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Relapse of leukemic cells that do not express the antigen targeted by chimeric antigen receptor (CAR) is still a risk. As is the potential for targeting hematopoietic stem cells (HSCs) that share the same antigen expression, off-tumor on-target toxicity. Further, CAR T cells that bind different epitopes of the same antigen can have different tumor-killing efficacies. Therefore, we screen murine single chain variable fragment (scFv) based for indirect affinity to identify a CAR that targets Acute myeloid leukemia (AML), while minimizing toxicities. Also, recent advances in CAR design have demonstrated that the requirement of two separate tumor antigens to be ligated by CARs can increase the specificity for tumor targets. So designing a CAR that only activates a T cell when it binds two separate AML antigens will allow T cells to enhance safety. Therefore, we set out to develop a affinity based multi-antigen CAR T cell therapy that targets well described antigens for AML, including CD33 and CD123. Mice were immunized with these antigens, spleens collected, and fused with myeloma cell lines. The antibodies of fused hybridomas were screened for binding and activation against antigens by high throughput flow cytometry. After screening, we derived multiple de novo CD33, and CD123 scFvs by sequencing. We incorporated CD33 and CD123 scFvs into standard mono-specific CARs utilizing a 41BB co-stimulatory domain to validate antigen-specificity. Gene transfer assessment of CAR T cells demonstrated about 50-80% transduction efficiency for CD33 and CD123 scFvs. There were no differences in CD4 and CD8 proportions in these CAR T cells. We next examined the CARs for their cytotoxic ability using a Real-Time Cell Analysis (RTCA) system. For the CD33 CARs, 2 (6A11-1 and 27A3-1) out of 5, and for the CD123 CAR, 2 (15A12-11 and 15 A12-12) out of 8, scFv sequences transduced into T cells were highly efficacious at killing target cells and generated significant amounts of cytokines such as IFN-g, TNF- a, and IL-6. CAR T cells with these same scFv sequences were able to proliferate better in response to targeted antigen. To find the best possible combination of CD33 and CD123 scFvs we double transduced T cells with four selected CD33 or CD123 scFvs each with only one co-stimulation domain either CD3z or 4-1BB in "AND" gate fashion. Clear differences in cytotoxic ability and cytokine production were observed. We selected 12 combination of CD33/123 CARs bi-specific CARs to evaluate in vitro efficacy, polyfunctionality, and safety. Finally, to find the best combination of CARs that would be less toxic to HSCs, we performed a Colony Forming Unit (CFU) assay with CD34+ bone marrow stem cells and found 5 bi-specific pairs that were less toxic to HSCs . Based on the CFU assay and PSI index, we were able to select the combination of CD33/123 scFvs that would target AML but minimize the killing of HSCs.

To investigate the risk factors of primary empty sella (PES) and its associations with cerebral small vessel diseases (CSVD).A total of 132 consecutive patients were recruited from Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University from December 2018 to January 2020, including 69 cases of PES, and age, gender-matched 63 subjects without PES. Demographics and clinical characteristics were recorded. Enlarged perivascular spaces (PVS) and white matter hyperintensities (WMH), which are image markers for CSVD, were assessed. Univariate logistic regression models and multivariate logistic regression models were performed to predict the independent risk factors of PES.There was a significant difference in baseline characteristics in terms of hypertension (p0.001) and pregnancy (p = 0.019) between PES and the control group; among markers of CSVD, whole WMH (p = 0.030) and periventricular hyperintensities (PVH) (p = 0.027) were significantly different; however, no significant differences concerning deep WMH, total PVS, basilar ganglia-PVS and centrum semiovale-PVS (p0.05). After adjusting relevant potential confounders, multivariate logistic regression revealed hypertension (OR=3.158, 95 %CI: 1.452∼6.865, p = 0.004) and pregnancy (OR=2.236, 95 %CI: 1.036-4.826, p = 0.040) were independent risk factors for PES.Hypertension and pregnancy are independent risk factors of PES. There is a possible correlation between PES and WMH, especially PVH, however, further studies are required to confirm these findings.

Halophenols are widely present in wastewater and groundwater, which threaten global water safety and human health. Importantly, hydrodechlorination (HDC) via heterogeneous catalysis is efficient and environmentally friendly. To further improve the catalytic performance and atomic economy, Pd-based single atom catalysts (SACs) were prepared through electrostatic attraction and used in the catalytic HDC for the first time. By substituting different defect-rich supports, the coordination environments of atomically dispersed Pd-atoms were mediated with different catalytic performance. The results demonstrate that the Pd/CeO2 SACs have the highest HDC activity and remain stable during cycle tests. The moderately intensive metal-support interactions (MIMSI) effect was identified as a critical factor for the higher activity of SACs, unlike the traditional strong metal-support interaction effect. The HDC process on Pd SACs was further investigated by combining kinetic experiments with density functional theory calculations, which facilitated the recognition of HDC intermediates and an understanding of the reaction mechanism.

