Modulating Cholesterol Metabolism via ACAT1 Knockdown Enhances Anti-B-Cell Lymphoma Activities of CD19-Specific Chimeric Antigen Receptor T Cells by Improving the Cell Activation and Proliferation.

CD19-specific CAR-T immunotherapy has been extensively studied for the treatment of B-cell lymphoma. Recently, cholesterol metabolism has emerged as a modulator of T lymphocyte function and can be exploited in immunotherapy to increase the efficacy of CAR-based systems. Acetyl-CoA acetyltransferase 1 (ACAT1) is the major cholesterol esterification enzyme. ACAT1 inhibitors previously shown to modulate cardiovascular diseases are now being implicated in immunotherapy. In the present study, we achieved knockdown of ACAT1 in T cells via RNA interference technology by inserting ACAT1-shRNA into anti-CD19-CAR-T cells. Knockdown of ACAT1 led to an increased cytotoxic capacity of the anti-CD19-CAR-T cells. In addition, more CD69, IFN-γ, and GzmB were expressed in the anti-CD19-CAR-T cells. Cell proliferation was also enhanced in both antigen-independent and antigen-dependent manners. Degranulation was also improved as evidenced by an increased level of CD107a. Moreover, the knockdown of ACAT1 led to better anti-tumor efficacy of anti-CD19 CAR-T cells in the B-cell lymphoma mice model. Our study demonstrates novel CAR-T cells containing ACAT1 shRNA with improved efficacy compared to conventional anti-CD19-CAR-T cells in vitro and in vivo.

Co-expression of IL-4/IL-15-based inverted cytokine receptor in CAR-T cells overcomes IL-4 signaling in immunosuppressive pancreatic tumor microenvironment.

The efficacy of CAR-T cell therapy has been hindered by several factors that are intrinsic to the tumor microenvironment. Many strategies are being employed to overcome these barriers and improve immunotherapies efficacy. Interleukin (IL)- 4 is a cytokine released by tumor cells inside the tumor microenvironment and it can oppose T cell effector functions via engagement with the IL-4 receptor on the surface of T cells. To overcome IL-4-mediated immunosuppressive signals, we designed a novel inverted cytokine receptor (ICR). Our novel CAR construct (4/15NKG2D-CAR), consisted of an NKG2D-based chimeric antigen receptor, co-expressing IL-4R as an extracellular domain and IL-15R as a transmembrane and intracellular domain. In this way, IL-4R inhibitory signals were converted into IL-15R activation signals downstream. This strategy increased the efficacy of NKG2D-CAR-T cells in the pancreatic tumor microenvironment in vitro and in vivo. 4/15NKG2D-CAR-T cells exhibited increased activation, degranulation, cytokine release, and cytotoxic ability of NKG2D-CAR-T cells against IL-4+ pancreatic cell lines. Furthermore, 4/15NKG2D-CAR-T cells exhibited more expansion, less exhaustion, and an increased percentage of less differentiated T cell phenotypes in vitro when compared with NKG2D-CAR-T cells. That is why IL-4R/IL-15R-modified CAR-T cells eradicated more tumors in vivo and outperformed NKG2D-CAR-T cells. Thus, we report here a novel NKG2D-CAR-T cells that could overcome IL-4-mediated immunosuppression in solid tumors.

ß2-Adrenergic Receptor Mediated Inhibition of T Cell Function and Its Implications for CAR-T Cell Therapy.

The microenvironment of most tumors is complex, comprising numerous aspects of immunosuppression. Several studies have indicated that the adrenergic system is vital for controlling immunological responses. In the context of the tumor microenvironment, nor-adrenaline (NA) is poured in by innervating nerves and tumor tissues itself. The receptors for nor-adrenaline are present on the surfaces of cancer and immune cells and are often involved in the activation of pro-tumoral signaling pathways. Beta2-adrenergic receptors (ß2-ARs) are an emerging class of receptors that are capable of modulating the functioning of immune cells. ß2-AR is reported to activate regulatory immune cells and inhibit effector immune cells. Blocking ß2-AR increases activation, proliferation, and cytokine release of T lymphocytes. Moreover, ß2-AR deficiency during metabolic reprogramming of T cells increases mitochondrial membrane potential and biogenesis. In the view of the available research data, the immunosuppressive role of ß2-AR in T cells presents it as a targetable checkpoint in CAR-T cell therapies. In this review, we have abridged the contemporary knowledge about adrenergic-stress-mediated ß2-AR activation on T lymphocytes inside tumor milieu.

An overview of viral mutagenesis and the impact on pathogenesis of SARS-CoV-2 variants.

