Chimeric antigen receptor (CAR)‐T cell therapy has drawn much attention due to its recent clinical success in B‐cell malignancies. In general, the CAR‐T cell discovery process consists of CAR identification, T‐cell activation, transduction, and expansion, as well as assessment of CAR‐T cytotoxicity. The current evaluation methods for the CAR‐T discovery process can be time‐consuming, low‐throughput and requires the preparation of multiple sacrificial samples in order to produce kinetic data. In this study, we employed the use of a plate‐based image cytometer to monitor anti‐CAIX (carbonic anhydrase IX) G36 CAR‐T generation and assess its cytotoxic potency of direct and selective killing against CAIX+ SKRC‐59 human renal cell carcinoma cells. The transduction efficiency and cytotoxicity results were analyzed using image cytometry and compared directly to flow cytometry and Chromium 51 (51Cr) release assays, showing that image cytometry was comparable against these conventional methods. Image cytometry method streamlines the assays required during the CAR‐T cell discovery process by analyzing a plate of T cells from CAR‐T generation to in vitro functional assays with minimum disruption. The proposed method can reduce assay time and uses less cell samples by imaging and analyze the same plate over time without the need to sacrifice any cells. The ability to monitor kinetic data can allow additional insights into the behavior and interaction between CAR‐T and target tumor cells. © 2020 International Society for Advancement of Cytometry

The article is concerned with health benefits of two main physiologically active ingredients namely, Isoflavones and γ-Aminobutyric acid, with emphasis on their fitness for fortification of yoghurt to be consumed as a functional food. Isoflavones (ISO) are part of the diphenol compounds, called "phytoestrogens," which are structurally and functionally similar to estradiol, the human estrogen, but much less potent. Because of this similarity, ISO were suggested to have preventive effects for many kinds of hormone-dependent diseases. In nature, ISO usually occur as glycosides and, once deconjugated by the intestinal microflora, the ISO can be absorbed into the blood. At present, it seems convincing their possible protective actions against various cancers, osteoporosis and menopausal symptoms and high levels of blood cholesterol as well as the epidemiological evidence. Γ-Aminobutyric acid (GABA), it is an amino acid that has long been reported to lower blood pressure by intravenous administration in experimental animals and in human subjects. GABA is present in many vegetables and fruits but not in dairy products. GABA was reported to lower blood pressure in people with mild hypertension. It was suggested that low-dose oral GABA has a hypotensive effect in spontaneously hypertensive. Yoghurt beyond its ability to be probiotic food via its culturing with the gut strains, it could further carry more healthy benefits when it was fortified with physiological active ingredients, especially GABA versus ISO preferring, whether, bacteriologically or biochemically, a fortification level of 50 mg ISO/kg or 200 mg GABA/kg.

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Background. Soft skimmed-milk cheese Kariesh is the most popular soft cheese in Egypt. In the past, Karish cheese was traditionally produced by the random fermentation of milk speared its cream layer by the gravity force. Recently, its production has been carried out by several manufacturing procedures using, ultrafi ltration (UF) -skimmed milk retentate, certain bacterial cultures, enzymatic coagulation, etc. Therefore, the biological and nutritional evaluations are required. The present study was conducted to evaluate the properties of such cheese coagulated using different procedures involving probiotic strains with emphasis on some chemical and biological attributes. Material and methods. Kariesh cheeses were manufactured without whey drainage from UF cow's skimmed milk concentrate coagulated either by 3% yoghurt bacterial starter culture (YC), 3% probiotic starter culture (ABT type) or 2.5% glucono delta lactone (GDL) added whether separately or incorporated with rennet (0.05 ml/kg) to achieve the acidic-enzymatic coagulation in comparison with those made conventionally using un-concentrated milk coagulated either by 2% of YC or ABT added whether separately or incorporated with rennet (0.25 ml/kg). Due to the fact that whey is naturally needed to drain in the conventional (C) procedure, the use of GDL as coagulant was eliminated. Results. The obtained results indicated that the application of UF-technique in Kariesh cheese industry was associated with signifi cant increments in the values of protein, ash and pH. Moreover, the electrophoretical patterns of UF-Kariesh cheese obtained with the incorporation between YC, ABT or GDL and rennet were distinguished with the presence of the band of glycomacropeptide, which is normally lost in the whey through the C-procedure. Furthermore, the food intake (FI), body weight gain (BWG), food effi ciency ratio (FER), triglycerides (TG), total cholesterol (TC), high density lipoproteins (HDL), total, as well as ionized Ca of blood and the count of lactic acid bacteria of feces of rats were signifi cantly heightened, while low density lipoprotein (LDL) level was lowered as they fed on UF-cheeses, especially when coagulated using ABT, those caused the strongest persistence against the rat colon tumor, which was induced by the injection with 1,2 di-methyl hydrazine. The use of ABT led to raise the dry matter (DM) and ash of Kariesh cheese. Also, the FI, BWG, FER, TG, total, as well as ionized Ca of blood and the count of lactic acid bacteria of feces of rats were signifi cantly increased, while TC, HDL and LDL of blood, as well as coliform count of feces of rats were signifi cantly declined. The incorporation of enzymatic with acidic coagulation in Kariesh cheese manufacture increased signifi cantly the values of ash and pH. However, the cheese fi gures of DM, protein and titratable acidity were decreased. Also, the values of FI, BWG, FER, TC, HDL, LDL and feces LAB of rats were lowered. Whilst, the values of TG, total as well as ionized Ca and feces coliform of rats...

