[Preparation of CD33 targeted bispecific- and trispecific-T cell engagers and their cytotoxicity on leukemia cells].

Objective: To investigate the effect of CD33-targeted bi-specific and tri-specific T-cell engagers on T-cell proliferation and explore their cytotoxicity on leukemia cells. Methods: The CD33-targeted bi-specific T-cell engager (CD33-BiTE) and tri-specific T-cell engager (CD33-TriTE) expression vectors were successfully constructed and expressed through a eukaryotic cell expression system. CD33-BiTE and CD33-TriTE were purified by affinity chromatography. The effects of CD33-BiTE and CD33-TriTE on T cells were analyzed through in vitro experiments. Results: ① CD33-BiTE and CD33-TriTE were successfully constructed and purified and could compete with flow cytometry antibodies for binding to the target cells. ② After 12 days of co-culture with CD33-BiTE and CD33-TriTE, the number of human T cells were expanded to 33.89±19.46 and 81.56±23.62 folds, respectively. CD33-TriTE induced a stronger proliferation of T cells than CD33-BiTE (P<0.05) . ③ Both CD33-BiTE and CD33-TriTE induced specific dose-dependent cytotoxicity on CD33(+) leukemia cells. ④ Compared to CD33-TriTE, leukemia cells were prone to express PD-L1 when co-cultured with T cells and CD33-BiTE. CD33-TriTE induced powerful cytotoxicity on leukemia cells with high PD-L1 expression. Conclusion: CD33-BiTE and CD33-TriTE expression vectors were constructed, and fusion proteins were expressed in eukaryotic cells. Our results support the proliferative and activating effects of BiTE and TriTE on T cells. Compared to that of CD33-BiTE, CD33-TriTE induced a stronger proliferative effect on T cells and a more powerful cytotoxicity on leukemia cells with high PD-L1 expression.

[Functional investigation of chimeric antigen receptor T cells targeting LMP1 antigen].

Objective: This study aimed to create a type of CAR-T cells that targets LMP1 antigen and study its immunotherapeutic effect on LMP1-positive hematological malignancies. Methods: To generate LMP1 CAR-T cells, a plasmid expressing LMP1 CAR was created using molecular cloning technology, and T cells were infected with LMP1 CAR lentivirus. The effects of LMP1 CAR-T cells on specific cytotoxicity against LMP1-positive tumor cell lines infected with the EB virus had been confirmed. Results: ① LMP1 protein expressing on EB virus-positive lymphoma cells surface was verified. ② The LMP1 CAR-expressing plasmid was created, and LMP1 CAR-T cells were obtained by infecting T cells with a lentivirus packaging system, with an infection efficiency of more than 80% . ③LMP1 CAR-T cells have a 4∶1 effect-to-target ratio in killing LMP1-positive lymphoma cells. The killing effect of LMP1 CAR-T cells on Raji cells was enhanced after 48 h of coculture, but there was no significant killing effect on Ramos, which are LMP1-negative lymphoma cells. ④After coculture with LMP1-positive lymphoma cells at a ratio of 1∶1 for 5 h, the degranulation effect was enhanced. The proportion of CD107a(+) T cells in the LMP1 CAR-T cell treatment group was significantly higher than that in the vector-T cell group [ (13.25±2.94) % vs (1.55±0.05) % , t=3.972, P=0.017]. ⑤After coculture with LMP1-positive lymphoma cells, the proportion of CD69(+) and CD25(+) T cells in the LMP1 CAR-T cell group was significantly higher than that in vector-T cell group [ (7.40±0.41) % vs (3.48±0.47) % , t=6.268, P=0.003; (73.00±4.73) % vs (57.67±2.60) % , t=2.842, P=0.047]. ⑥After coculture with LMP1-positive lymphoma cells, cytokine secretion in the LMP1 CAR-T cell group was higher than that in the vector-T cell group [interferon-gamma: (703±73) ng/L vs (422±87) ng/L, t=2.478, P=0.068; tumor necrosis factor-alpha: (215±35) ng/L vs (125±2) ng/L, t=2.536, P=0.064]. Conclusion: In this study, we found that the LMP1 protein is only found on the surface of the EBV-positive tumor cell. Simultaneously, we created an LMP1 CAR-expressing plasmid and obtained LMP1 CAR-T cells by infecting T cells with a lentivirus packaging system. Furthermore, we demonstrated that LMP1 CAR-T cells could specifically kill LMP1-positive tumor cells in vitro. The degranulation and activation effects of LMP1 CAR-T cells were enhanced after coculture with LMP1-positive tumor cells, indicating a potential clinical application.

