The immunosuppressive characteristics of FB1 by inhibition of maturation and function of BMDCs

Fumonisin B₁ (FB₁) is one kind of mycotoxins that has the neurotoxicity, carcinogenicity, hepatotoxicity and immunotoxicity produced by the fungus Fusarium verticillioides, which commonly infects corn and other crops and is harmful to animal and human health upon consumption of FB₁-contaminated feed or food. However, the mechanism of immunotoxicity, especially the immunosuppression induced by FB₁ is still unclear. The most pivotal cells in the induction of immune responses are dendritic cells (DCs). In this study, we used murine bone marrow-derived dendritic cells (BMDCs) as a model system to elucidate the effect of FB₁ on the function of BMDCs through biological methods. We found that FB₁ reversed the morphological changes and enhanced the endocytosis of FITC-dextran in LPS-treated BMDCs. At the same time, FB₁ decreased the LPS-induced expressions of MHC II, C[1]D80 and CD86 molecules in BMDCs (p < 0.05), as well as the T-cell stimulatory capacity of BMDCs (p < 0.01). Moreover, the secretions of IL-6, IL-10 and IL-12, but not TNF-α induced by LPS exposure were suppressed by FB₁ in a dose dependent (p < 0.01). It was considered that the immunosuppressive effects of FB₁ were mainly caused by changing the morphology and interfering with the process of antigen uptake, processing and presentation. The results highlighted that FB₁ had the capacity to modulate the immune responses of BMDCs.     

Aflatoxin B1 modulates the insulin-like growth factor-2 dependent signaling axis

Although aflatoxin B₁ (AFB₁) is known as a mycotoxin that induces hepatocellular carcinoma (HCC), its effects on HCC cells have not been sufficiently investigated.The HCC cell lines HepG2, Huh-6, Huh-7, and PLC were cultured (5 × 10⁵ cells/ml) and various concentrations of AFB₁ were added. The expression levels of the α-fetoprotein (AFP), insulin-like growth factor-2 (IGF-2), and insulin-like growth factor-1 receptor (IGF-1R) genes in each sample were determined by real-time PCR, with the following results:(1) The level of AFP expression in HepG2 increased at 5–50 ng/ml of AFB₁ in a dose-dependent manner. The AFP expression level in Huh-6 increased at 0.01–5 ng/ml of AFB₁ in a dose-dependent manner and decreased to half controls level at 50 ng/ml of AFB₁. The AFP expression level in Huh-7 decreased to one-third the original level at 0.5–50 ng/ml of AFB₁. The AFP expression level in PLC decreased at 0–0.5 ng/ml of AFB₁ in a dose-dependent manner, and decreased to one-third at concentrations of AFB₁ between 0.5 and 50 ng/ml.(2) The IGF-2 and IGF-1R expression levels in Huh-6 increased more than 10-fold at 0.5–5 ng/ml of AFB₁, but decreased to half at 50 ng/ml of AFB₁. The IGF-2 and IGF-1R expression levels in other cell lines increased in a dose-dependent manner.AFB₁ induced translations of IGF-2 and IGF-1R and cell proliferation: When 50 ng/ml AFB₁ was administrated, cell numbers were 2.0-, 1.7-, and 1.5-fold higher than those of controls after 3 days of culture in HepG2, Huh-7, and PLC, respectively. Particularly, in Huh-6, it increased 2.5-fold higher than those of controls following 5 ng/ml AFB₁ administration. The ratio of fold-change phospho-IGF-1R in all cell lines that were treated with AFB₁, increased 1.1–1.5-fold.These results indicate that AFB₁ may enhance HCC cell proliferation through an IGF-2-dependent signal axis, although it remains to be investigated whether those effects are associated with human hepatocarcinogenesis resulting from AFB₁ exposure.     

