[Activity comparison of humanized CD19 CAR-T cells with murine CD19 CAR-T on Nalm-6 cells and xenograft tumor model].

Objective: To compare the activity difference of the high affinity humanized CD19 chimeric antigen receptor (CAR)-T cells and murine CD19 CAR-T cells. Methods: Peripheral venous blood T cells from 8 healthy volunteers were collected and infected with humanized and murine CD19 CAR lentivirus. Human and murine CD19 CAR-T cells were prepared and cell proliferation was detected by cell counting kit-8 (CCK-8) method. The cytotoxicity of CD3(+) T cells, humanized and murine CD19 CAR-T cells to NALM-6 cells was detected by lactate dehydrogenase assay. Thirty BAL B/c nude mice transplanted with NALM-6 cells were randomly divided into 3 groups with 10 mice in each group and injected humanized CD19 CAR-T cells, mouse CD19 CAR-T cells and control CD3(+) T cell via tail vein, respectively. The proportion of NALM-6 cells in peripheral blood and the proportion of CD19 CAR-T cells in T cells from the vein of the inner canthus were detected by flow cytometry. The overall survival of BAL B/c nude mice was observed. Results: The proliferation of mouse and humanized CD19 CAR-T cells were (68.50±0.93)% and (80.63±1.41)%, respectively (t=20.353, P<0.001) after cultured in vitro for 24 hours, and were (91.38±1.41)% and (148.13±1.25)%, respectively (t=85.364, P<0.001) after cultured for 48 hours. When the effect to target ratio was 1∶1, there was no difference between the humanized and murine CD19 CAR-T cell group after co-culture for 24 hours (P=0.169), while the killing activity of humanized CD19 CAR-T cells against NALM-6 cells was higher than that of murine CD19 CAR-T cells (P<0.01) after 48 hours of co-culture. When the effect to target ratio was 4∶1, the cytotoxicity of humanized CD19 CAR-T cells against NALM-6 cells was higher than that of murine CD19 CAR-T cells in co-culture for 24 and 48 hours (P<0.01). On the seventh day of CD19 CAR-T cell therapy, the proportion of NALM-6 cells in the peripheral blood of BAL B/c nude mice decreased to the lowest level in the humanized CD19 CAR-T cell group and the murine CD19 CAR-T cell group. After 21 days, the proportion of NALM-6 cells in the murine CD19 CAR-T cell group was higher than that in the humanized CD19 CAR-T cell group (P(21 d)=0.001, P(28 d)<0.001, P(35 d)<0.001). The proportion of humanized and murine CD19 CAR-T cells in the peripheral blood reached the peaks after 7 days of therapy, and the proportion of humanized CD19 CAR-T cells was higher than that of murine CAR-T cells (P(7 d)=0.002). The CD19 CAR-T cells disappeared in the peripheral blood in the murine CD19 CAR-T cell group after 14 days of therapy, while in the humanized CD19 CAR-T cell group it disappeared after 21 days of therapy. The median survival of BAL B/c nude mice in the murine CD19 CAR-T cell group and the humanized CD19 CAR-T cell group was 42 days and 63 days, respectively (χ(2)=15.382, P<0.001). Conclusions: High affinity humanized CD19 CAR-T cells have stronger proliferation, higher cytotoxicity and longer survival time compared with those of murine CD19 CAR-T cells. The results indicate that the clinical efficacy of humanized CD19 CAR-T cells would be better than that of murine CD19 CAR-T cells.

[In vitro studies on the transfer of CAR into leukemia cells due to their residue in the autologous CAR-T cell preparation system for acute B-cell acute lymphoblastic leukemia].

