[Spatial and Temporal Distribution and Source Variation of Heavy Metals in Cultivated Land Soil of Xiangzhou District Based on EBK Interpolation Prediction and GDM Model].

In order to explore the spatial and temporal changes in spatial patterns and source changes in heavy metals in Xiangzhou District, 395 and 326 soil samples were collected from cultivated soil in Xiangzhou District in November 2009 and November 2019, respectively. The contents of Cr, Pb, As, Hg, and Cd during these two years were measured. The spatial pattern and variation distribution of five types of heavy metals during these two years were obtained by using the empirical Bayesian Kriging (EBK) method. The effect (q-statistic) of 19 environmental factors and 5 types of heavy metals was calculated by using the geographical detector model (GDM), and the changes over the two years were compared. The results showed that compared with that in 2009, the heavy metal contents of Cr, Pb, Hg, and As in Xiangzhou District were decreased as a whole in 2019, whereas the Cd content increased overall. The spatial differentiation of heavy metals in the soil in Xiangzhou District in 2019 was more complicated than that in 2009. Pb, Hg, and Cd in the south and Hg in the central urban area and surrounding areas also increased. The content of each element decreased to the north and northwest. Compared with that in 2009, the explanatory power of natural factors and the distance between pollution enterprises on the single factor of the five soil heavy metal contents in 2019 decreased, and the influence on the contents under the control of single factors decreased significantly. The superposition influence of human activity factors increased, especially the distance between residential land, road, and land for pollution enterprises and environmental factors on soil heavy metal elements. These results indicated that the changes in soil heavy metal sources in 2019 tended to be complex, with structural factors as the main influencing factor. The influence of the emission of polluting enterprises on heavy metal elements decreased, whereas the influence of human activities on heavy metal content increased.

QSAR study on MHC class I a alleles based on the novel parameters of amino acids.

MHC-epitope binding plays a key role in the cellular immune response. Accurate prediction of MHC-epitope binding affinity can greatly expedite epitope screening by reducing costs and experimental effort. In this paper, 13 T descriptors， which derived from 544 physicochemical properties of the natural amino acids, were used to characterize 4 MHC class I alleles epitope peptide sequences, the optimal QSAR models were constructed by using stepwise regression combines with multiple linear regression (STR-MLR). For HLA-A*0201, HLA-A*0203, HLA-A*0206 and HLA-A*1101 alleles, the leave one out cross validation values (Q(2)(train)) were 0.581, 0.553, 0.525 and 0.588, the correlation coefficients (R(2)(train)) of training datasets were 0.607, 0.582, 0.556 and 0.606, the correlation coefficients (R(2)(test)) of test datasets were 0.533, 0.506, 0.501 and 0.502, respectively. The results showed that all models can obtain good performance for prediction and explain the mechanism of interaction between MHC and epitope. The descriptors will be useful in structure characterization and activity prediction of peptide sequences.

1,25-dihydroxyvitamin D3 pretreatment enhances the efficacy of allergen immunotherapy in a mouse allergic asthma model.

BACKGROUND: Allergen-specific immunotherapy can induce immune tolerance to specific allergens by regulating immune status of individuals. However, its clinical application is limited due to individual differences in efficacy among patients and un-confirmed safety. 1,25 Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) has been shown to be involved in a variety of physiological processes, including immune response regulation. In the present study we explored the role of 1,25(OH)(2)D(3) pretreatment for immunotherapy. METHODS: Seventy-five BALB/c mice were randomly divided into five groups (15 mice per group). The mouse allergic asthma model was established by intra-peritoneal injection of ovalbumin (OVA, 10 µg) and aluminium hydroxide (2 mg) as an adjuvant. Intra-peritoneal injection of 50 ng of 1,25(OH)(2)D(3) served as a pretreatment, subcutaneous injection of OVA (100 µg) as an immunotherapy, and 1% OVA inhalation as a challenge. Histopathological analysis was performed on four mice per group. The number of cells and their classification in bronchoalvolar lavage (BAL) fluid were assayed. Levels of serum OVA-specific immunoglobulin E (sIgE) and IFN-γ, IL-4, IL-5 and IL-10 in BAL fluid were measured by ELISA. RESULTS: After 1,25(OH)(2)D(3) pretreatment, immunotherapy could significantly inhibit the infiltration of inflammatory cells into lung tissues and BAL fluid of mice with allergic asthma when compared with un-treated animals (eosinophils: (7.46 ± 1.34) × 10(4)/ml vs. (13.41 ± 1.67) × 10(4)/ml, P < 0.05). In addition, levels of IL-4 ((36.91 ± 7.87) pg/ml vs. (43.70 ± 6.42) pg/ml, P > 0.05) and IL-5 ((41.97 ± 7.93) pg/ml vs. (60.14 ± 8.35) pg/ml, P < 0.05) in BAL fluid and serum sIgE ((0.42 ± 0.05) vs. (0.75 ± 0.06) OD units, P < 0.05) were profoundly reduced. However, the IL-10 level in BAL fluid was significantly increased ((67.74 ± 6.57) pg/ml vs. (44.62 ± 8.81) pg/ml, P < 0.05). CONCLUSIONS: These results indicated that 1,25(OH)(2)D(3) pretreatment enhanced the inhibitory effects of immunotherapy on allergic airway inflammation. In the treatment of allergic diseases, 1,25(OH)(2)D(3) pretreatment may be beneficial for improving the efficacy of immunotherapy.