Protective role of sodium selenite on histopathological lesions,decreased T-cell subsets and increased apoptosis of thymus in broilers intoxicated with aflatoxin B1

For evaluating the ability of selenium (Se) in counteracting the adverse effects of aflatoxin B₁ (AFB₁), two hundred 1-day-old male Avian broilers, divided into five groups, were fed with basal diet (control group), 0.3 mg/kg AFB₁ (AFB₁ group), 0.3 mg/kg AFB₁ + 0.2 mg/kg Se (+Se group I), 0.3 mg/kg AFB₁ + 0.4 mg/kg Se (+Se group II) and 0.3 mg/kg AFB₁ + 0.6 mg/kg Se (+Se group III), respectively. Compared with control group, the decreased relative weight of thymus and percentages of mature thymocytes, congestion in medulla and much debris in cortex of thymus, and the increased apoptotic thymocytes were observed in AFB1 group. However, supplied dietary sodium selenite could increase the relative weight of thymus and percentages of mature thymocytes, and alleviate histopathological lesions. Compared with AFB1 group, the percentages of apoptotic thymocytes detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method and flow cytometry method in three +Se groups were decreased, the expression of Caspase-3 and Bax, through quantitative real-time PCR and immunohistochemical method, in three +Se groups were decreased, while the expression of Bcl-2 was increased. The results indicate that sodium selenite supplied in the diet, through a mechanism of apoptosis regulation, may ameliorated AFB₁-induced lesions of thymus and accordingly improve the impaired cellular immune function.     

Effect of the combined probiotics with aflatoxin B1-degrading enzyme on aflatoxin detoxification,broiler production performance and hepatic enzyme gene expression

In order to degrade aflatoxin B₁ (AFB₁), AFB_l-degrading microbes (probiotics) such as Lactobacillus casei, Bacillus subtilis and Pichia anomala, and the AFB_l-degrading enzyme from Aspergillus oryzae were selected and combined to make feed additive. Seventy-five 43-day-old male Arbor Acres broilers were randomly divided into 5 groups, 15 broilers for each group. The broilers were given with 5 kinds of diets such as the basal diet, 400 μg/kg AFB₁ supplement without feed additive, and 200, 400, 800 μg/kg AFB₁ supplement with 0.15% feed additive. The feeding experimental period was 30 d, which was used to determine production performance of broilers. In addition, serum, liver and chest muscle were selected for measuring AFB₁ residues, gene expressions, microscopic and antioxidant analyses. The results showed that adding 0.15% feed additive in broiler diets could significantly relieve the negative effect of AFB₁ on chicken’s production performance and nutrient metabolic rates (P < 0.05). It could also improve AFB₁ metabolism, hepatic cell structure, antioxidant activity, and many hepatic enzyme gene expressions involved in oxidoreductase, apoptosis, cell growth, immune system and metabolic process (P < 0.05). It could be concluded that the feed additive was able to degrade AFB₁ and improve animal production.     

Aflatoxins B1, B2 and G1 modulate cytokine secretion and cell surface marker expression in J774A.1 murine macrophages

Aflatoxins are fungal products which occur in food and feed. They are potent hepatocarcinogens, and are known to cause immunosuppression. We investigated the effect of aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂) and aflatoxin G₁ (AFG₁) exposure, alone and in combination, on the secretion of key pro- and anti-inflammatory cytokines from the murine macrophage cell line, J774A.1. Exposure of macrophages to low doses of aflatoxin (0.01 or 0.1 ng/mL) resulted in a statistically significant change in the secretion of a number of cytokines following stimulation with lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls. Specifically, treatment with AFB₁ or AFB₂ alone significantly decreased (P < 0.01) the secretion of the anti-inflammatory cytokine interleukin (IL) 10 (IL-10), while the secretion of the pro-inflammatory cytokine IL-6 was significantly increased (P < 0.01). In addition, aflatoxin exposure affected expression levels of key cell surface markers involved in the inflammatory response. Toll-like receptor 2 (TLR2) and Cluster of Differentiation 14 (CD14) expression levels decreased significantly (P < 0.01), but Toll-like receptor 4 (TLR4) expression was unaffected. This data provides further insight into the mechanisms by which aflatoxins modulate the host immune response to exert their immunosuppressive activity.     