[Relationship between sugary drinks and diabetes of adults in Wuhai city].

OBJECTIVE: To explore the relationship between sugary drinks and diabetes of adults in Wuhai city. METHODS: A multiple stage stratified cluster sampling was conducted on 8 131 residents who were between 35 and 79 years by cross-sectional study in Wuhai city. Questionnaires, physical measurements and laboratory tests were used to collect information on demographic information, dietary behavior, health status, blood glucose indicators. Besides, other covariate information was also collected by these ways. The analysis was carried out by chi-square test, trend chi-square test and multifactor Logistic regression. RESULTS: The detection rates of impaired fasting glucose and diabetes of people who were 35 years old and above in Wuhai city were 6.0% and 18.4%, respectively, and they both increased with age (P<0.01, P< 0.01). The detection rates of impaired fasting glucose and diabetes of the men were both far higher than the women (P< 0.01, P< 0.01). For the men, the detection rate of diabetes increased with age (Ptrend<0.01), but for the women, and the detection rate of impaired fasting glucose and diabetes both increased with age (Ptrend<0.01, Ptrend<0.01). The consumption rate of sugary drinks of the people who were 35 years old and above in Wuhai city was 30.2%. And after all the subjects were classified into three groups, A (0- mL/d), B (16- mL/d), and C (237- mL/d) according to the daily different drinking quantities, statistical results found that group A accounted for 75.4%, group B for 21.5%, and group C for 3.1%. In group A, for comparison, the impaired fasting glucose OR values of group B and group C were 1.4 and 2.2, respectively. And diabetes OR values of group B and group C were 1.2 and 2.1 respectively compared with group A, and the trend of OR values increased both had statistical significance (Ptrend <0.01, Ptrend < 0.01). Also, after adjusting for other covariates in multifactor Logistic regression, the OR values of impaired fasting glucose in group B and group C were 1.6 and 3.8 respectively, and the OR values of diabetes were 1.9 and 4.9 respectively, compared with group A, and besides, the trend of OR values increased both still had statistical significance (Ptrend <0.01, Ptrend < 0.01). CONCLUSION: Increased consumption of sugary drinks will increase the risk of impaired fasting glucose and diabetes. Residents in Wuhai city should control their consumption of sugary drinks.

[Associations of sedentary behavior and physical activity with dyslipidemia].

OBJECTIVE: To analyze associations of sedentary behavior and physical activity with dyslipidemia among residents in Wuhai city. METHODS: Data about social demographic characteristics, life style, health status and other covariate required for analysis in this study was obtained from a cross-sectional study on a total of 11 497 18-79 years old residents in Wuhai City by questionnaire, body mea-surement and laboratory examination. In this study, sedentary behavior and physical activity were evaluated using international physical activity questionnaire long version (IPAQ). IPAQ is widely used all over the world, and its reliability and validity have been tested in Chinese population. 2016 Chinese Guideline for the Management of Dyslipidemia in Adults was used to define dyslipidemia in this study. RESULTS: According to IPAQ scoring protocol, 124 participants were excluded as a result of reporting more than 960 min of physical activity per day. 50.58% of 11 373 participants included in the analysis reported more than 4 hours of sedentary behavior per day in this study, thus 49.42% participants reported no more than 4 hours of sedentary behavior per day; the proportions of these 11 373 participants who reached Low level physical activity, Moderate level physical activity and high level physical activity were 23.43%, 37.29% and 39.28% respectively; and the detection ratios of new cases and prevalent cases of dyslipidemia in Wuhai City were 20.46% and 16.13% respectively. After controlling for confounders in this study, we found out that sedentary behavior increased the risk of new cases of dyslipidemia in women (OR=1.17, 95% CI: 1.00-1.36), and increased the risk of prevalent cases of dyslipidemia in both men (OR=1.21, 95% CI: 1.02-1.44) and women (OR=1.24, 95% CI: 1.04-1.48); as for association of physical activity with dyslipidemia, association was found between high level physical activity and prevalent cases of dyslipidemia in men in this study (OR=0.78, 95% CI: 0.62-0.98), suggested that high level physical activity may help to reduce the risk of prevalent cases of dyslipidemia in men. CONCLUSION: Our results from this cross-sectional study in Wuhai City suggested that sedentary behavior increased the risk of dyslipidemia; by contrast, physical activity may help to reduce the risk of dyslipidemia.