[Insertion of TC motif into hepatitis B virus core protein c/e1 site does not affect the expression of S and e antigen].

OBJECTIVE: To investigate whether insertion of TC motif into hepatitis B virus (HBV) core protein c/e1 site affects the expression of S and e antigen. METHODS: Different oligonucleotides encoding TC motif were inserted into the c/e1 site of the core gene of a 1.3 copy wild-type HBV genome vector. HepG2 cells were divided into several groups of cells to transiently transfect with the wild-type and mutant HBV vectors, respectively. In each group, the expression level of core protein inside cells was detected by western blotting, and the levels of S and e antigen in culture medium were analyzed by ELISA assay. RESULTS: Western blotting showed that these TC-tagged core proteins were expressed at similar level of wild-type one. ELISA assay indicated that the level of S and e antigen in culture medium of different groups were not significantly different. CONCLUSION: Insertion of TC motif into HBV core protein c/e1 site does not interference with the expression of viral protein encoded by HBV genome.

Effects of two novel nucleoside analogues on different hepatitis B virus promoters.

AIM: To explore the effects of the nucleoside analogues beta-L-D4A and beta-LPA on hepatitis B virus (HBV) promoters. METHODS: Four HBV promoters were amplified by polymerase chain reaction (PCR) and subcloned into the expression vector pEGFP-1. The four recombinants controlled by HBV promoters were confirmed by restriction analysis and sequencing. Human hepatoma HepG2 cells transfected with the recombinant plasmids were treated with various concentrations of beta-L-D4A and beta-LPA. Then, enhanced green fluorescent protein (EGFP)-positive cells were detected by fluorescence microscopy and using a fluorescence activated cell sorter (FACS). RESULTS: Four HBV promoters were separately obtained and successfully cloned into pEGFP-1. Expression of EGFP under the control of the surface promoter (Sp) and the X promoter (Xp) was inhibited by beta-L-D4A in a dose-dependent manner, while expression of EGFP under the control of the core promoter (Cp) and Xp was inhibited by beta-LPA in a dose-dependent manner. CONCLUSION: The two novel nucleoside analogues investigated here can inhibit the activities of HBV promoters in a dose-dependent manner. These findings may explain the mechanisms of action by which these two novel compounds inhibit HBV DNA replication.

[Effects of immunotherapy on the expression of costimulatory molecules on dendritic cells in a mouse model of asthma].

