Homeobox B5 suppression attenuates proliferation and elevates apoptosis in hepatoma cell lines through ERK/MDM2 signalling.

Homeobox B5 (HOXB5), a member of the HOX gene family, is an important gene in tumourigenesis. However, its role in hepatocellular carcinoma (HCC) cell proliferation and apoptosis remains unclear. In this study, we investigated the role and regulation mechanism of HOXB5 in HCC cell lines Hep3B and LM6. The data indicated high expression of HOXB5 in HCC tissues and cell lines. In HCC cells, inhibition of HOXB5 by transfection with HOXB5 siRNA significantly constrained cell viability, and Bcl-2 levels, and it increased cell apoptosis, cytochrome c levels, BAX levels, and caspase-3 activity. On the contrary, HOXB5 overexpression increased proliferation and Bcl-2 levels but inhibited BAX levels and caspase-3 activity in these cells. HOXB5 downregulation attenuated activation of extracellular signal-regulated kinase (ERK) and expression of the murine double minute 2 (MDM2) oncogene. Incubation with the ERK activator, phorbol 12-myristate 13-acetate (40 µmol/L), for 12 hours reversed the effects of HOXB5 inhibition on MDM2 expression, cell proliferation, and apoptosis in HCC cells. Overall, this study demonstrated that HOXB5 inhibition regulated MDM2 expression by controlling ERK activation and that it modulated proliferation and apoptosis in HCC cells.

Integrated Satellite, Unmanned Aerial Vehicle (UAV) and Ground Inversion of the SPAD of Winter Wheat in the Reviving Stage.

Chlorophyll is the most important component of crop photosynthesis, and the reviving stage is an important period during the rapid growth of winter wheat. Therefore, rapid and precise monitoring of chlorophyll content in winter wheat during the reviving stage is of great significance. The satellite-UAV-ground integrated inversion method is an innovative solution. In this study, the core region of the Yellow River Delta (YRD) is used as a study area. Ground measurements data, UAV multispectral and Sentinel-2A multispectral imagery are used as data sources. First, representative plots in the Hekou District were selected as the core test area, and 140 ground sampling points were selected. Based on the measured SPAD values and UAV multispectral images, UAV-based SPAD inversion models were constructed, and the most accurate model was selected. Second, by comparing satellite and UAV imagery, a reflectance correction for satellite imagery was performed. Finally, based on the UAV-based inversion model and satellite imagery after reflectance correction, the inversion results for SPAD values in multi-scale were obtained. The results showed that green, red, red-edge and near-infrared bands were significantly correlated with SPAD values. The modeling precisions of the best inversion model are R² = 0.926, Root Mean Squared Error (RMSE) = 0.63 and Mean Absolute Error (MAE) = 0.92, and the verification precisions are R² = 0.934, RMSE = 0.78 and MAE = 0.87. The Sentinel-2A imagery after the reflectance correction has a pronounced inversion effect; the SPAD values in the study area were concentrated between 40 and 60, showing an increasing trend from the eastern coast to the southwest and west, with obvious spatial differences. This study synthesizes the advantages of satellite, UAV and ground methods, and the proposed satellite-UAV-ground integrated inversion method has important implications for real-time, rapid and precision SPAD values collected on multiple scales.

CXCR5+ CD8+ T cells potently infiltrate pancreatic tumors and present high functionality.