Cytotoxicity and immunotoxicity of cyclopiazonic acid on human cells

In this study, in vitro cytotoxicity and immunotoxicity of the mycotoxin cyclopiazonic acid (CPA) was evaluated on human cells. To evaluate cytoxicity, several cellular targets were used (CD34+, monocytes, THP-1 and Caco-2). Monocytes were more sensitive to CPA than the THP-1 monocytic cell line after 48 h of incubation in the tested conditions. Half maximal inhibitory concentration (IC50) were determined to be 8.5 × 10⁻⁸ and 1.75 × 10⁻⁷ M for monocytes and THP1, respectively, while IC50 > 1.25 × 10⁻⁷ M was observed for Caco-2 and CD34+ cells. The CPA effect on macrophage differentiation was also examined at non-cytotoxic concentrations. The monocyte differentiation process was markedly disturbed in the presence of CPA. After 6 days of culture, CD71 expression was downregulated, while CD14 and CD11a expressions did not change. Moreover, activated macrophages showed a raised burst activity and TNF-α secretion. Overall, the results indicated that CPA exhibited toxicity on various human cellular models. Moreover, at non-cytotoxic concentrations, CPA disturbed human monocytes differentiation into macrophages. This work contributes to understanding the immunosuppressive properties of this food-related toxin.     

Suppressive effect of β,β-dimethylacryloyl alkannin on activated dendritic cells in psoriasis by the TLR7/8 pathway

β,β-dimethylacryloyl alkannin (DMA) is a key component of Lithospermum and possesses good efficacy for treating psoriasis. DMA inhibits activated dendritic cells (DCs), but the mechanism is unknown. Therefore, this study aimed to explore the modulation of the TLR7/8 pathway by DMA in psoriasis-activated DCs. Models of psoriasis-like skin lesions were established using BALB/c mice; 8 mice were treated with DMA (2.5 mg/kg). Bone marrow cells were isolated and induced into DCs using R848, a TLR7/8 agonist. Splenic CD11c + cells were detected by flow cytometry. Skin CD11c + cells were detected by immunofluorescence. TLR7, TLR8, MYD88, and IRAKM proteins were detected by Western blot. The effects of DMA on surface molecules of DCs were observed by flow cytometry. mRNA expression of inflammatory factors was detected by qRT-PCR. Secreted cytokines were detected by cytometric bead array. Compared with the model group, psoriasis-like skin lesions were alleviated by DMA, the splenic CD11c + cells were significantly decreased (P < 0.01), and CD11c + cell numbers in skin lesions were decreased (P < 0.01). Expression levels of TLR7, MYD88, and IRAKM were significantly decreased (P < 0.05). R848-stimulated DCs showed increased expression of I-A/I-E, CD80, and CD86 (P < 0.01), increased IL-23 and IL-1β mRNA and secretion (P < 0.05), and increased TLR7, TLR8, MYD88, and IRAKM expression (P < 0.01); DMA inhibited all of these effects of the TLR7/8 pathway activation by R848 (P < 0.05). In conclusion, DMA could inhibit psoriasis-activated DCs via the TLR7/8 pathway.     

A comprehensive analysis of transfection-assisted delivery of iron oxide nanoparticles to dendritic cells