OBJECTIVE: To establish a mouse model of ovalbium in (OVA) specific immunotherapy, and to explore the effects of specific immunotherapy on the expression of CD(80) and CD(86) on spleen-derived dendritic cells (DC) in a mouse asthmatic model. METHODS: A hundred and twenty BALB/c mice were randomly divided into three groups, with 40 mice each. The asthma model (group A) was established by intraperitoneal injection of 10 microg OVA and 1% OVA aerosol challenges. Consecutive subcutaneous injection of 1 mg OVA in the dorsal aspect of the foot was then given to establish the model of OVA specific immunotherapy (group B), while PBS (0.01 mol/L) was given in group C to serve as the control. The pathological changes of lung tissues were studied by HE stain. The serum OVA-specific IgE level, and IL-2 and IL-4 production in the splenic T cells were determined by enzyme-linked immunosorbent assay (ELISA). The expression of CD(80) and CD(86) on DC, which were isolated from the spleen, were detected by fluorescein-activated cell sorter. (3)H-thymidine ((3)H-TdR) incorporation assay was used to determine the response of splenic T and the secretion of interleukin-4 (IL-4) and IL-5 was assayed with the use of ELISA after coculture of T cells with DC. RESULTS: (1) The infiltration of eosinophils and lymphocytes within epithelium and lamina propria was present in group B, but was milder than that in group A. The absorbance at 490 nm (A(490)) of serum specific IgE in group A 712 +/- 129, was more than that in group C (20 +/- 13, P < 0.05), but there was no significant difference between group B (124 +/- 59, P > 0.05) and C. The production of IL-2 by splenic T cells in group B (8 +/- 3) pg/ml was less than that in group A [(22 +/- 8) pg/ml, P < 0.05], but there was no significant difference between group B and group C [(6 +/- 4) pg/ml, P > 0.05]. The production of IL-4 by splenic T cells in group B (8.4 +/- 4.3) pg/ml was less than that of group A [(32.4 +/- 12.1) pg/ml, P < 0.05], but there was no significant difference between group B and group C [(5.1 +/- 1.1) pg/ml, P > 0.05]. (2) The expression of CD(86) on splenic DC cells from group B (58.23%) was significantly lower than that from group A (77.59%) and group C (77.84%), while the expressions of CD(80) in group A (96.98%) and group B (95.63%) were higher than that in group C (77.37%). (3) When untreated T cells were cocultured with DC from the individual groups and stimulated by OVA, the secretion of IL-4 from T cells in group B [(10.8 +/- 2.3) pg/ml] was significantly lower than that in group A [(17.3 +/- 4.7) pg/ml, P < 0.05], but there was no significant difference when compared to group C [(5.7 +/- 2.7) pg/ml, P > 0.05], and the secretion of IL-5 from T cells in group B [(18.8 +/- 3.8) pg/ml] was significantly lower than that in group A [(35.7 +/- 7.9) pg/ml, P < 0.05], but there was no significant difference when compared to group C [(11.0 +/- 2.2) pg/ml, P > 0.05]. When untreated T cells were cocultured with DC from the individual groups and stimulated by OVA, the stimulating index of T cells in group B (3.8 +/- 0.7) was significantly lower than that in group A (11.5 +/- 3.2, P < 0.05), but there was no significant difference when compared to group C (5.8 +/- 1.5, P > 0.05). CONCLUSIONS: A mouse model of asthma-specific immunotherapy was successfully established. Downregulation of CD(86) on the surface of DC might be the underlying mechanisms by which specific immunotherapy promotes Th1 to Th2 polarization and induces T cell anergy.

[Immunological efficiency of DNA vaccine targeting dendritic cells against tumor].

AIM: To construct the DNA vaccine containing ovalbumin (OVA) and Fc fusion gene targeting dentritic cells (DCs) and evaluate its anti-tumor efficiency in an E.G7-OVA-bearing tumor model. METHODS: Constructed OVA-Fc-pcDNA3.1 plasmids were transfected into CHO cells with lipofectamine and the in vitro expression of OVA-Fc was determined by flow cytometry and ELISA. (51)Cr-relaese assay was used to determine the anti-tumor activity of cytotoxic T lymphocytes (CTLs) from splenocytes of immunized mice. According to tumor volume and survival time of the mice, the therapeutic effect of this vaccine was evaluated in E.G7-OVA-bearing tumor model. RESULTS: DNA sequencing and restriction endonuclease digestion analysis indicated that the recombinant expression vector OVA-Fc-pcDNA3.1 had been constructed successfully. OVA-Fc expression could be detected in CHO cells transfected with OVA-Fc-pcDNA3.1 by ELISA and flow cytometry. The DNA vaccine containing OVA-Fc fusion gene inhibited tumor's growth and prolonged the time of tumor-bearing mice due to elicit the CTL-mediated anti-tumor immunity. CONCLUSION: OVA-Fc-pcDNA3.1 DNA vaccine targeting dendritic cells can elicit the CTL-mediated anti-tumor immunity, which lays the foundation for further clinical experiments.

[Exploration of mechanisms underlying T-cell anergy induced by immunotherapy in allergic murine asthma model].