Despite continued improvement in conventional therapy, pancreatic cancer continues to be one of the deadliest tumors worldwide with abysmal 5-year survival rate. New immunotherapeutic strategies that aim at improving antitumor cytotoxic CD8+ T cell responses are being developed in solid tumors. To assist the development of immunotherapies, we investigated the CD8+ T cells in pancreatic cancer patients. Compared to healthy individuals, pancreatic cancer patients presented a significant enrichment in the frequency of CD8+CXCR5+ T cells. In the tumor microenvironment, the frequencies of CD8+CXCR5+ T cells were further increased. In most cases, over half of tumor-infiltrating CD8+ T cells were CD8+CXCR5+ T cells. Compared to the circulating population, the tumor-infiltrating CD8+CXCR5+ T cells expressed higher levels of PD-1 and TIM-3. Functional analyses demonstrated that upon CD3/CD28 activation, the percentages of TNF-expressing and IFN-γ-expressing cells in CD8+CXCR5+ T cells were significantly higher than that in CD8+CXCR5- T cells. CD8+CXCR5+ T cells also presented enhanced cytotoxicity than CD8+CXCR5- T cells. Upon PD-1 and TIM-3 blockade, the functions of CD8+CXCR5+ T cells were further improved. The disease-free survival of pancreatic cancer patients following tumor resection was positively correlated with the frequencies of circulating and tumor-infiltrating CD8+CXCR5+ T cells. Together, our study identified that CD8+CXCR5+ T cells were a potent subset of CD8+ T cells that were highly enriched in pancreatic cancer patients and could respond to anti-PD-1/anti-TIM-3 blockade by further upregulation in function.

Determination of two potential toxicity metabolites derived from the disruption of the pksCT gene in Monascus aurantiacus Li As3.4384.

BACKGROUND: We previously demonstrated that disruption of the pksCT gene of Monascus led to a greater than 98% decrease in its citrinin production capacity in Monascus (PHDS26). Two potentially toxic compounds, monascopyridine A (MPA) and monascopyridine B (MPB), were found in the fermentation products of the pksCT gene-disrupted Monascus. Moreover, a rapid and reliable high-performance liquid chromatography method was developed for the simultaneous determination of MPA and MPB. We studied the effects of various extraction parameters and designed an orthogonal experiment to investigate the importance of each factor. RESULTS: The optimal extraction conditions were: methanol concentration, 90%; extraction temperature, 40 °C; extraction time, 10 min; two extraction cycles; and a solid-liquid ratio of 1:25. Under the optimal chromatographic conditions, good linearity was reached over the concentration ranges 0.5-200 µg mL-1 and 0.5-300 µg mL-1 for MPA and MPB, respectively, and the corresponding determination coefficients were 0.9999 and 0.9997. The percentage relative standard deviation values of within-day and between-day precision for MPA were 2.0% and 2.1%, respectively; the corresponding values for MPB were 4.8% and 4.6%. The average recovery for MPA and MPB was 99.9% and 94%, respectively. CONCLUSION: Maximum MPA and MPB yields (2073.7 and 1961.7 µg g-1 , respectively) were observed after 16 days of cultivation. © 2017 Society of Chemical Industry.

Urchin-like gold nanoparticle-based immunochromatographic strip test for rapid detection of fumonisin B1 in grains.

An immunochromatographic strip (ICS) using urchin-like gold nanoparticles (UGNs) for sensitive detection of fumonisin B1 (FB1) was developed to meet the requirement for rapidly monitoring FB1 in grain samples. The sensitivity of the ICS was 5.0 ng/mL, which represents a fourfold increase in sensitivity over conventional strip preparation using colloidal gold as the antibody-labeled probe. Analysis of FB1 in grain samples showed that data obtained from the strip tests were in a good agreement with those obtained from HPLC and enzyme-linked immunosorbent assays (ELISAs). This qualitative test did not require any specialized equipment, and the detection time was less than 5 min, which is suitable for on-site testing of FB1 in grain samples. Overall, to our knowledge, this is the first report of using a UGN as the antibody-labeled probe for sensitive detection of FB1 in grains using an ICS. Graphical Abstract Preparation of ICS using conventional colloidal gold and urchin-like gold nanoparticle, respectively.

Systemic TNFα gene therapy synergizes with liposomal doxorubicine in the treatment of metastatic cancer.

