[Construction and Optimization of CD19 Chimeric Antigen Receptor T Cells Derived from C57BL/6J Mice].

OBJECTIVE: To explore the stimulation conditions, optimal culture time and infection time of C57BL/6J mice CD3+ T cells in vitro, so as to improve the infection efficiency of CD19 chimeric antigen receptor T cells (mCD19 CAR-T). METHODS: Purified C57BL/6J mice CD3+ T cells were cultured in anti-CD3/CD28 coated, anti-CD3 coated+soluble anti-CD28 and anti-CD3 coated, respectively. The cells were stimulated in above three conditions for 12 h and 24 h, following with 24 h, 48 h and 72 h incubation and then the number of cell clones was recorded. C57BL/6J mice CD3+ T cells were stimulated for 12 h, 24 h, and 36 h under the above three conditions, then interleukin (IL)-2 (100 U/ml) was added. The number of cell clones was recorded under microscope at 24 h, 48 h, and 72 h of culture. After 24 h of stimulation, CD3+ T cells derived from C57BL/6J mice were infected with retrovirus for 48 h to establish mCD19 CAR-T cells, and the percentage of GFP+ CAR-T cells was detected by flow cytometry. RESULTS: The infection efficiency of mCD19 CAR-T cells derived from C57BL/6J mice was only 5.23% under the optimized conditions of mCD19 CAR-T cells derived from BALB/c mice. The number of clones of C57BL/6J mice CD3+ T cells was the highest in anti-CD3 coated+soluble anti-CD28 group after stimulated for 24 h and followed cultured for 48 h. After 24 hours of stimulation under the above conditions and 48 hours of culture with IL-2, the number of T cell proliferating clones in the anti-CD3 coated+soluble anti-CD28 group was significantly increased compared with the same group without IL-2, and the infection efficiency of CAR-T cells in this group reached 17.63%±4.17%. CONCLUSION: The optimal conditions for constructing CAR-T cells from C57BL/6J mice CD3+ T cells are different from those of BABL/c mice. T cells stimulated by anti-CD3 coated+soluble anti-CD28+IL-2 can obtain mCD19 CAR-T cells with the highest efficiency after retrovirus infection.

Correlation analysis between the in vivo bioavailability and in vitro bioaccessibility of nitro PAHs in soil: Application of simplified FOREhST in vitro methods based on the Chinese pharmacopoeia.

In this study, the relative bioavailability (RBA) of nitrated polycyclic aromatic hydrocarbons (NPAHs) in soil samples (n = 30) was assessed using an in vivo mouse model. Based on the correlation between the bioaccessibility data obtained from the Tenax improved traditional Fed ORganic Estimation human Simulation Test (FOREhST) in vitro method (TITF) and the bioavailability data obtained from in vivo experiments, the TITF method was further optimized and simplified by referring to the "Pharmacopoeia of the People's Republic of China: Volume IV, 2020" to adjust the formulation and parameters of the gastrointestinal fluid (GIF) in order to establish a simpler and lower cost in vitro method for the determination of the bioaccessibilities of NPAHs. The dose-accumulation relationship of the in vivo experiment showed that the linear dose-response was better in adipose tissue (R2 = 0.77-0.93), and the accumulation of NPAHs in adipose tissue was higher than that in kidney or liver tissue. Depending on the mouse adipose model, the NPAHs-RBA ranged from 1.88 % to 73.92 %, and a strongly significant negative relationship (R2 = 0.94, p < 0.05) was found between the NPAHs-RBA and Log Kow. The simplified experiment of the TITF showed that the composition of the GIF medium had a significant effect on the bioaccessibilities of NPAHs. The NPAH bioaccessibilities measured by the Tenax improved simplified FOREhST method (TISF) (9.0-36.5 %) were higher than that of the traditional FOREhST method (6.8-22.8 %) but significantly lower than that of the TITF method (16.8-55.2 %). With an increase in the bile concentration in the GIF (from 6 to 10 g/L), the bioaccessibilities of NPAHs increased from 9.0 to 36.5 % to 12.9-42.4 %. The accuracies of the four in vitro methods for predicting the bioavailabilities of NPAHs was in the following order: Tenax improved simplified FOREhST method with increased bile concentration (TITF-IB) (R2 = 0.54-0.87) ≈ TITF (R2 = 0.55-0.85) > TISF (R2 = 0.41-0.77) > FOREhST (R2 = 0.02-0.68). These results indicated that the simple in vitro method could also effectively predict the bioavailabilities of NPAHs.

Human nails as a valuable noninvasive alternative for estimating exposure to parabens.