Objective: To investigate the characteristics and cytotoxicity in vitro of the residual leukemia cells in the culture system that caused the accidental transfer of CD19 chimeric antigen receptor (CAR) into leukemia cells during the preparation of autologous CD19 CAR-T cells of relapsed/refractory B-cell acute lymphoblastic leukemia. Methods: ①Peripheral blood mononuclear cells (PBMC) of 30 patients with relapsed/refractory B-cell acute lymphoblastic anemia (R/R B-ALL) who accepted CD19 CAR-T cell therapy and six healthy volunteers were collected. ②The residual leukemia cells were analyzed by flow cytometry in the system after the PBMCs of R/R B-ALL patients were sorted by CD3 magnetic beads. ③ CD3(+) T cells from patients and healthy volunteers were transfected with CD19 CAR and CD22 CAR lentivirus to prepare CD19 CAR-T and CD22 CAR-T cells. ④The Nalm-6 cell line was resuscitated and the Nalm-6 cells with CD19 CAR lentivirus were transfected to prepare CD19 CAR-Nalm-6 cells. The patient's primary ALL cells were transfected with CD19 CAR lentivirus at the same time. ⑤The transfection rates were analyzed by flow cytometer, the cell proliferation was analyzed by the CCK-8 method, and the cell-killing activities were detected by the lactate dehydrogenase method. Results: ① Among the 30 R/R B-ALL patients who received CD19 CAR-T cell therapy, two patients had 2.04% and 3.32% residual leukemia cells in CD3(+) T cells. After 4 days in culture, the residual leukemia cells disappeared and could not be detected by a flow cytometer with prolonged cultivation in vitro. ② The proliferation of CD19 CAR-Nalm-6 cells was higher than that of the Nalm-6 cells. ③ The killing activity of the CD19 CAR-T cells on Nalm-6 cells was higher than that of the CD19 CAR-Nalm6 cells at a target ratio of 1∶1 on 24, 48, 72 h, respectively. The cytotoxicity of CD22 CAR-T cells on CD19 CAR-Nalm-6 cells was significantly higher than that of CD19 CAR-T cells. ④ The cytotoxicity of CD22 CAR-T alone on CD19 CAR-Nalm-6 cells was higher than that of CD19 CAR-T combined with CD22 CAR-T at the same target ratio. Conclusion: The residual leukemia cells in the culture system in the preparation of CD19 CAR-T cells may lead to the introduction of CD19 CAR into leukemia cells and results in the failure of the CD19 CAR-T cell therapy. Detecting the residual leukemia cells in the culture system via flow cytometry before transfection with CD19 CAR lentivirus is needed. Thus, CD22 CAR-T cell therapy could be used as one of the salvage treatments.

[Analysis on poor efficacy factors in the treatment of recurrent/refractory B-cell lymphoma with CD19 CAR-T cells].

Objective: To investigate the factors influencing the efficacy of CD19 chimeric antigen receptor T (CAR-T) cells in the treatment of patients with relapsed refractory B cell lymphoma and to provide evidence for further improvement of CAR-T efficacy. Methods: A total of 34 patients with relapsed and refractory B-cell lymphoma were recruited from the Department of Hematology of Tianjin First Central Hospital from February 2017 to January 2019. All patients received CD19 CAR-T cell therapy. These patients were evaluated for efficacy, factors with poor efficacyand adverse effects. Results: The overall response rate was 58.8% (20/34) and the complete remission rate was 41.2% (14/34) after infusion of CD19 CAR-T cells in 34 patients with relapsed refractory B cell lymphoma. According to the efficacy of CAR-T cells, patients were divided into two groups, 20 in the effective group and 14 in the poorly effective group. The median am ount of CD19 CAR-T cell infusions in these two groups were 8.6 (5.0-12.7)×10(6)/kg and 9.7 (5.8-15.0) × 10(6)/kg, respectively, and the difference was not statistically significant (P=0.654). The percentage of CD19 CAR-T cells in the effective group and the poorly treated group was 10.28% (3.92%-44.16%) and 4.05% (0.92%-28.63%), respectively.The effective group had a higher proportion of CAR-T cells than the poorly treated group, but the difference was not statistically significant (P=0.371).The presence of massive mass was an unfavorable factor affecting the efficacy of CD19 CAR-T cells and the difference was statistically significant (P=0.001). Logistic regression multivariate analysis showed that the characteristics of massive tumors were still independent prognostic factors for poor efficacy of CD19 CAR-T cells (P=0.005, OR=0.039). Of all 34 patients, there were 70.6% (24/34) who showed varying degrees of adverse reactions after the infusion of CD19 CAR-T cells, mainly cytokines release syndrome (CRS). The median time of occurrence of fever was on the third day after infusion (0-11th) day. 16 patients were with grade 1 CRS, 7 with grade 2, and 1 with grade 3. After glucocorticoids and support treatment, they all showed improvements. Conclusions: CD19 CAR-T cell therapy has achieved a certain effect in CD19(+)B cell lymphoma, but has poor efficacy on some patients. Large mass tumors may be an adverse factors to CAR-T cell treatment.