Background CD19 chimeric antigen receptor (CAR) T cell therapies have been successful in B cell malignancies. Recent reports about CD19 CAR T cell therapy in B-cell acute lymphoblastic leukemia suggest that the median event-free survival of children and young adult patients is longer than that of adult patients. Since the reason is unclear, we compared the functions of CAR-T derived from young or aged mice and also healthy human donors. Methods Young and aged B6 mice spleens (6-12 vs. ≥72 weeks) or young and aged human PBMCs (20-26 vs. 53-60 years) were used for mouse or human CAR-T preparation. 4 types of mouse CD19 CAR and 2 types of human CD19 CAR were evaluated in T cells. Results Aged mouse CAR-T predominates with CD8+ and effector-like phenotypes at the expense of CD4+ and memory-like phenotypes. Compared to young mouse CAR-T, aged mouse CAR-T exhibited superior cytotoxicity for mouse CD19+ artificial antigen presenting cell (aAPC). Using our immune competent in vivo murine model, aged mouse CAR-T was short-lived and expanded poorly despite superior in vitro cytotoxicity. RNA-Seq suggestes that young mouse CAR-T is advantageous for cell proliferation and regulation of cell differentiation whereas aged mouse CAR-T up-regulates gene expression pathways that regulate responses to stimulus and exocytosis. Furthermore, compared to mouse CAR-T, human CAR-T is complementary with immune phenotypes after human CD19+ aAPC stimulation. Conclusions Aged donor derived CAR-T exhibited enhanced effector functions but shorter persistence and less memory-like phenotypes. Our results suggest that the difference of clinical outcomes may be due to an age-dependent CAR-T cell phenotype that is reflected by its unique gene expression pattern, secretory profile, and/or transcription factor balance. In our future directions we are identifying potential methods to improve the function of aged donor derived CAR-T.

Rosemary and its polyphenolic diterpene, carnosic acid, are known to possess antioxidant activity and are used as a natural antioxidant food preservative. The intake of vegetables and certain plant components has been reported to play a major role in promoting gastrointestinal health. In the current study, the anticancer activity of rosemary extract and carnosic acid was evaluated to understand the potential implications on gastrointestinal health. We also evaluated the anti-cancer activity of rosemary extract in a nude mouse model. Rosemary extract and carnosic acid, increased apoptosis, and decreased viability in colon cancer cell lines. Rosemary extract and carnosic acid significantly upregulated the expression of Nrf2 in colon cells and inhibited a HCT116 xenograft tumor formation in mice. These results are especially significant as rosemary extract is increasingly being incorporated into food products across the United States and Europe as a food preservative.

Activated T cells differentiate into functional subsets with distinct metabolic programs. Glutaminase (GLS) converts glutamine to glutamate to support the tricarboxylic acid cycle and redox and epigenetic reactions. Here, we identify a key role for GLS in T cell activation and specification. Though GLS deficiency diminished initial T cell activation and proliferation and impaired differentiation of Th17 cells, loss of GLS also increased Tbet to promote differentiation and effector function of CD4 Th1 and CD8 CTL cells. This was associated with altered chromatin accessibility and gene expression, including decreased PIK3IP1 in Th1 cells that sensitized to IL-2-mediated mTORC1 signaling. In vivo, GLS null T cells failed to drive Th17-inflammatory diseases, and Th1 cells had initially elevated function but exhausted over time. Transient GLS inhibition, however, led to increased Th1 and CTL T cell numbers. Glutamine metabolism thus has distinct roles to promote Th17 but constrain Th1 and CTL effector cell differentiation.

A widely-used method for generating dendritic cell (DC) is to culture bone marrow cells in granulocyte-macrophage colony-stimulating factor (GM-CSF)-containing medium for 6-10 days. Usually, non-adherent cells are used as qualified dendritic cells while the adherent ones are discarded as "non-dendritic cells" or macrophages. In this study, we show that the adherent cells are nearly identical to the non-adherent cells in both dendritic cell surface markers expression and main dendritic cell-related functions, hence to prove that these "junk cells" are actually qualified dendritic cells.