Exposure of human to parabens (commonly used preservatives) is inevitable due to their extensively applied in numerous consumer products. Thus, a reliable noninvasive matrix reflecting long-term exposure to parabens is essential for human biomonitoring study. Human nails are potentially a valuable alternative for measuring intergrated exposure to parabens. In this work, we collected 100 paired nail and urine samples from university students in Nanjing, China, and measured simultaneously for six parent parabens and four metabolites. Methylparaben (MeP), ethylparaben (EtP), and propylparaben (PrP) were three predominant paraben analogue in both matrices, with the median concentrations being 12.9, 0.753, and 3.42 ng/mL in urine, and 1540, 154, and 961 ng/g in nail, respectively, while 4-hydroxybenzoic acid (4-HB) and 3,4-dihydroxybenzoic acid (3,4-DHB) were the most abundant metabolites (median values of 143 and 35.9 ng/mL, respectively) in urine. Gender-related analysis suggested that females exposed to more higher parabens than males. Significantly positive correlations were found between levels of MeP, PrP, EtP, and OH-MeP (r = 0.54-0.62, p < 0.01) in paired urine and nail samples. Our result here suggests that human nails, as an emerging biospecimen, are a potentially valuable biological matrix to evaluate human long-term exposure to parabens.

Bioaccessibility and bioavailability of NPAHs in soils using in vitro-in vivo assays: Comparison of laboratory and outdoor environmental aging effect.

Aging process is one of the most important factors that markedly reduces bioaccessibility and bioavailability (bioac-bioav) of organic contaminants. However, only few data on comparison of the effects of laboratory artificial aging (LAA) and outdoor environmental aging (OEA) processes on nitrated polycyclic aromatic hydrocarbons (NPAHs) bioac-bioav are available. In the current study, oral bioac-bioav of NPAHs in LAA and OEA soils (aging time intervals: 0, 45, 90, 120 and 150 d) were measured by in vitro traditional Fed ORganic Estimation human Simulation Test (FOREhST) and Tenax improved FOREhST (TI-FOREhST) methods, and in vivo mouse model. Tenax significantly increased the bioaccessibility of NPAHs in freshly spiked and aging soils from 0.3-40.9 % to 15.6-95.3 %, and 0.3-40.9 % to 1.0-84.5 %, respectively. Aging significantly reduced the NPAHs bioaccessibility (from 36.5 % to 10.7 %, and 12.1 % to 5.1 % as measured by FOREhST and TI-FOREhST, respectively) and bioavailability (from 27.7 % to 9.9 %, as measured by mouse model). The changes in bioac-bioav were mainly observed within the first 120 d of aging. The statistical analyses of NPAHs bioac-bioav showed no significant difference (p > 0.05) among the aging time intervals in LAA and OEA soils, which demonstrated that the LAA can relatively represent the OEA. Determination of TOC content in LAA and OEA soil can intuitively reflect whether the difference of NPAHs bioac-bioav between two aging treatment groups is significant. The mean bioaccessibility of NPAHs in soil measured by TI-FOREhST (mean 20.6 %) is closer to the bioavailability measured by mouse model (mean 19.4 %), indicating that Tenax improved in vitro method is more reliable than traditional methods, to predict the bioavailability of NPAHs.

Assessment of DDT and its Metabolites Bioaccessibility in Historically Contaminated Soils Using Unfed and Fed in Vitro Methods.

Bioaccessibility of hydrophobic organic contaminants (HOCs) from unintentional ingestion of soil is increasingly assessed with in vitro gastrointestinal models incorporating a sorption sink. In this study, the bioaccessibility of DDTs in contaminated soils (n = 11) was determined using "unfed" unified bioaccessibility method (UBM) and fed organic estimation human simulation test (FOREhST) with/without Tenax as an absorbent. By adding Tenax, the bioaccessibility of DDTs determined using UBM was significantly increased from 4.9-30.6% to 31.6-86.0%. In contrast, the bioaccessibility of DDTs determined using FOREhST without/with Tenax were similar with values of 20.0-60.9% vs 31.5-47.6%, implying that the influence of food components on the absorption efficiency of the sink should not be overlooked. Much high fraction of DDTs (bioaccessibility: 11.7-24.8%) remained in FOREhST supernatant after Tenax collection, suggesting that prediction of bioavailability through bioaccessibility obtained by absorbent needs to be treated with caution when bioaccessibility is determined using a "fed state" in vitro method.

Role of the initial degree of anaemia and treatment model in the prognosis of gastric cancer patients treated by chemotherapy: a retrospective analysis.