Polylysine (PL) has been used to facilitate dendritic cell (DC) uptake of super paramagnetic iron oxide (SPIO) nanoparticles for use in magnetic resonance imaging (MRI). In this work, we examined the effect of PL on cell toxicity and induction of cell maturation as manifested by the up-regulation of surface molecules. We found that PL became toxic to bone marrow-derived DCs (BMDCs) at the 10 μg/ml threshold. Incubation of BMDCs with 20 μg/ml of PL for 1 h resulted in approximately 90% cell death. However, addition of SPIO nanoparticles rescued DCs from PL-induced death as the combination of SPIO with PL did not cause cytotoxicity until the PL concentration was 1000 μg/ml. Prolonged exposure to PL induced BMDC maturation as noted by the expression of surface molecules such as MHC class II, CD40, CCR7 and CD86. However, the combination of SPIO and PL did not induce BMDC maturation at 1 h. However prolonged exposure to SPIO nanoparticles induced CD40 expression and protein expression of TNFα and KC. The data suggest that the use of PL to enhance the labeling of DCs with SPIO nanoparticles is a dedicated work. Appropriate calibration of the incubation time and concentrations of PL and SPIO nanoparticles is crucial to the development of MRI technology for noninvasive imaging of DCs in vivo.From the Clinical EditorThe authors of this study present detailed data on toxicity and efficiency of polylysine-facilitated uptake of USPIO-s by dendritic cells for cell-specific MR imaging.     

Polybacterial immunomodulator Respivax restores the inductive function of innate immunity in patients with recurrent respiratory infections

Respivax (BulBio-NCIPD Ltd.) is an oral polybacterial immunomodulator intended for treatment and prevention of non-specific respiratory tract infections. We studied for the first time its effects on the inductive mechanisms of innate immunity, in the course of 3-month immunoprophylaxis of 25 patients with recurrent and chronic respiratory infections. The expression of pattern-recognition receptors on peripheral blood (PB) monocytes and plymorphonuclear cells (PMNs), the antigen-presenting and co-stimulatory potential of peripheral blood monocytes and dendritic cells; and the stimulated Th1/Th2 cytokine production were determined by flow cytometry. As compared to healthy controls, patients were characterized with down-regulation of TLR2 and TLR4/CD14 complex on PB monocytes (p < 0.01), decreased share of CD14+CD16+ DCs precursors (p < 0.01), decreased CD86 expression on PB DCs (p < 0.05) and a Th2 shift of cytokine profile. Respivax modulated differentially the surface expression of pattern-recognition receptors on PB monocytes, increasing TLR2 and CD14 without affecting TLR4 expression. Further on, Respivax enhanced the differentiation of mature CD86^high dendritic cells (DCs). Importantly, Respivax promoted a Th1 shift of cytokine profile and restored the Th1/Th2 cytokine balance without pro-inflammatory effects. Noteworthy, Th1/Th2 ratios in the patient's group correlated positively with the levels of TLR2 (R = 0.5, p < 0.001) and CD14 expression (R = 0.4, p < 0.05). We conclude that Respivax treatment restores the inductive function of innate immunity at three key levels: antigen recognition and presentation, co-stimulation of naïve T cells, and Th1/Th2 balance. This results, at least in part, from a differential modulation effect on the expression of pathogen-recognition receptors.     

APL-1, an altered peptide ligand derived from human heat-shock protein 60, selectively induces apoptosis in activated CD4 + CD25 + T cells from peripheral blood of rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a chronic T-cell mediated autoimmune disease that affects primarily the joints. The induction of immune tolerance through antigen-specific therapies for the blockade of pathogenic CD4 + T cells constitutes a current focus of research. In this focus it is attempted to simultaneously activate multiple regulatory mechanisms, such as: apoptosis and regulatory T cells (Tregs). APL-1 is an altered peptide ligand derived from a novel CD4 + T-cell epitope of human heat-shock protein of 60 kDa, an autoantigen involved in the pathogenesis of RA. Previously, we have reported that APL-1 induces CD4 + CD25^highFoxp3 + Tregs in several systems. Here, we investigated the ability of APL-1 in inducing apoptosis in PBMCs from RA patients, who were classified as active or inactive according to their DAS28 score. APL-1 decreased the viability of PBMCs from active but not from inactive patients. DNA fragmentation assays and typical morphological features clearly demonstrated that APL-1 induced apoptosis in these cells. Activated CD4 + CD25 + T cells but not resting CD4 + CD25 − T cells were identified as targets of APL-1. Furthermore, CD4 + T-cell responses to APL-1 were found to be dependent on antigen presentation via the HLA-DR molecule. Thus, APL-1 is a regulatory CD4 + T cell epitope which might modulate inflammatory immune responses in PBMCs from RA patients by inducing CD4 + CD25^highFoxp3 + Tregs and apoptosis in activated CD4 + T cells. These results support further investigation of this candidate drug for the treatment of RA.     