Tumor necrosis factor alpha (TNFα) is a potent antitumoral cytokine, either killing tumor cells directly or affecting the tumor vasculature leading to enhanced accumulation of macromolecular drugs. Due to dose limiting side effects systemic administration of TNFα protein at therapeutically active doses is precluded. With gene vectors, tumor restricted TNFα expression can be achieved and in principle synergize with chemotherapy. Synthetic gene carriers based on polyamines were intravenously injected, which either passively accumulate within the tumor or specifically target the epidermal growth factor receptor. A single intravenous injection of TNFα gene vector promoted accumulation of liposomal doxorubicine (Doxil) in murine neuroblastoma and human hepatoma by enhancing tumor endothelium permeability. The expression of transgenic TNFα was restricted to tumor tissue. Three treatment cycles with TNFα gene vectors and Doxil significantly delayed tumor growth in subcutaneous murine Neuro2A neuroblastoma. Also tumors re-growing after initial treatment were successfully treated in a fourth cycle pointing at the absence of resistance mechanisms. Systemic Neuro2A metastases or human LS174T colon carcinoma metastases in liver were also successfully treated with this combined approach. In conclusion, this schedule opens the possibility for the efficient treatment of tumors metastases otherwise not accessible for macromolecular drug carriers.

[Soil Carbon Pool Allocation Dynamics During Soil Development in the Lower Yangtze River Alluvial Plain].

The allocation dynamics of soil carbon pools during soil development and land use are the key to revealing the carbon cycle process. To clarify the distribution of the soil carbon pool and its change trend, a soil reclamation chronosequence (0 a, 60 a, 160 a, 280 a, 1 000 a, and 1 500 a reclamation) was established in a typical alluvial plain in the Lower Yangtze River, and the content and density of soil organic carbon (SOC), soil inorganic carbon (SIC), particulate organic carbon (POC), and mineral-associated organic carbon (MAOC), along with carbon sequestration potential (CSP) indicators of topsoil under different land use types were measured and analyzed. The results showed that after approximately 1 500 a reclamation, the SOC content developed from the Yangtze River alluvial deposits generally increased by 4.9% after the initial decline, whereas the SIC content decreased to 0.2% from 25.8% of the total carbon content due to its rapid leaching. The MAOC content was normally higher than that of POC, and MAOC was contributing 48.0%-79.7% of the SOC accumulation. In this region, the soil organic carbon density (SOCD) accounted for 57.4%-100% of the total carbon density, the soil carbon sequestration levels (CSL) ranged from 18.6% to 56.1%, and CSP under paddy-dryland rotation increased by 20.8% compared to that under dryland. The C/N ratio and total nitrogen content are key factors in explaining soil carbon accumulation processes, and the reclamation year plays an important role in evaluating soil carbon sequestration levels. After long-term utilization, the cultivated soil in the Yangtze River floodplain must be carefully managed through balanced fertilization to maintain soil productivity, promote the accumulation of SOC, and avoid the decline in soil carbon sequestration capacity.

Generation of a tumor- and tissue-specific episomal non-viral vector system.

BACKGROUND: A key issue for safe and reproducible gene therapy approaches is the autologous and tissue-specific expression of transgenes. Tissue-specific expression in vivo is either achieved by transfer vectors that deliver the gene of interest into a distinct cell type or by use of tissue-specific expression cassettes. Here we present the generation of non-viral, episomally replicating vectors that are able to replicate in a tissue specific manner thus allowing tissue specific transgene expression in combination with episomal replication. The episomal replication of the prototype vector pEPI-1 and its derivatives depends exclusively on a transcription unit starting from a constitutively active promoter extending into the scaffold/matrix attachment region (S/MAR). RESULTS: Here, we exchanged the constitutive promoter in the pEPI derivative pEPito by the tumor specific alpha fetoprotein (AFP) or the muscle specific smooth muscle 22 (SM22) promoter leading to specific transgene expression in AFP positive human hepatocellular carcinoma (HUH7) and in a SM22 positive cell line, respectively. The incorporation of the hCMV enhancer element into the expression cassette further boosted the expression levels with both promoters. Tissue specific-replication could be exemplary proven for the smooth muscle protein 22 (SM22) promoter in vitro. With the AFP promoter-driven pEPito vector hepatocellular carcinoma-specific expression could be achieved in vivo after systemic vector application together with polyethylenimine as transfection enhancer. CONCLUSIONS: In this study we present an episomal plasmid system designed for tissue specific transgene expression and replication. The human AFP-promoter in combination with the hCMV enhancer element was demonstrated to be a valuable tissue-specific promoter for targeting hepatocellular carcinomas with non-viral gene delivery system, and tissue specific replication could be shown in vitro with the muscle specific SM22 promoter. In combination with appropriate delivery systems, the tissue specific pEPito vector system will allow higher tissue-specificity with less undesired side effects and is suitable for long term transgene expression in vivo within gene therapeutical approaches.