[PD-1 expression, mRNA level and cytotoxicity changes in CD19CAR-T cells].

Objective: To observe the changes of PD-1 expression, mRNA level and cytotoxic activity of CD19 CAR-T cells during the culture process of CAR-T cells. Methods: The peripheral blood T cells of 6 lymphoma patients with high expression of PD-1 and 6 healthy volunteers were the source of CAR-T cells. The expression of PD-1 was analyzed by flow cytometry. The mRNA level of PD-1 was analyzed by PCR. The cell proliferation was analyzed by CCK-8 assay. The cytotoxicity was analyzed by LDH assay. Results: ①The transfection efficiency of high PD-1 expression T cells and healthy volunteer T cells were as the same (P>0.05) . ②The cell proliferation capacity of CD19 CAR-T cells from high PD-1 expression T cells or healthy volunteer T cells, with or without PD-1 inhibitor were as the same (P>0.05) . ③The cytotoxicity to lymphoma cells of high PD-1 expression T cells and CAR-T cells were lower than that of these two T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer T cells (P<0.001) . There was no difference of the cytotoxicity between the CAR-T cells from high PD-1 expression T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer (P>0.05) . ④There was no difference of the expression of PD-1 in all CAR-T cell groups during the culture process (P>0.05) . There was no difference of mRNA level of PD-1 in all groups during the culture process (P>0.05) . ⑤The PD-1 expression of CAR-T cells increased by the time of culture after contacting with lymphoma cells (P<0.001) . The PD-1 inhibitors could antagonize this effect. There was no difference of mRNA level of PD-1 in all groups after contacting with lymphoma cells (P>0.05) . Conclusion: The PD-1 expression of CAR-T cells from high PD-1 expression T cells increased by the time of culture after contacting with lymphoma cells. However, the mRNA level of PD-1 of all groups did not change, even if PD-1 inhibitor was applied.

[The evaluation of modified cell infusion method to reduce febrile non-hemolytic transfusion reaction in CD(19) chimeric antigen receptor T cell threapy].

Objective: To retrospectively analyze the efficacy and safety of modified cell infusion method in reducing the incidence of febrile non-hemolytic transfusion reaction (FNHTR). Methods: A total of 69 patients were enrolled in the clinical trial of CD(19) chimeric antigen receptor T (CAR-T) cell treatment from February 2017 to October 2018. Study group received the modified cell infusion method, that 1×10(6) CAR-T cells were re-suspended in 2 mg human serum albumin with total volume of 20 ml and injected intravenously. The control group was intravenously administrated with CAR-T cell in 100 ml normal saline. The incidence of FNHTR, cytokine releasing syndrome (CRS) grade, cytokine level and efficacy were compared. Results: (1)The incidence of FNHTR in the study group was 21.1%, significantly lower than that in the control group (71%)(P=0.000). (2)There was no statistical difference in cell proliferation between the study group and the control group on day 4, 7, 14 and 21 after CAR-T cell infusion (P=10.223, 3.254, 5.551, 7.605). (3)There was no statistical difference in CRS grading between the study group and the control group (P=0.767). There was no statistical difference in the levels of interleukin 2 receptor (IL-2R), IL-6, tumor necrosis factor (TNF)-α between the two groups. (4)The C-reaction protein (CRP) level of the study group was lower than that of the control group on day 4 and 7 (P=0.026, 0.007). (5)There was no statistical difference of response rates in acute lymphocytic leukemia (ALL) and non-Hodgkin lymphoma (NHL) patients between the two groups (P(ALL)=0.842; P(NHL)=0.866). Conclusion: The modified cell infusion method in CD(19) CAR-T cell treatment reduces the incidence of treatment-related FNHTR. It does not affect the proliferation of CAR-T cells in vivo, the grading of CRS and the response rates.