Physiologically based toxicokinetics of serum aflatoxin B1-lysine adduct in F344 rats

Aflatoxin B₁-lysine adduct (AFB-Lys) is a reliable biomarker for aflatoxin exposure; however, a systematic toxicokinetic evaluation has not been reported. In this study, male F344 rats were orally exposed to single, or repeated, doses of AFB₁ and the toxicokinetics of serum AFB-Lys that followed treatments were investigated. A single-dose of AFB₁ increased serum AFB-Lys levels rapidly peaking at 4 h, followed by first-order elimination, through which the half-life was estimated to be 2.31 days. A physiologically based pharmacokinetic model showed that approximately 3.00–3.90% and 1.12–1.98% of the administered AFB₁ doses were converted to serum AFB-Lys adducts at 2 h and 24 h post treatment, respectively. Repeated AFB₁ exposure at 5–25 μg/kg body weight linearly increased serum AFB-Lys levels for 5 weeks in animals, resulting in a 1–1.5 times higher AFB-Lys level overall. This indicates the potential of this adduct as a reliable biomarker for repeated low dose exposure. Higher dose exposure at 75 μg/kg increased the level of AFB-Lys to a maximum at 2 weeks, followed by a gradual decrease to near plateau level up to 5 weeks. In conclusion, this study systematically evaluated the toxicokinetics of serum AFB-Lys adduct in F344 rats using a physiologically based pharmacokinetic model and robust statistical modeling analysis and provided a firm and clear understanding of the toxicokinetics of this biomarker.     

Effect of vitamin D3 on chemokine levels and regulatory T-cells in pulmonary tuberculosis

1,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃] the active form of vitamin D₃ acts as an immunomodulator in various immune cells. The present study is aimed to study the effect of 1,25(OH)₂D₃ on chemokine levels and regulatory T-cells in 51 healthy controls (HCs) and 50 pulmonary tuberculosis (PTB) patients. Peripheral blood mononuclear cells were cultured with culture filtrate antigen (CFA) of Mycobacterium tuberculosis in the presence or absence of 1,25(OH)₂D₃ at 10^− 7 M concentration for 72 h and the percentage positive regulatory T-cell subsets were studied using flow cytometry. The chemokine levels were estimated in the culture supernatants by ELISA. 1,25(OH)₂D₃ significantly upregulated the frequency of regulatory T-cell subsets while suppressed the production of chemokine levels in CFA stimulated cultures of HCs and PTB patients (p < 0.05). Correlation analysis revealed a significant negative correlation between CD4 + Foxp3 + regulatory T-cells and MCP-1, MIP-1β and IP-10 in CFA stimulated with 1,25(OH)₂D₃ treated cells (p < 0.05). The results suggested that 1,25(OH)₂D₃ upregulated regulatory T-cells and act as anti-inflammatory by downregulating chemokine levels which could be beneficial to protect the host from inflammation and tissue damage during infection.     

Loxoribine,a selective Toll-like receptor 7 agonist,induces maturation of human monocyte-derived dendritic cells and stimulates their Th-1- and Th-17-polarizing capability