Clear cell renal cell carcinoma (ccRCC) is the major type of RCC and is among the 10 most common cancers in both men and women. ccRCC is characterized by the loss of chromosome 3p, where the von-Hippel-Lindau (VHL) gene is found. Its gene product, pVHL suppresses the hypoxia inducible factor (HIF) transcription factors and the deletion of VHL leads to HIF hyperactivation, upregulating the expression of many downstream genes involved in angiogenesis, metabolism, and cell-cycle regulation. Two gene products, carbonic anhydrase IX (CAIX) and CD70 which we are pursuing as therapeutic targets are downstream genes of the HIF-1α pathway and thus commonly overexpressed in ccRCC. Despite the development of checkpoint blockade inhibitors (CBIs) for the treatment of this disease, curative therapies are rare. Chimeric Antigen Receptor (CAR)-T cells are a new type of “living drug”, in which T cells are engineered to express a single chain variable antibody fragment (scFv) linked to an intracellular signaling motif that includes CD3ζ (z) activation domain (first generation), with CD28 or 41BB (second generation), or both (third generation) costimulatory domains. CAR-T cells have proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. However, these results have not been translatable to solid tumors due to inefficient homing of CAR-T cells, the suppressive tumor microenvironment (TME), and on-target off-tumor toxicities resulting from the sharing of CAR-T target epitopes on healthy tissues. To translate CAR-T cell therapy to ccRCC, we developed dual-targeted CAR-T cells against both CAIX and CD70, in which dual CAR can target double positive (CAIX+ CD70+) and single positive (CAIX+ CD70-, CAIX- CD70+) tumor cell populations to address this heterogeneity. By IHC staining, we demonstrated that these two target antigens are overexpressed in ccRCC and represent circa 90% of the tumor cell population regardless of disease stages. Here we build on our previous findings and demonstrate that 41BB CAR and CD4/CD8 CAR-T cell mixtures exhibit the superior efficacy and persistence in our ccRCC orthotopic NSG-SGM3 mouse model compared to other CAR constructs and CD8 alone. A panel of dual-targeted CARs have been encoded in our 41BB CAR construct and assessed in vitro and in vivo by using Celigo imaging cytometry assay and humanized ccRCC orthotopic NSG-SGM3 mouse model respectively. Dual-targeted anti-CAIX/CD70 CD4/8 CAR-T cells outperformed singly-targeted CAR-T cells with elevated efficacy. In summary, anti-CAIX/CD70 CAR-T cells hold a great promise to achieve ccRCC cures. Citation Format: Yufei Wang, Marion Grimaud, Alicia Buck, Atef Fayed, Matthew Chang, Rebecca Jennings, Maura Sticco-Ivins, Miriam Ficial, Leo L. Chan, Sabina Signoretti, Quan Zhu, Wayne A. Marasco. Develop dual-targeted CAR-T cells to achieve RCC cures [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6606.