[Efficacy and safety of CD19 chimeric antigen receptor T cells for the treatment of 22 patients with B-cell lymphoma].

Objective: To investigate the efficacy and safety of CD19 chimeric antigen receptor T (CAR-T) lymphocytes for the treatment of B cell lymphoma. Methods: A total of 22 patients with B-cell lymphoma from February 1, 2017 to July 1, 2018 were reviewed to evaluate the efficacy and adverse reactions of CD19 CAR-T. Results: Of 22 patients with B-cell lymphoma received CD19 CAR-T cells, the median dose of CAR-T cells was 7.2 (2.0-12.0) ×106/kg. Nine of 12 cases of relapse refractory patients were overall response. Complete remission (CR) occurred in 2 of 12 patients, partial remission (PR) in 7 of 12 patients. The overall response in minor residual disease positive (MRD) group was 8 of 10 patients. CD19 CAR-T cells proliferated in vivo and were detectable in the blood of patients. The peak timepoints of CAR-T cells proliferated in the relapsed refractory and MRD positive groups were 12 (5-19) and 4.5 (1-12) days after treatment respectively, and among peripheral blood cells, CAR-T cells accounted for 10.10% (3.55%-24.74%) and 4.02% (2.23%-28.60%) of T lymphocytes respectively. The MRD positive patients achieved sustained remissions during a median follow-up of 8 months (rang 3-18 months) . None of all the patients relapsed during a median follow-up time of 10 months (3-18 months) . However, 7 PR responders of the relapsed refractory patients maintained a good condition for 1.5-6.0 months. One patient bridged to hematopoietic stem cell transplantation, another one sustained remission for 12 months. Cytokine-release syndrome (CRS) occurred in 14 patients with grade 1-2 CRS in MRD positive group and grade 3 CRS in relapsed refractory group. Conclusions: CAR-T cell therapy not only played a role in the rescue treatment of relapsed and refractory patients, but also produced a surprising effect in the consolidation and maintenance of B-cell lymphoma. CD19 CAR-T cells might be more effective in the treatment of MRD positive B-cell lymphoma patients than in the refractory or relapsed cases. High response rate was observed with fewer adverse reactions.

[Effect of PD-1 inhibitor Nivolumab on the proliferation and cytotoxicity of anti-CD19 chimeric antigen receptor T cells].

Objective: To Evaluation the effect of PD-1 inhibitor Nivolumab on the proliferation and cytotoxicity of anti-CD19 chimeric antigen receptor T cells (CD19-CAR-T) in vitro. Methods: Five patients with high PD-1 expression in peripheral blood and five healthy volunteers were selected. These peripheral blood mononuclear cells were used as the source of T cells to prepare CD19-CAR-T cells. Different doses (72, 36, 18 µg/ml) of Nivolumab was added on day 8 to the culture medium. Patient T cells incubated with 72 µg/ml Nivolumab and CD19-CAR-T cells of healthy volunteers were used as controls. CCK-8, lactate dehydrogenase (LDH) cytotoxicity assay and ELASA were used to detect the proliferation capacity, the specific cytotoxicity and the inflammatory factor secretion. Results: ①T cells from patients with high expression of PD-1 as the source of CD19-CAR-T cells did not affect transfection rate compared with that of healthy volunteers [(32.80±7.22)% vs (35.10±5.84)%, t=-0.554, P=0.593]. ②Incubation of CD19-CAR-T cells with 72 µg/ml Nivolumab did not affect CD19-CAR-T cell proliferation, but its cytotoxicity was significantly higher than that of CD19-CAR-T cells alone or patients' T cells +72 µg/ml Nivolumab (all P<0.001), there was no significant difference in the killing activity between the 72 µg/ml and 36 µg/ml Nivolumab treated CD19-CAR-T cells on Pfeiffer cells (P=0.281, 0.267, respectively), and they were all higher than those of 18 µg/ml Nivolumab treated CD19-CAR-T cells (all P<0.001). ③Different doses of PD-1 inhibitor Nivolumab combined with CD19-CAR-T cells does not affect the secretion of IFN-γ and IFN-α (all P>0.05). Conclusion: Combination of 36 µg/ml PD-1 inhibitor and CD19-CAR-T cells could reduce the drug toxicity and enhance the cytotoxicity.