AIM: To explore the mechanisms underlying the T cell anergy induced by immunotherapy for the treatment of allergic asthma. METHODS: Murine model of allergic asthma was established through the OVA sensitization and challenge. Three injections of 1 mg OVA were used as immunotherapy. Pathological examination of lung tissues, cell count and differential count of bronchoalveolar lavage fluid (BALF), determination of serum OVA-specific IgE, and measurement of IL-2 and IL-4 production and proliferation of the splenic T cells to OVA were preformed to analyze the effect of immunotherapy on asthma. The expression of CTLA-4 on splenic T cells was detected by FACS. RESULTS: Asthma-specific immunotherapy could alleviate lung inflammation, decrease eosinophil infiltration in BALF (P<0.01) and serum OVA-specific IgE, inhibit the production of IL-2 and IL-4 and the proliferation of splenic T cells to OVA (P<0.01), and up-regulate the expression of CTLA-4 on splenic T cells. CONCLUSION: The up-regulation of CTLA-4 expression on T cells may be involved in the T cell anergy induced by asthma-specific immunotherapy.

[The mechanism and implication of costimulatory molecules in dendritic cells in the development of asthma].

OBJECTIVE: To exploring the role of costimulatory molecules in the development of asthma and its mechanisms. METHODS: Murine asthma model was established with ovalbumin (OVA) sensitization and challenge, and the model was confirmed by histological analysis of lung tissues, cell numbers and differentiations of bronchoalveolar lavage, serum OVA-specific IgE level and interleukin (IL)-4 and IL-5 production by splenic T cells. The purity of spleen-derived dendritic cells was assayed with fluorescein-actived cell sorter (FACS) by analyzing CD(11c) molecule. FACS was also used to measure the expression of CD(80) and CD(86) on spleen-derived dendritic cell (DC) from OVA-sensitized and challenged mice. Finally, the production of IL-4 and IL-5 in naive T cells after stimulation with spleen-derived DC from OVA-sensitized and challenged mice were determined with ELISA. RESULTS: Histological analysis of lung tissues, components of broncho-alveolar fluid, the level of serum OVA-specific IgE, and the production of IL-4 and IL-5 were all consistent with the characteristic of a murine asthma model. The expression of CD(80) on spleen-derived DC from OVA-sensitized and challenged mice was increased significantly compared with that from PBS-treated mice, while there was no difference in CD(86) expression. On the other hand, DC from OVA-sensitized and challenged mice stimulated the production of IL-4 and IL-5 by naïve T cells. CONCLUSION: Our results suggest that DC, via upregulation of CD(80), might play a pivotal role in the maintenance and amplification of allergic immune response, namely the Th2 immune response.

[In vitro induction of immune response by dendritic cells pulsed with TRAG-3-derived cytotoxic T lymphocyte epitope].

OBJECTIVE: To explore the in vitro immune response to taxol resistance associated antigen 3 (TRAG-3)-derived cytotoxic T lymphocyte (CTL) epitope-pulsed dendritic cell (DC). METHODS: The HLA-A2.1 restricted CTL epitope of TRAG-3 was previously identified to be a nonameric peptide sequence from 58 to 66 amino acid residues (TRAG-3(58-66)). The peptide was synthesized according to Fmos procedure, purified and molecular weight determined. Peripheral blood mononuclear cells (PBMC) of normal HLA-A2.1(+) individuals were used to isolate DC and DCs' phenotype identified by flow cytometry. Induction of CTL was achieved by in vitro stimulation of HLA-A2.1(+) PBMC with peptide-pulsed DC. CTL activity against HLA-A2.1(+)/TRAG-3(+) melanoma cell line LB373-MEL was assessed by (51)Cr release assay and IFN-gamma released by ELISA. RESULTS: The synthetic nonameric peptide was above 90% pure and the determined values of molecular weight were conformed to their theoretical values. The phenotype of the isolate DCs was characteristic for mature ones. PBMC stimulated in vitro by TRAG-3-derived epitope-pulsed DCs for three times could specifically kill the target cells and secreted high concentration of IFN-gamma. CONCLUSION: TRAG-3-derived epitope-pulsed DC can elicit specific anti-tumor immune response in vitro. Clinical trials using it as tumor vaccine may be feasible as specific immunotherapy for patients with TRAG-3 expressing cancers.