Recently, a guanosine analog, 7-allyl-7,8-dihydro-8-oxo-guanosine (loxoribine), has been identified as a selective Toll-like receptor (TLR)7 agonist. Bearing in mind the controversy regarding the expression of TLR7 by human myeloid dendritic cells (DCs) and its significance for functions of these cells, the goal of this study was to investigate the effect of loxoribine on differentiation, maturation and functions of human monocyte-derived (Mo)DCs. Immature MoDCs were obtained by cultivation of monocytes for 6 days with recombinant granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. These cells were stimulated with loxoribine (250 μM) for an additional 48 h. Phenotypic properties of MoDCs were determined by flow cytometry, cytokine production was assayed by ELISA, whereas their allostimulatory capability was tested using a mixed leukocyte reaction. We showed that loxoribine up-regulated the expression of TLR7, CD40, CD54, CD80, CD83 and CCR7 and stimulated the production of IL-12, IL-23, IL-27 and IL-10 by MoDCs, whereas the level of interferon (IFN)-β was not modulated. Allogeneic CD4⁺T cells in co-culture with loxoribine-treated MoDCs proliferated more strongly, at lower DC/CD4⁺T-cell ratio (1:80), and secreted significantly higher levels of IL-17 and IFN-γ compared to the cultures with control MoDCs. The stimulatory effect of loxoribine on T helper (Th)1 polarization capability of MoDCs was further potentiated by ligation of CD40. In conclusion, our results show that loxoribine stimulated differentiation, maturation, allostimulatory as well as Th1 and Th17 polarization capability of human MoDCs and suggests that these effects might be associated with up-regulation of TLR7 expression, but not increased IFN-β production.     

MicroRNA-106b regulates pro-allergic properties of dendritic cells and Th2 polarisation by targeting early growth response-2 in vitro

Recent evidence has suggested that miRNA is implicated in the immune response of allergic and inflammatory diseases. However, little is known about its role in the mechanism that underlies the establishment of pro-allergic DCs in allergic rhinitis is not fully understood. This study assessed whether and how microRNA (miR)-106b regulates the pro-allergic properties of DCs upon allergen stimulation in vitro. Bone marrow-derived dendritic cells (BMDCs) were generated and stimulated with ovalbumin (OVA) to identify the miRNA expression profile. After transfection with miR-106b mimics and inhibitors OVA-activated BMDCs were further evaluated for surface marker expression using flow cytometry, cytokine production using ELISA and subsequent effects on Th2 cell polarisation using flow cytometry. Moreover, the upstream controllers and potential target proteins of miR-106b were examined in a western blot analysis. Results showed that MiR-106b expression was significantly inhibited in activated BMDCs upon OVA stimulation (p < 0.05). Surface marker expression (e.g., MHC class II, CD80 and CD86) was significantly upregulated after the transfection of an miR-106b inhibitor (p < 0.05), and the proportion of GATA-3⁺ T cells was significantly increased among CD4⁺ T cells that were cocultured with miR-106b inhibitor-pretreated BMDCs (p < 0.05). Conversely, IL-12 production from OVA-activated BMDCs and the proportion of T-bet⁺ T cells increased significantly in a coculture of CD4⁺ T cells and miR-106b mimics-transfected BMDCs (p < 0.05). The early growth response (Egr)-2 was identified via luciferase reporter assays as a target gene of miR-106b, and significant Egr-2 upregulation was observed in OVA-activated BMDCs following transfection with a miR-106b inhibitor (p < 0.05). In conclusion, our results suggest that miR-106b negatively regulates the pro-allergic properties of BMDCs and subsequent Th2 polarisation upon OVA stimulation and might represent a promising therapeutic target for allergic inflammation.     

Ozonolysis efficiency and safety evaluation of aflatoxin B1 in peanuts

Ozonolysis efficiency of aflatoxin-contaminated peanuts (ACPs) was investigated, and the safety of ACPs untreated/treated by ozone was evaluated after 28-day intragastrically administration in male and female Wistar rats. 89.40% of aflatoxin B₁ (AFB₁) in peanuts was decomposed by ozone with a concentration of 50 mg/L, flow rate of 5 L/min for 60 h. After 60 h, the ozonolysis efficiency of AFB₁ was not further improved. In the subchronic toxicity experiment, all rats did not have unusual changes in behavior, and no signs of intoxication were observed except for several dead rats due to inappropriate gavage or anesthesia. The results of subchronic toxicity indicated that rats fed on ACPs alone had significantly decreased in body weight gain and feed conversion efficiency. Most serum biochemical indexes of rats had apparently changed compared with those in the negative control, and gender difference significantly affected most indexes of subchronic toxicity except for the ratios of organ to body weight and histopathological observation. Rats fed on ACPs treated by ozone showed significant beneficial health effects. All the results suggested that the deleterious effects of AFB₁ could be highly reduced by ozone, and ozone itself did not show any toxic effects on animals in this processing.     