Geochemical behavior and source analysis of rare earth elements in intensive agriculture soils through high-resolution sampling.

Rare Earth Elements (REEs) are crucial for a low-carbon economy but overuse pollutes ecosystems. Studying their behavior and source in agricultural soils provides vital insights for soil management. To address this, we collected an extensive dataset comprising 2901 topsoil and 733 subsoil samples from an agriculturally dominant region in eastern China, characterized by uniform parent material. This comprehensive dataset enabled a thorough examination of the geochemical behavior and sources of REEs in the region. Our findings revealed the feasibility of utilizing La, Ce, and Y to predict ΣREE, LREE, HREE, and LREE/HREE ratios, thereby enhancing the REEs database for eastern China. The average ΣREE concentration in the study area measured 193.0 µg/g, with LREEs constituting 89 % of this total, representing a slight enrichment relative to the Upper Continental Crust. Notably, the electron-donating ability of Ce exhibited a correlation with Mn, indicating its potential as an indicator of redox reactions. The results obtained through structural equation modeling and random forest analysis identified Al, Ti, and K as the most influential environmental factors affecting ΣREE concentrations. Additionally, the indirect impact of K on ΣREE through pH was established. The strong correlation observed between major elements and Heavy Rare Earth Elements (HREEs) shed light on the mechanisms responsible for the notable fractionation of REEs in topsoil. Furthermore, our investigation underscored the substantial influence of pedogenetic processes on the enrichment and leaching of REEs. Source analysis pinpointed significant contributors to REEs, including fertilizers, pesticides, domestic wastewater, coal fly ash, and the native soil parent material. In conclusion, our study emphasizes the pressing need for judicious control of anthropogenic REE inputs. This step is pivotal not only for ensuring the sustainable utilization of soil but also for ultimately safeguarding ecosystems and environmental quality.

A Selective Separation Mechanism for Mono/divalent Cations and Properties of a Hollow-Fiber Composite Nanofiltration Membrane Having a Positively Charged Surface.

Positively charged nanofiltration (NF) technology is considered a green and low-cost method for mono/divalent cation separation. Nevertheless, the separation rejection mechanisms of these NF membranes have yet to be extensively investigated. In this work, we fabricated a thin-film composite (TFC) hollow-fiber (HF) NF membrane with a positively charged surface via modification of the nascent interfacial polymerization layer using a branched polyethyleneimine (BPEI)/ethanol solution. Then, we extensively investigated its selective separation mechanism for mono/divalent cations. We proposed and proved that there exists a double-charged layer near the membrane surface, which helps to repel the divalent cations selectively via Donnan exclusion while promoting the fast penetration of monovalent cations. Meanwhile, the membrane skin layer is loose and hydrophilic due to the loose BPEI structure and the abundance of amine groups, as well as the changed fabrication conditions. In this way, we achieved very good mono/divalent cation selectivity and relatively high water permeance for the as-prepared HF NF membrane. We also obtained good anti-fouling, anti-scaling, and acid resistance, and long-term stability as well, which are urgently needed during practical application. Furthermore, we successfully amplified this HF NF membrane and proved that it has broad application prospects in mono/divalent cation separation.

Hollow Fiber Membrane for Organic Solvent Nanofiltration: A Mini Review.

Organic solvents take up 80% of the total chemicals used in pharmaceutical and related industries, while their reuse rate is less than 50%. Traditional solvent treatment methods such as distillation and evaporation have many disadvantages such as high cost, environmental unfriendliness, and difficulty in recovering heat-sensitive, high-value molecules. Organic solvent nanofiltration (OSN) has been a prevalent research topic for the separation and purification of organic solvent systems since the beginning of this century with the benefits of no-phase change, high operational flexibility, low cost, as well as environmental friendliness. Especially, hollow fiber (HF) OSN membranes have gained a lot of attention due to their high packing density and easy scale-up as compared with flat-sheet OSN membranes. This paper critically reviewed the recent research progress in the preparation of HF OSN membranes with high performance, including different materials, preparation methods, and modification treatments. This paper also predicts the future direction of HF OSN membrane development.