Viruses are submicroscopic, obligate intracellular parasites that carry either DNA or RNA as their genome, protected by a capsid. Viruses are genetic entities that propagate by using the metabolic and biosynthetic machinery of their hosts and many of them cause sickness in the host. The ability of viruses to adapt to different hosts and settings mainly relies on their ability to create de novo variety in a short interval of time. The size and chemical composition of the viral genome have been recognized as important factors affecting the rate of mutations. Coronavirus disease 2019 (Covid-19) is a novel viral disease that has quickly become one of the world's leading causes of mortality, making it one of the most serious public health problems in recent decades. The discovery of new medications to cope with Covid-19 is a difficult and time-consuming procedure, as new mutations represent a serious threat to the efficacy of recently developed vaccines. The current article discusses viral mutations and their impact on the pathogenicity of newly developed variants with a special emphasis on Covid-19. The biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), its mutations, pathogenesis, and treatment strategies are discussed in detail along with the statistical data.

Isoprenaline and salbutamol inhibit pyroptosis and promote mitochondrial biogenesis in arthritic chondrocytes by downregulating ß-arrestin and GRK2.

Rheumatoid arthritis and osteoarthritis overlap many molecular mechanisms of cartilage destruction. Wear and tear in cartilage is chondrocyte-mediated, where chondrocytes act both as effector and target cells. In current study, role of ß2-AR was studied in chondrocytes both in vitro and in vivo. High grade inflammation in vitro and in vivo disease models led to decline in anti-inflammatory ß2-AR signaling and use of ß2-AR agonist attenuated arthritis symptoms. Detailed analysis in chondrocytes revealed that Isoprenaline (ISO) and Salbutamol (SBT) increased cell viability and relative Bcl-2 expression, meanwhile, decreased proteins levels of TNF-α, IL-6 and IL-8 in arthritic chondrocytes when compared with control, respectively. SBT preserved physiological concentration of antioxidant enzymes (CAT, POD, SOD and GSH) in cartilage homogenates and ISO inhibited IL-1ß-mediated genotoxicity in arthritic chondrocytes. Moreover, ß2-AR agonist increased mitochondrial biogenesis and proteoglycan biosynthesis by upregulating the gene expression of PGC1-α, NRF2 and COL2A1, Acan, respectively. ISO and SBT inhibited extracellular matrix (ECM) degradation by downregulating the gene expression of MMP1, MMP3, MMP9 and ADAMTS5 in vitro and in vivo study. In mechanism, ß2-AR agonists decreased ß-arrestin and GRK2 pathway, and as a result mice receiving SBT did not exhibit severe disease. Hence our data suggest ß2-AR agonist administered at disease onset can inhibit receptor internalization by downregulating the expression of ß-arrestin and GRK2 in chondrocytes.

Hyperglycemia-associated Alzheimer's-like symptoms and other behavioral effects attenuated by Plumeria obtusa L. Extract in alloxan-induced diabetic rats.

Diabetes mellitus is a chronic metabolic complaint with numerous short- and long-term complications that harm a person's physical and psychological health. Plumeria obtusa L. is a traditional medicine used in the treatment of diabetes to reduce complications related to behavior. Plumeria is a genus with antipsychotic activities. The objective of this study was to examine the effects of a methanolic extract of Plumeria obtusa L. in the attenuation of diabetes, on symptoms of Alzheimer disease, and on other associated behavioral aspects. A single dose of alloxan was administered to an experimental group of rats to induce development of diabetes (150 mg/kg, intraperitoneal) and the rats were then administered selected doses of methanolic extract of Plumeria obtusa L. (Po.Cr) or glibenclamide (0.6 mg/kg) for 45 consecutive days. Behavioral effects were evaluated using three validated assays of anxiety-related behavior: the open field test, the light and dark test, and the elevated plus maze. Anti-depressant effects of Plumeria obtusa L. were evaluated using the forced swim test (FST) and memory and learning were assessed using the Morris water maze (MWM) task. Po.Cr was also evaluated for phytochemicals using total phenolic content (TPC), total flavonoid content (TFC), and high-performance liquid chromatography assays, and antioxidant capability was assessed through assays of DPPH radical scavenging, total oxidation capacity, and total reducing capacity. In the alloxan-induced model of diabetes, the administration of Po.Cr and glibenclamide for 45 days produced a marked decrease (p < 0.001) in hyperglycemia compared to control animals. Po.Cr treatment also resulted in improvement in indicators, such as body weight and lipid profile (p < 0.05), as well as restoration of normal levels of alanine transaminase (ALT) (p < 0.001), a biomarker of liver function. Diabetic rats presented more Alzheimer-like symptoms, with greater impairment of memory and learning, and increased anxiety and depression compared to non-diabetic normal rats, whereas treated diabetic rats showed significant improvements in memory and behavioral outcomes. These results demonstrate that Po.Cr reversed alloxan-induced hyperglycemia and ameliorated Alzheimer-related behavioral changes, which supports additional study and assessment of conventional use of the plant to treat diabetes and associated behavioral complications.

Suppression of 4.1R enhances the potency of NKG2D-CAR T cells against pancreatic carcinoma via activating ERK signaling pathway.