Theophylline: a review of population pharmacokinetic analyses.

WHAT IS KNOWN AND OBJECTIVE: Numerous population pharmacokinetic studies of theophylline have been conducted in paediatric and adult patients. The purpose of this review was to summarize the published studies concerning population pharmacokinetics of theophylline in patients of different ages and discuss factors that might cause the large variability in the pharmacokinetics of theophylline. METHODS: A literature search was conducted in PubMed using the following keywords: 'theophylline', 'population pharmacokinetic(s)' and 'nonlinear mixed effect model'. Additionally, the relevant references listed in the retrieved articles were manually reviewed. All of the studies that reported the population pharmacokinetics of theophylline in humans were included in this review. However, articles were excluded if they were not written in English. RESULTS AND DISCUSSION: Sixteen articles were included in this review. Among them, 11 were conducted on paediatric patients, and five were conducted on adults. A one-compartment model with first-order elimination was employed in most of the included articles. A nonlinear mixed effect modelling approach (NONMEM) was the most commonly used software to develop a population pharmacokinetic model. Body weight and age (post-conceptional age and post-natal age) were the most important factors associated with the clearance (CL) of theophylline in paediatric patients. Body weight (ideal body weight and lean body mass), age and smoking status were most frequently used to estimate the CL of theophylline in adults. The median (range) estimate values of CL for paediatric and adult patients were 0·062 (0·0056-0·0949) L/h/kg and 0·053 (0·0493-0·0517) L/h/kg, respectively. The median values of the interindividual variability of CL were 33·5% in adults and 25·8% in paediatric patients. The mean values of the residual variability were 21% in paediatric patients and 14·3% in adults. WHAT IS NEW AND CONCLUSION: This review concludes that body weight and age were the most important factors associated with the clearance of theophylline in paediatric patients. Body weight, age and smoking were most frequently used to estimate the clearance of theophylline in adults. Future studies are warranted to detect the influence of new factors, such as cytochrome P450 (CYP) 1A2 gene polymorphisms, on the pharmacokinetics of theophylline because some pharmacokinetic variability was not fully explained.

[Establishment of macrophage model of iron overload in vitro and the injury induced by oxidative stress on macrophage with iron overload].