Graphene Quantum Dots-Doped Thin Film Nanocomposite Polyimide Membranes with Enhanced Solvent Resistance for Solvent-Resistant Nanofiltration.

The core of the organic solvent nanofiltration (OSN) technology is solvent-resistant nanofiltration (SRNF) membranes. Till now, relative poor performance of solvent resistance is still the bottleneck of industrial application of SRNF membranes. This work reports a novel polyimide (PI)-based thin-film nanocomposite (TFN) membrane which was embedded with graphene quantum dots (GQDs) and showed an improved solvent resistance for OSN application. This kind of SRNF membrane, termed (PI-GQDs/PI)XA, was synthesized via serial processes of interfacial polymerization (IP), imidization, cross-linking, and solvent activation. The IP process was performed between an aqueous m-phenylenediamine solution doped with GQDs, having an average size of 1.9 nm, and an 1,2,4,5-benzenetetracarboxylic acyl chloride n-hexane solution on the PI substrate surface. The prepared (PI-GQDs-50/PI)X SRNF membranes without organic solvent activation achieved an ethanol permeance of nearly 50% higher than those of the GQD-free membranes under the same preparation conditions, while no compromise of the dye rejection was observed. Further, after the solvent activation using N, N-dimethylformamide (DMF) at 80 °C for 30 min, the ethanol permeance achieved about an 8-folds increment, from 2.84 to 22.6 L m-2 h-1 MPa-1. Interestingly, the rejection of rhodamine B also increased from 97.8 to 98.6%. A long-term permeation test of more than 100 h using rose bengal (RB, 1017 Da)/DMF solution at room temperature demonstrated that the synthesized (PI-GQDs-50/PI)XA membranes could maintain the DMF permeance and the RB rejection as high as 18.3 L m-2 h-1 MPa-1 and 99.9%, respectively. Moreover, the immersion test of the prepared (PI-GQDs-50/PI)XA SRNF membranes in both DMF and ethanol at room temperature for about one year also demonstrated the long-term organic solvent stability, indicating their good potential for OSN application.

Cimetidine enhances immune response of HBV DNA vaccination via impairment of the regulatory function of regulatory T cells.

Cimetidine (CIM), a histamine 2-receptor antagonist, is postulated to enhance immune responses owing to its inhibitory effects on suppressor T cells. In this report, we evaluated effects of cimetidine on the potency of antigen-specific immunity generated by DNA vaccine encoding hepatitis B surface antigen (HBsAg, pcD-S2). Our data demonstrate that CIM as adjuvant significantly increased HBsAg-specific cell-mediated and humoral immunities that were characterized by higher Ig2a/IgG1 ratio. In addition, CIM significantly promotes an elevated level of IL-4 and IFN-gamma in antigen-specific CD4(+) T cells and a robust antigen-specific cytotoxic response in the animals immunized with pcD-S2 plus CIM. Further, CIM induces pro-inflammatory cytokine expression such as the IL-12 and down-regulates anti-inflammatory cytokine expression such as IL-10 and TGF-beta, which may lead to an impairment of CD4(+)CD25(+) Treg cell-mediated suppression. Collectively these findings suggest that CIM enhances the immune responses of HBV DNA vaccine through the stimulation of pro-inflammatory and inhibition of anti-inflammatory cytokine expression patterns.

Spatiotemporal variability of soil nutrients and the responses of growth during growth stages of winter wheat in northern China.