Clinical scale electroloading of mature dendritic cells with melanoma whole tumor cell lysate is superior to conventional lysate co-incubation in triggering robust in vitro expansion of functional antigen-specific CTL

Recent commercial approval of cancer vaccine, demonstrating statistically significant improvement in overall survival of prostate cancer patients has spurred renewed interest in active immunotherapies; specifically, strategies that lead to enhanced biological activity and robust efficacy for dendritic cell vaccines. A simple, widely used approach to generating multivalent cancer vaccines is to load tumor whole cell lysates into dendritic cells (DCs). Current DC vaccine manufacturing processes require co-incubation of tumor lysate antigens with immature DCs and their subsequent maturation. We compared electroloading of tumor cell lysates directly into mature DCs with the traditional method of lysate co-incubation with immature DCs. Electroloaded mature DCs were more potent in vitro, as judged by their ability to elicit significantly (p < 0.05) greater expansion of peptide antigen-specific CD8⁺ T cells, than either lysate-electroloaded immature DCs or lysate-co-incubated immature DCs, both of which must be subsequently matured. Expanded CD8⁺ T cells were functional as judged by their ability to produce IFN-γ upon antigen-specific re-stimulation. The electroloading technology used herein is an automated, scalable, functionally closed cGMP-compliant manufacturing technology supported by a Master File at CBER, FDA and represents an opportunity for translation of enhanced potency DC vaccines at clinical/commercial scale.     

Evaluation of an ompA-based phage-mediated DNA vaccine against Chlamydia abortus in piglets

Chlamydia abortus (C. abortus) is an obligate intracellular pathogen that causes abortion in pigs and poses a zoonotic risk in pregnant women. Although attenuated and inactivated vaccines are available, they do not provide complete protection in animals underlining the need to develop new vaccines. In this study, we tested the hypothesis that intramuscular immunization with an ompA-based phage-mediated DNA chlamydial vaccine candidate will induce significant antigen-specific cellular and humoral immune responses. Thus, groups of piglets (five per group) were immunized intramuscularly with the phage-MOMP vaccine (λ-MOMP) or a commercial live-attenuated vaccine (1B vaccine) or a GFP-expressing phage (λ-GFP) or phosphate buffered saline (PBS) (control) and antigen-specific cell-mediated and humoral immune responses were evaluated. By day 63 post-immunization, the λ-MOMP vaccine elicited significantly higher (P < 0.05) levels of antigen-specific serum IgG antibody responses than the 1B vaccine or control did. Also, piglets immunized with λ-MOMP vaccine had significantly higher (P < 0.05) MOMP-specific lymphocyte proliferative responses compared to those immunized with the 1B vaccine or control. Furthermore, the total T-cell numbers (CD3 +) and the proportion of CD4 + and CD8 + T-cell subsets as well as the ratio of CD4 +/CD8 + T cells elicited following immunization were comparable between the λ-MOMP- and 1B-vaccinated animals on both days 63 and 70. Interestingly, although the proportion of CD3+CD4−CD8− double negative T cells on day 63 was significantly higher (P < 0.05) in the 1B vaccine group compared to the λ-MOMP-immunized group, there was a significant decrease in the proportion of this T-cell population on day 70 in the 1B compared to the λ-MOMP vaccinated group. These results indicate that the λ-MOMP DNA vaccine is capable of inducing antigen-specific cellular and humoral immune responses that may provide protective immunity against a live challenge infection with C. abortus.     