[Expression of signaling pathway of mammalian target of rapamycin in articular cartilage cell induced by interleukin 1ß].

Objective: To observe effect of interleukin(IL)-1ß on the expression of signaling pathway of mammalian target of rapamycin(mTOR) of articular cartilage. Methods: Articular cartilage of rats was isolated under sterile technique, cells were digested by type Ⅱ collagenase and trypsin and cultured in vitro, pre-culture the Ⅱ cells for three days, different concentrations of IL-1ß were added for 24 hours.The cells were stained with toluidine blue and HE, to observe morphological changes of cells.RT-PCR was used to detect the mRNA expression of typeⅡcollagen gene, aggrecan gene, mTOR gene and P70S6K gene, Western blotting was used to detect the expression of protein related to mTOR. Results: With increasing concentrations of IL-1ß, the phenotype of cells appeared polygon into a spindle, the mRNA expression of gene of type Ⅱ collagen (the control group: 0.821±0.014; 1 ng/ml: 0.614±0.014; 10 ng/ml: 0.549±0.009; 100 ng/ml: 0.520±0.008), aggrecan(0.867±0.005; 0.857±0.001; 0.554±0.008; 0.538±0.004) and mTOR(0.845±0.015; 0.785±0.009; 0.569±0.025; 0.518±0.014) reduced, but P70S6K(0.465±0.024; 0.566±0.022; 0.663±0.022; 0.896±0.015) increased by PCR .Expression of protein detected by Western blotting was similar to the trend of PCR. Conclusion: mTOR signaling pathway may play an important role on the degeneration of articular cartilage, regulating mTOR signaling pathway may provides a new idea of delaying the degeneration process of cells.

A cyclometallated iridium(III) complex as a c-myc G-quadruplex stabilizer and down-regulator of c-myc oncogene expression.

A new cyclometallated iridium(III) complex with the 2,2'-biquinoline N-donor ligand has been synthesized and characterized. The interaction and affinity of the complex towards c-myc G-quadruplex and duplex DNA have been investigated using UV/Vis spectroscopy and gel mobility shift assay. These studies revealed that complex 1 binds to c-myc G-quadruplexes (Pu22 and Pu27) with high affinity but does not interact with duplex DNA either by intercalation or groove binding. The ability of 1 to stabilize c-myc G-quadruplex DNA in vitro has also been examined through a PCR stop assay and a cell-based luciferase reporter assay. Complex 1 displays promising cytotoxic activity against the HeLa cell line with sub-micromolar potency.

B7-1 (CD80) and B7-2 (CD86) have complementary roles in mediating allergic pulmonary inflammation and airway hyperresponsiveness.