OBJECTIVE: To establish macrophage iron overload model in vitro by co-culture macrophages with iron, and to explore the effect of iron overload on cell reactive oxygen species (ROS) and the impact of ROS on macrophages. METHOD: Iron overload group were treated with different concentrations (0, 5, 10, 20, 40, 80 µmol/L respectively) of ferric ammonium citrate (FAC). The control group was the group of macrophages without FAC treatment. We detected the number and state of cells, metabolic activity, the change of phagocytosis, the levels of ROS and reactive nitrogen, and changes of related oxidative stress signaling pathways in different groups. Changes in the above indexes were detected after application of deferasirox (DFX) to remove iron and the antioxidant N -acetylcysteine (NAC) to clear excess oxidative stress. RESULTS: (1)The levels of labile iron pool (LIP) in macrophages co-cultivated with iron was increased with the increase of iron concentration in a dose-dependent manner. The LIP levels was the highest in the macrophages treated with 80 µmol/L. (2)The increase of FAC concentration, the metabolic activity of macrophages in the 5 FAC-treated groups decreased to 51.58%, 40.98%, 16.23%, 3.46%, and 0.05% of the activity level of the control group (all P< 0.05). The group with the metabolic activity decreased to 16.23% (20 µmol/L) was selected as the iron overload group for the following experiments. (3)Compared with the control group, the number of macrophages in the iron overload group reduced to 32.80% (P<0.05), and the state of cells changed from adherence to partial suspension. The phagocytosis of macrophages in the iron overload group reduced to 20.40% of the control group (P<0.05). (4)Our further experiment showed that the levels of ROS and the activity nitrogen in the iron overload group increased by 7.71-and 1.45-fold compared with the control group (both P<0.05). The RT-PCR showed up-regulated mRNA expression of genes related with ROS production, i. e. nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX 4) gene related with ROS production and inducible nitric oxide synthase (iNOS) gene related with reactive nitrogen production, down-regulated mRNA expression of glutathione peroxidase 1 (GPX1) gene which participated in ROS clearance. Moreover, mRNA expression of phosphatidylinositol-3-kinase (PI3K) gene involved in oxidative stress signaling pathway in the iron overload group was up-regulated, while fork head protein O3 (FOXO3) which regulated oxidative stress through negative feedback showed a down-regulation level of mRNA expression compared with the control group. (5)After iron chelation and antioxidant treatment, the above-mentioned damage in the iron overload group were partially reversed. CONCLUSIONS: The damages of iron overload on macrophages may be mediated by inducing oxidative stress and activating oxidative stress signaling pathways. Our established model provides a method to explore the mechanism of iron overload on macrophage, and may shed some new light on possible therapeutic target in treating iron overload patients.

Fluorimetry determination of trace Ce3+ with EDTP.

The use of ethylenediaminetetrakis(methylphosphonic) acid (EDTP) in the fluorimetry determination of trace amounts of Ce3+ ions is described. The fluorescence intensity of Ce3+ was greatly enhanced when an 1:1 complex with EDTP in solution of pH 7-8 formed. The apparent excitation and emission wavelength used were 313 and 397 nm, respectively. The fluorescence intensity varied linearly with the concentration of Ce3+ in the range of 1 x 10(-8) -1 x 10(-4) mol l(-1). The quenching effects of coexist ions (other rare earth ions, Fe3+ and some inorganic anions) were studied. This technique has unique advantage in eliminating disturb of coexist.

Study on the binding of cerium(III) ion and spermine to TRNA(Phe).

Fluorescence of Ce(III) aqua ion at pH 6.0 is found to be in good linear relationship with its concentration, and the intensity is strong enough to be employed to determine its concentration. TRNA(Phe) evidently quench this fluorescence. Fluorescence titration experiments were performed to Ce(III)-tRNA(Phe) system, the binding number and association constant was estimated with a Scatchard plot. Two classes of binding sites with association constant of 5.2 x 10(7) and 4.4 x 10(6) M, respectively, were found. Addition of spermine slightly decrease binding number and association constant of Ce(III) ion.

Urinary and biliary metabolites of pyrene in rainbow trout (Oncorhynchus mykiss).

1. Pyrene was administered i.p. as a single dose to trout (Oncorhynchus mykiss). Urine was collected continuously for 3 days and bile sampled at the end of this period. Pyrene metabolites in these biological fluids were identified by 1H-nmr spectrometry, glc-ms and hplc-ms. 2. 1-Hydroxypyrene was the major oxidation metabolite in the urine and bile. Small amounts of 1,6-dihydroxypyrene and a putative 1,8-dihydroxypyrene metabolite also were detected. Unchanged pyrene was not found in any of these biological fluids. 3. Both free and conjugated metabolites of pyrene were found in the bile and urine. The majority of the pyrene metabolites in the bile were conjugated with glucuronic acid or sulphate.

[Study on dissolution rate of injection of microencapsulated compound megestrol acetate].

Dissolution rate of megestrol acetate in the injection of microencapsulated compound megestrol acetate was determined by the first derivative spectrum amplitude method. The experimental results reveal that t50 of the microencapsulated sample (I) is about 60.16 d, while the broken microencapsulated sample (II) is about 15.89 d and the unmicroencapsulated sample (III) about 15.87 d. The difference is regarded as of obvious significance (P less than 0.01). The values of the dissolution rate were linear with the parameters of rabbits and women both in vivo.