Studying soil nutrient variability and its effect on the growth and development of crops under a traditional tillage mode is the foundation for comprehensively implementing precision agriculture policies at the field scale and ensuring excellent crop management. In this paper, a 28.5 hm2 winter wheat field under the traditional cultivation model in Tianzhuang town of Huantai County was selected as the research area. The mesh point method was utilized for sampling (60×60 m), and the characteristics of soil available nitrogen (AN), available phosphorus (AP) and available potassium (AK) variations in the before sowing, reviving, jointing, and filling stages of winter wheat were analyzed using geostatistical and GIS methods. Moreover, Pearson correlation analysis was used to study the response of wheat growth and development to soil nutrient variations. As the growth stages progressed, 1) each nutrient showed the characteristics of low-high-low and moderate variability. The highest AN and AK contents were found at the reviving stage, while AP reached a turning point at the jointing stage. The order of variability of each nutrient was AN>AP>AK. 2) The nutrient variations first increased and then decreased and showed medium to strong spatial correlation. The three nutrients were strongly spatially correlated in the before sowing stage and moderately spatially correlated during the reviving stage. During the jointing and filling stages, AN had moderate spatial correlation, and AP and AK had strong spatial correlation. The spatial correlation of each nutrient was the weakest in the reviving stage, and the spatial correlation of AN was strongest in the before sowing stage, while the spatial correlations of AP and AK were strongest in the jointing stage. The spatial correlation of each soil nutrient decreased from the before sowing stage to the reviving stage and from the jointing stage to the filling stage, and the spatial correlation increased from the reviving stage to the jointing stage. 3) The soil nutrient content first increased and then decreased, and the grades of the nutrients gradually decreased. 4) The correlation between soil nutrients and wheat growth gradually increased. AN had the highest correlation with wheat growth, followed by AK and AP. The effect of soil nutrients on the growth of wheat at the reviving stage was greater than the effect of nutrients in the current stage. The growth of wheat at the jointing stage was mainly influenced by nutrients in the current stage, while the growth of wheat at the filling stage was influenced by the nutrient contents of both the previous and current stages. Thus, the date of fertilizer supplementation should be postponed properly. In this study, the soil nutrient dynamics and their influence on the growth of wheat during the winter wheat growth period under the traditional field model were well described, and these results could provide a theoretical basis for the precision management of soil nutrients in the northern winter wheat area where the planting environment and cultivation management are relatively uniform.

Sequential administration of cytokine genes to enhance cellular immune responses and CD4 (+) T memory cells during DNA vaccination.

Antigen specific memory T cells (Tm) have shown to be an important factor in protecting hosts against subsequent infection by previously encountered pathogens. During T-cell activation, several cytokines including IL-6, IL-7 and IL-15, play crucial roles in the development of T cells into memory T cells. With the aim of generating specific Tm, we examined a strategy of sequential administration of molecular adjuvants. In this strategy a DNA vaccine encoding the VP1 capsid protein of foot and mouth disease virus (designated pcD-VP1) was co-delivered to mice along with an IL-6 expressing plasmid (pVAX-IL-6) as an initial molecular adjuvant and boosted with either an IL-7 or IL-15 expressing plasmid, (pVAX-IL-7 or proVAX-IL-15) as the secondary adjuvant. During the pcD-VP1 immunization, we demonstrated that the groups primed with IL-6 and boosted with either IL-7 or IL-15 resulted in the enhancement of cellular and humoral immune responses, maturation of dendritic cells (DCs) and macrophages, and a higher frequency of CD4 (+) Tm (characterized by expressing CD44 (high) CD62L (low) markers, compared with the other groups). Thus, we took advantage of the different effects of cytokines on T cell development, not only to induce a higher level of immune responses after vaccination, but also to generate a higher ratio of CD4 (+) Tm in this sequential cytokine prime-boost study. This would then lead to the mounting of an effective long-term antigen specific immune response.

Expression and subcellular localization of menin in human cancer cells.

The aim of this study was to elucidate the expression and localization of menin, a protein encoded by the multiple endocrine neoplasia type I (MEN1) gene, in 13 human cancer cell lines. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expression of the menin gene. The localization of the menin protein was detected by immunofluorescence microscopy. Western blotting was used to determine the quantity of menin in the nucleus, cytosol and membrane of the cells. RT-PCR revealed that menin was expressed in all the cell lines examined in this study. Immunofluorescence microscopy revealed that menin was located primarily in the nucleus. In the GES-1 (transformed human gastric epithelium), MCF-7 (breast cancer), SGH44 (brain glioma) and HeLa (cervical cancer) cell lines, menin was also found to be localized to the membrane, cytosol and nucleus. Moreover, in SGH44 cells more menin was located in the cytosol than the nucleus. Similar findings were obtained by western blotting. In the GES-1 and MKN-28 cells undergoing octreotide treatment, cytoplasmic menin was significantly increased compared with the control groups. Therefore, we suggest that menin is expressed in a number of human cancer cell lines and that the cytosolic distribution increases when the cells undergo octreotide treatment, indicating a new role for menin.