We examined the roles of B7-1 (CD80) and B7-2 (CD86) in a model of allergic pulmonary inflammation and airway hyperresponsiveness (AHR) by using mice with germline deletions of the B7-1 and/or B7-2 molecules. Multiple parameters of the allergic response were affected to varying degrees by the absence of B7-1 and/or B7-2. Mice lacking both B7-1 and B7-2 had no elevation of serum immunoglobulin E, lack of airway eosinophilia, and no AHR. These same disease parameters were also reduced in mice lacking either B7-1 or B7-2. Lack of B7-1 and/or B7-2 resulted in an increase in T-helper 1 cytokine production. Our observations suggest that whereas B7-2 is quantitatively more significant in the induction of this response, B7-1 and B7-2 may have complementary roles in mediating the development of allergic pulmonary inflammation.

NF-kappa B/Rel transcription factors: c-Rel promotes airway hyperresponsiveness and allergic pulmonary inflammation.

The NF-kappa B/Rel family of transcription factors induces many genes involved in immune and inflammatory responses. Mice with germline deletions of individual NF-kappa B/Rel subunits have different phenotypes, suggesting that the NF-kappa B/Rel transcription factors have different functions. We tested whether c-Rel promotes allergic asthma using a murine model of allergen-induced pulmonary inflammation and airway hyperresponsiveness. Our investigation focused on c-Rel, which is expressed in lymphoid cells and is important for lymphocyte activation. In response to allergen sensitization and challenge, c-Rel-deficient mice did not develop increases in pulmonary inflammation, bronchoalveolar lavage fluid eosinophilia, or total serum IgE. c-Rel deficiency also prevented the induction of airway hyperresponsiveness. Allergen-treated wild-type mice had increased DNA binding to an NF-kappa B consensus site. Chemokine expression was altered in allergen-treated c-Rel-deficient mice. Monocyte chemoattractant protein-1, which is regulated by NF-kappa B, was decreased in allergen-treated c-Rel-deficient mice relative to wild-type controls. The increase in NF-kappa B/Rel transcription factors after allergen challenge in wild-type mice and the decrease in allergen reactivity found in c-Rel-deficient mice indicate that c-Rel promotes allergic inflammation. Alteration of pulmonary chemokine expression in c-Rel-deficient mice may inhibit allergen-induced pulmonary inflammation and airway hyperresponsiveness.

Presence of antibodies to heat stress proteins and its possible significance in workers exposed to high temperature and carbon monoxide.

Antibodies to the ubiquitous group of stress proteins known as heat shock proteins (Hsps) have been found to be associated with a number of diseases in humans. Hsps are known to be induced by certain xenobiotics, some of which are common in the working environment. The biological significance of the presence of such autoantibodies is presently unclear. In the present study, we used immunoblotting to investigate the presence of antibodies against the different stress proteins, Hsp27, Hsp60, Hsp71, Hsc (heat shock cognate) 73 and Hsp89 alpha and beta in groups of workers exposed to high temperature or carbon monoxide. These data were related to a detailed clinical evaluation and to various laboratory measurements including electrocardiogram (ECG), B echogram, white blood cell counts and typing, the activity of alanine aminotransferase (ALT), acid phosphatase (ACP) and alkaline phosphatase (ALP) and lymphocyte DNA damage. Antibodies to Hsp27 and Hsp71 were found more frequently in the high temperature and carbon monoxide-exposed groups than in controls (P < 0.05). The carbon monoxide-exposed group showed the highest incidence of anti-Hsp antibodies. Anti-Hsp60 antibodies were only detected in workers exposed to high temperature or carbon monoxide. The percentage of workers with abnormal ECG, B echogram changes and displaying hepatitis B antigen (HBsAg) was higher in the carbon monoxide group than in the control group (P < 0.05). There was a significant increase in the activity of ALT in the high temperature and carbon monoxide groups and in the activities of ACP and ALP in the carbon monoxide group (P < 0.05). The extent of DNA damage measured in lymphocytes was higher in workers from the high temperature and carbon monoxide-exposed groups. We suggest that the increased frequency of antibodies to Hsps is the result of these damages of the release of denatured Hsps and of a decrease in the phagocytic ability of macrophages in these workers. The data gathered in the present study show a statistical relation between the occurrence of antibodies against Hsps and the frequency of health problems in workers and suggest a potential role for the antibodies as useful biomarkers to assess whether workers are experiencing environmental stress.