Modulation of regulatory T cells by intranasal allergen immunotherapy in an experimental rat model of airway allergy

Allergic airway diseases such as asthma and allergic rhinitis are increasing in prevalence worldwide. The theory of an altered Th1/Th2 balance in allergic diathesis has recently been termed a “procrustean paradigm” as it failed to explain many preclinical findings. Regulatory T cells (Treg) have now been shown to be critical in T-cell homeostasis and in the maintenance of peripheral tolerance to allergens. Allergen specific immunotherapy (SIT) has been shown to induce regulatory T cells in allergic patients. Among various types of SIT, intranasal immunotherapy had not been studied in detail for the treatment of allergic airway diseases. So, there was a need to study the contribution of regulatory T cells and their mechanistic pathways following intranasal immunotherapy in-vivo. It had been previously shown that intranasal allergen immunotherapy using Alstonia scholaris pollen extract abrogates allergic airway inflammation with decline in IgE and Th2 cytokine levels. The present study for the first time offers a multi-targeted approach towards attenuation of airway allergy by the generation of CD4 + CD25 + Foxp3 + T cells and other subsets of Treg cells like Tr1 cells, Th3 cells, CTLA4 + Treg cells, and also modulation of various Treg cell surface molecules like GITR, OX40, CD39 and CD73 by intranasal immunotherapy in the same animal model. This animal experiment will thus help to chart out newer molecular targets for treating allergic asthma or rhinitis.     

Daphnoretin modulates differentiation and maturation of human dendritic cells through down-regulation of c-Jun N-terminal kinase

Daphnoretin, an active constituent of Wikstroemia indica C.A. Meys, has been shown possessing anti-cancer activity. In this study, we examined the effect of daphnoretin on differentiation and maturation of human myeloid dendritic cells (DCs). After treatment with daphnoretin (0, 1.1, 3.3, 10 and 30 μM) to initiate monocytes, the recovery rate of DCs was reduced in a dose-dependent manner. The mature DCs differentiated in the presence of daphnoretin had fewer and shorter dendrites. Daphnoretin modulated DCs differentiation and maturation in terms of lower expression of CD1a, CD40, CD83, DC-SIGN, and HLA-DR. Daphnoretin inhibited the allostimulatory activity of DCs on proliferation of naive CD4⁺ CD45⁺ RA⁺ T cell. On the mitogen-activated protein kinase, daphnoretin down-regulated the lipopolysaccharide-augmented expression of phosphorylated c-Jun N-terminal kinase (pJNK), but not p38 and extracellular signal-regulated kinase 1/2 (ERK1/2). Activation of JNK by anisomycin reversed the effect of daphnoretin on daphnoretin-inhibited pJNK expression and dendrite formation of DCs. In disease model related to maturation of DCs, daphnoretin suppressed the acute rejection of skin allografts in mice. Our results suggest that daphnoretin modulated differentiation and maturation of DCs toward a state of atypical maturation with impaired allostimulatory function and this effect may go through down-regulation of phosphorylated JNK.     

Effects of Zizyphus lotus L. (Desf.) polyphenols on Jurkat cell signaling and proliferation

We assessed the effects of Zizyphus lotus L. (Desf.) polyphenols (ZLP) on T-cell signaling and proliferation. Our results showed that ZLP exerted no effect on the increases in intracellular free calcium concentrations, [Ca^2 +]_i, in human Jurkat T-cells. However, ZLP modulated the thapsigargin-induced increases in [Ca^2 +]_i in these cells. ZLP treatment was found to decrease the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). In addition, ZLP induced a rapid (t_1/2 = 33 s) and dose-dependent decrease in intracellular pH (pH_i) in human Jurkat T-cells. Furthermore, ZLP significantly curtailed T-cell proliferation by diminishing their progression from S to G2/M phase of cell cycle, and the expression of interleukin-2 (IL-2) mRNA. Taken together, the results of the present study demonstrate that ZLP modulate cell signaling and exert immunosuppressive effects in human T-cells.