NanoPARCEL: a method for controlling cellular behavior with external light.

We developed a simple preparation procedure for the protein encapsulated nanoparticle and used the nanoparticle for spatiotemporal activity control of various proteins. We succeeded in the local protein activation within cells by light using the nanoparticle.

Cimetidine augments Th1/Th2 dual polarized immune responses to recombinant HBV antigens.

Cimetidine (CIM) is a histamine H2 receptor inverse agonist used primarily as an anti-stomach acids drug, but recent studies showed that it may also modulate immune responses. To evaluate its potential usefulness as an adjuvant, we determined its immune modulating effects on subunit immunization using an HBV-derived recombinant protein antigen rHBsAg. CIM activated the PI3K-Akt signaling pathway in DCs. As an adjuvant, it activated immunogenic DCs, deactivated tolerogenic T cells (nTreg), and augmented both Th1- and Th2-polarlized immune responses to rHBsAg. As a result, it enhanced both antibody- and cytotoxic T cell-mediated immune responses. Importantly, in comparison with the FDA-approved human adjuvant alum, CIM is superior in its ability to block IL-10 up-regulation and potentiate Th1/Th2 dual polarization. These results suggest that CIM may be a better adjuvant for therapeutic vaccines against chronic viral infection, such as the HBV infection, where dual polarization should allow more effective elimination of infected host cells.

PyNTTTTGT prototype oligonucleotide IMT504, a novel effective adjuvant of the FMDV DNA vaccine.

Synthetic oligodeoxynucleotides (ODNs) such as CpG can stimulate B and plasmacytoid dendritic cells in vertebrate immune systems. Several studies showed that non-CpG ODNs could also induce strong stimulation of B and T cells. PyNTTTTGT ODNs, non-CpG ODNs, can activate and cause immunoglobulin secretion by B cells and proliferation of T cells in vivo. By using PyNTTTTGT ODNs as an adjuvant for a FMDV DNA vaccine, we found that levels of antibody production, T-cell proliferation, and CTL activity were significantly increased compared with the DNA vaccine alone. Compared with the adjuvant effects of CpG ODNs on DNA vaccination, similar levels of antibody production and T-cell proliferation, and higher levels of CTL activity and IFN-gamma expression in CD8 T cells were induced by the IMT504 ODNs. On the other hand, RT-PCR results show that IMT504 ODN may activate the DNA sensor of DAI (DNA-dependent activator of IFN regulatory factors) and partially stimulate TLR9. At this point, the PyNTTTTGT prototype IMT504 ODN can reasonably be predicted to be a good adjuvant for FMDV DNA vaccine in small animals, but its efficacy in larger animals remains to be explored.

The effects of IL-6 and TNF-alpha as molecular adjuvants on immune responses to FMDV and maturation of dendritic cells by DNA vaccination.

Various approaches have been developed to improve efficacy of DNA vaccination, such as the use of plasmid expressing cytokine as a molecular adjuvant. In this study, we investigated whether co-inoculation of a construct expressing either IL-6 or TNF-alpha as the molecular adjuvant with FMDV DNA vaccine, pcD-VP1, can increase immune responses. Compared to the group immunized with pcD-VP1 alone, the co-inoculation with either molecular adjuvant induced a higher ratio of IgG2a/IgG1, higher levels of expression of IFN-gamma in CD4+ and CD8+ T cells, IL-4 in CD4+ T cells, and in vivo antigen-specific cytotoxic response. Both adjuvants induced maturation of dendritic cells, suggesting a correlation between the initiating innate response and subsequent activating adaptive immune responses. Together, the results demonstrate that IL-6 and TNF-alpha used as molecular adjuvants can enhance the antigen-specific cell-mediated responses elicited by VP1 DNA vaccine.