The combined effects of high temperature and carbon monoxide on heat stress response.

In this study, we have examined the effects of exposure to high temperature, carbon monoxide or a combination of both conditions in a model system, the rat and in industrial workers. In the rat liver, HSP70 mRNA and HSP70 synthesis were measured by dot hybridization and western blot. The results showed that after a heat stress HSP70 mRNA and its product, HSP70 increased significantly and there was a synergism in the combined effects of high temperature and carbon monoxide exposure on the induction of HSP70 mRNA and HSP70 synthesis. Heat played a major role in this induction. The presence of antibodies to human HSP27, HSP60, HSP70, HSC73, HSP89 alpha and beta in workers exposed to heat, carbon monoxide was also measured by western blot using purified HSPs as antigens. Plasma free amino acids were measured in the same group of workers. The incidence of antibodies to HSP27 and HSP70 was significantly higher in the workers working in an environment with extreme heat, and high carbon monoxide emission than in a control group. The carbon monoxide exposed group showed the highest incidence of antibodies to HSPs. Although our previous results indicated that workers had an insufficient protein intake, plasma free amino acids tended to increase, especially in methionine and tryptophan two kinds of amino acids which are absent from the main stress protein, HSP70. These results suggest that the major problems that these workers may face are how to facilitate the use of plasma free amino acids and reduce the inhibition of synthesis of normal proteins when they are exposed to occupational harmful factors. These results also add new information on the measurement of HSPs as a potential biomonitor to assess whether organisms are experiencing metabolic stress within their environment.

Early health effects and biological monitoring in persons occupationally exposed to tetraethyl lead.

Dependent on the level of occupational exposure to tetraethyl lead, the occurrence of early signs of toxicity and the urinary excretion of triethyl lead, diethyl lead and total lead compounds were investigated. This was done in the following cohorts in the province of Hubei, China: 277 workers at gasoline depots exposed to gasoline, 36 traffic policemen exposed to automobile exhaust and 342 public office workers (virtually non-exposed controls). Mean external tetraethyl lead exposure concentrations were 84.8 micrograms/m3 (as Pb) for the gasoline depot workers, 5.2 micrograms/m3 for traffic police and 1.1 microgram/m3 for the controls. No significant subclinical indications of organic lead toxicity were found in the group of traffic policemen compared with the controls. In the cohort of gasoline workers, however, there was a statistical increase (vs controls) in the frequency of appearance of tremor and of sinus bradycardia. When the cohort of gasoline workers was divided into subgroups of different ranges of exposure, dose-dependence was noted. In general, the urinary excretion of triethyl lead was very low compared to that of diethyl lead, which appears to be a sensitive and specific indicator of exposure to tetraethyl lead; total lead excretion did not correlate well with actual external tetraethyl lead exposure. On the basis of these data it seems that current occupational exposure limits for tetraethyl lead are inadequate and need to be revised. In addition, a biological limit, based on urinary diethyl lead excretion, may be proposed.

[DNA methylation of monohalogenated methanes of F344 rats]. / DNA-Methylierung von monohalogenierten Methanen in F-344 Ratten.

Monohalogenated methanes (methyl chloride, methyl bromide and methyl iodide) are mutagenic and carcinogenic. The possible mechanism of these effects, DNA methylation, was studied. DNA adducts from organs of F344 rats exposed to these chemicals were separated and identified with high performance liquid chromatography (HPLC) and gas-chromatography/mass-spectrometry (GC/MS). DNA adducts, 7-methylguanine (7-MeG) and O6-Methylguanine(0(6)-MeG), incorporation of 14C into de novo synthesis of nucleobases could be observed in enzymatic DNA hydrolysates by HPLC and determination of the radioactivity in the fractions. The formation of DNA adducts in the studied organs was only quantitatively different. The formation of O6-MeG was further proved by analysing the acidic hydrolysates using HPLC with non-radioactive O6-MeG as internal standard. 7-MeG and 3-MeA were identified with GC/MS analysis.