were treated with HC (1, 5, 10, 25 and 50 µg/ml), and cell viability was evaluated with WST 1 assay kit. Cell cycle analysis was carried out by flow cytometry, and pro/anti-apoptotic proteins were measured using Western blot. The effect of topical and intraperitoneal (IP) treatment with HC in mice was assessed using the DMBA/TPA skin carcinogenesis model. Cisplatin (2.5 mg/kg; IP) was used as a positive control. Papilloma incidence, yield and volume were monitored, and isolated papillomas were assessed for their pro/anti-apoptotic proteins and morphology. RESULTS: ß-2-himachalen-6-ol showed a dose-dependent decrease in cell survival with an IC50 and IC90 of 8 and 30 µg/ml, respectively. Flow cytometry analysis revealed that treatment with 10 µg/ml HC significantly increased the number of cells undergoing late apoptosis (28%), while 25 µg/ml caused a larger cell shift towards late apoptosis (46.6%) and necrosis (39%). A significant decrease in protein levels of p53 and Bcl-2 and a significant increase in p21 and Bax were observed. Also, there was a significant decrease in p-Erk and p-Akt protein levels. The treatment of mice (IP and topical) with HC caused a significant decrease in papilloma yield, incidence and volume. Similar effects were observed with cisplatin treatment, but HC-treated groups exhibited twofold to threefold increase in survival rates. Similar patterns in the pro- and anti-apoptotic proteins were observed in mice treated with HC, except for a significant increase in p53 protein. CONCLUSIONS: In conclusion, HC treatment induced cell cycle arrest (low dose) and promoted apoptosis partly via inhibition of the MAPK/ERK and PI3K/AKT pathways with no significant toxicity to laboratory mice.

Antitumor activity of ß-2-himachalen-6-ol in colon cancer is mediated through its inhibition of the PI3K and MAPK pathways.

Previous studies in our laboratory showed that Daucus carota oil extract (DCOE) possesses in vitro and in vivo anticancer activities. Chemical analysis of DCOE led to the isolation of ß-2-himachalen-6-ol (HC) which exhibited potent anticancer activity against colon, breast, brain and skin cancer cells. The present study investigates the anticancer activity of HC against SW1116 colon cancer cell lines, and evaluates its effect in a 1,2-dimethylhydrazine (DMH) colon carcinogenesis black6 mice model. The SW1116 colon cancer cell line was treated with HC (1, 5, 10 and 25 µg/ml) and cell viability was evaluated with WST 1 assay kit. Cell cycle analysis was carried out by flow cytometry, and pro/anti-apoptotic proteins were measured using western blot. The effect of intraperitoneal (IP) treatment with HC (10, 25 and 50 µg/ml) in mice was assessed using the DMH colon carcinogenesis model with Cisplatin (2.5 µg/kg; IP) as a positive control. Blood samples were collected for assessment of liver toxicity and colon tumor incidence and size were studied histologically. HC showed a dose-dependent decrease in cell survival with an IC50 of 18 and 14.5 µg/ml after 24 and 48 h respectively. Flow cytometry analysis revealed that 10 µg/ml HC increased the number of cells undergoing necrosis (18.05%) and late apoptosis (15.66%). At HC 25 µg/ml more cells shifted toward necrosis (58.01%) and late apoptosis (30.47%). Western blot analysis revealed a significant decrease in p-Erk, p-Akt, pro-caspase-3 and Bcl-2 and an increase in p53, p21, Bax and PARP proteins. Mice treatment (IP) with HC caused a significant decrease in tumor incidence and size. Similar effects were observed with cisplatin treatment. In conclusion, HC treatment (low dose) induced cell cycle arrest and promoted apoptosis via inhibition of the MAPK/ERK and PI3K/AKT pathways. HC treatment also had antitumor effect in vivo with no significant toxicity to laboratory mice.

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.

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.

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.

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(-) CD8(hi) 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 CD44(hi) 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.

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 NS1223₋1230 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.

Leishmania major: low infection dose causes short-lived hyperalgesia and cytokines upregulation in mice.

Neural involvement was traditionally associated with leprosy. However, more recent studies have shown the presence of a persistent hyperalgesia in cutaneous leishmaniasis caused by the infection of BALB/c mice with a high dose of Leishmania major. In this study, we report the presence of hyperalgesia within the first two weeks of infection caused by a low dose of the parasite. Using BALB/c mice, we demonstrate the presence of hyperalgesia during the first 10 days of infection as assessed by thermal pain tests. After 10 days these decreased pain thresholds start to recover resulting in similar levels to those in uninfected controls during the third week of infection. This hyperalgesia is accompanied by a sustained upregulation of interleukin-1beta (IL-1beta) and an early upregulation of interleukin-6 (IL-6) which is restored to normal levels after five days of infection. In conclusion, this study shows that, during early infection, the low dose of L. major causes hyperalgesia accompanied by an upregulation of IL-1beta and IL-6 and that these effects are reversed within the first two weeks of infection.