BACKGROUND: Anaemia is highly prevalent in gastric cancer (GC) patients. The role of initial haemoglobin levels in predicting the prognosis of GC patients treated by chemotherapy has not been well determined. Our present study aims to evaluate the relationship between the degree of anaemia and the overall survival (OS) and progression-free survival (PFS) of patients with GC. METHODS: Our retrospective study enrolled 598 patients who were treated with chemotherapy when the recurrent or metastatic GCs were unsuitable for surgical resection. Univariate and multivariate analyses were performed to identify risk factors that had the potential to affect patient prognosis. Additionally, the relationship between clinicopathological characteristics, including treatment method, and degree of cancer-related reduction in haemoglobin was further analysed. RESULTS: Our results revealed that patients with HBini level ≤ 80 g/L had a trend toward a shortened median OS and PFS (p = 0.009 and p = 0.049, respectively). Interestingly, we also found that HBdec ≥ 30 g/L was associated with a significantly shortened median OS and PFS (p = 0.039 and p = 0.001, respectively). Multivariate analysis showed that HBini levels ≤80 g/L could be used as an independent prognostic factor for recurrent and metastatic GC. More importantly, HBdec ≥ 30 g/L and treatment response were also significantly associated with OS and PFS. Furthermore, the degree of haemoglobin decrease was associated with chemotherapy including platinum and the number of chemotherapy cycles. CONCLUSION: Our study concludes that the initial degree of anaemia and a decrease in haemoglobin of ≥30 g/L can serve as biomarkers to predict prognosis in recurrent or metastatic GC patients, while chemotherapy treatment rather than red blood cell (RBC) transfusion can improve their prognosis. Additionally, platinum should not be recommended for treating severely anaemic GC patients.

Elevated RBP4 plasma levels were associated with diabetic retinopathy in type 2 diabetes.

The retinol-binding protein 4 (RBP4) has been postulated to play a role in glucose homeostasis, insulin resistance, and diabetes mellitus in human and animal studies. The aim of the present study was to evaluate the role of RBP4 in Chinese patients with type 2 diabetes mellitus with and without diabetic retinopathy (DR). Plasma RBP4 concentrations were tested in 287 patients with type 2 diabetes. At baseline, demographic and clinical information including presence of DR and vision-threatening DR (VTDR) was collected. The relationship between RBP4 and DR (VTDR) was investigated using logistic regression. Patients with DR or VTDR had significantly higher plasma levels of RBP4 on admission (P<0.0001). Receiver operating characteristics (ROCs) to predict DR and VDTR demonstrated areas under the curve for RBP4 of 0.79 (95% confidence interval (CI): 0.73-0.85) and 0.90 (95% CI: 0.85-0.94), respectively, which were superior to other factors. For each 1 µg/ml increase in plasma level of RBP4, the unadjusted and adjusted risk of DR would be increased by 8% (with the odds ratio (OR) of 1.08 (95% CI: 1.05-1.13), P<0.001) and 5% (1.05 (1.02-1.11), P=0.001), respectively. It was 12% (with the OR of 1.12 (95% CI: 1.07-1.18), P<0.001) and 9% (1.09 (1.05-1.15), P<0.001) for VTDR. The present study shows that elevated plasma levels of RBP4 were associated with DR and VDTR in Chinese patients with type 2 diabetes, suggesting a possible role of RBP4 in the pathogenesis of DR complications. Lowering RBP4 could be a new strategy for treating type 2 diabetes with DR.

Transcatheter Arterial Chemoembolization Combined with Simultaneous Computed Tomography-guided Radiofrequency Ablation for Large Hepatocellular Carcinomas.

BACKGROUND: Currently, the treatment of large hepatocellular carcinoma (HCC) is still a challenging problem. Transcatheter arterial chemoembolization (TACE) is the main treatment for intermediate end-stage HCC, while it is only a palliative and not a curative treatment due to the existence of residual tumors, and radiofrequency ablation (RFA) has limitations in complete ablation of large HCC. We hypothesized that TACE combined with simultaneous RFA (herein referred to as TACE + RFA) could improve the efficacy and survival of large HCC. This study aimed to investigate the feasibility, efficacy, and safety of TACE + RFA on single large HCC. METHODS: A total of 66 patients with single large HCC (≥5 cm in diameter) were recruited between February 2010 and June 2016. TACE was first performed and computed tomography was performed immediately after TACE, and the lesions with poor lipiodol deposition were subjected to simultaneous RFA. The success rate, technique-related complications, liver and kidney functions, serum alpha-fetoprotein (AFP) levels, progression-free survival (PFS), median survival time (MST), focal control rate, and long-term survival rate were evaluated. RESULTS: TACE + RFA were performed smoothly in all the patients with the success rate of 100%. Intra- and post-operative severe complications were not observed. There were no marked differences in mean alanine transaminase or aspartate transaminase before TACE + RFA compared with 7 days after TACE + RFA (all P > 0.05). In 57 AFP-positive patients, the levels of serum AFP were reduced by 100.0%, 100.0%, and 94.7% at 1, 3, and 6 months after TACE + RFA, respectively; the tumor control rates (complete remission + partial remission) were 100.0% (66/66), 92.4% (61/66), 87.9% (58/66), and 70.1% (39/55) at 1, 3, 6, and 12 months after TACE + RFA, respectively. Patients were followed up for 7-82 months after TACE + RFA. The MST was 18.3 months, PFS was 14.2 ± 6.2 months, and the 1-, 3-, and 5-year survival rates were 93.2% (55/59), 42.5% (17/40), and 27.2% (9/33), respectively. CONCLUSION: TACE + RFA is safe, feasible, and effective in enhancing the focal control rate and survival rate of patients with large HCC.