Pancreatic carcinoma (PC) is one of the most common malignancies. Chimeric antigen receptor (CAR)-modified T cells has achieved remarkable efficacy in the treatment of hematological malignancies. However, lack of tumor-specific targets and the existence of inhibitory factors limit the function of CAR T cells when treating solid tumors. 4.1R has been reported to suppress the anti-tumor activity of T cell responses. In this study, we investigated the anti-tumor activity of 4.1R deletion in natural killer group 2D (NKG2D)-CAR T cells against PC. The CAR T cells were obtained by transfecting T cells with lentiviral vector carrying NKG2D-CAR, NC-NKG2D-CAR, or KD2-NKG2D-CAR. In vitro, NKG2D-CAR T cells showed higher cytotoxicity than Mock T cells. However, compared to NKG2D-CAR T cells, furtherly higher cytotoxicity against PC cells in a dose-dependent manner was found in KD2-NKG2D-CAR T cells. In addition, the proliferation rate and cytotoxic activity of KD2-NKG2D-CAR T cells were significantly higher than those of NKG2D-CAR T cells. Besides, the inhibitory receptors PD-1 and TIM-3 were expressed in lower level on KD2-NKG2D-CAR T cells. In vivo, KD2-NKG2D-CAR T cells suppressed tumor growth more effectively in a xenograft model compared to NKG2D-CAR T cells. Mechanistically, 4.1R regulated CAR T cell function via activating ERK signaling pathway. Therefore, the study provides a new idea to enhance the anti-tumor efficiency of CAR T therapy.

CD19-CAR-T Cells Bearing a KIR/PD-1-Based Inhibitory CAR Eradicate CD19+HLA-C1- Malignant B Cells While Sparing CD19+HLA-C1+ Healthy B Cells.

B cell aplasia caused by "on-target off-tumor" toxicity is one of the clinical side effects during CD19-targeted chimeric antigen receptor (CAR) T (CD19-CAR-T) cells treatment for B cell malignancies. Persistent B cell aplasia was observed in all patients with sustained remission, which increased the patients' risk of infection. Some patients even died due to infection. To overcome this challenge, the concept of incorporating an inhibitory CAR (iCAR) into CAR-T cells was introduced to constrain the T cells response once an "on-target off-tumor" event occurred. In this study, we engineered a novel KIR/PD-1-based inhibitory CAR (iKP CAR) by fusing the extracellular domain of killer cell immunoglobulin-like receptors (KIR) 2DL2 (KIR2DL2) and the intracellular domain of PD-1. We also confirmed that iKP CAR could inhibit the CD19 CAR activation signal via the PD-1 domain and CD19-CAR-T cells bearing an iKP CAR (iKP-19-CAR-T) exerted robust cytotoxicity in vitro and antitumor activity in the xenograft model of CD19+HLA-C1- Burkitt's lymphoma parallel to CD19-CAR-T cells, whilst sparing CD19+HLA-C1+ healthy human B cells both in vitro and in the xenograft model. Meanwhile, iKP-19-CAR-T cells exhibited more naïve, less exhausted phenotypes and preserved a higher proportion of central memory T cells (TCM). Our data demonstrates that the KIR/PD-1-based inhibitory CAR can be a promising strategy for preventing B cell aplasia induced by CD19-CAR-T cell therapy.

Co-Expression of IL-7 Improves NKG2D-Based CAR T Cell Therapy on Prostate Cancer by Enhancing the Expansion and Inhibiting the Apoptosis and Exhaustion.

Chimeric antigen receptor (CAR) T-cell therapy is a promising approach in treating solid tumors but the therapeutic effect is limited. Prostate cancer is a typical solid malignancy with invasive property and a highly immunosuppressive microenvironment. Ligands for the NKG2D receptor are primarily expressed on many cancer cells, including prostate cancer. In this study, we utilized NKG2D-based CAR to treat prostate cancer, and improved the therapeutic effect by co-expression of IL-7. The results showed that NKG2D-CAR T cells performed significantly increased cytotoxicity against prostate cancer compared to non-transduced T cells in vitro and in vivo. Moreover, the introduction of the IL-7 gene into the NKG2D-CAR backbone enhanced the production of IL-7 in an antigen-dependent manner. NKG2DIL7-CAR T cells exhibited better antitumor efficacy at 16 h and 72 h in vitro, and inhibited tumor growth in xenograft models more effectively. In mechanism, enhanced proliferation and Bcl-2 expression in CD8+ T cells, decreased apoptosis and exhaustion, and increased less-differentiated cell phenotype may be the reasons for the improved persistence and survival of NKG2DIL7-CAR T cells. In conclusion, these findings demonstrated that NKG2D is a promising option for CAR T-cell therapy on prostate cancer, and IL-7 has enhanced effect on NKG2D-based CAR T-cell immunotherapy, providing a novel adoptive cell therapy for prostate cancer either alone or in combination with IL-7.

Effects of sesame seed extract as a natural antioxidant on the oxidative stability of sunflower oil.

Natural and de-novo biosynthesized phyto-compounds have gained much significance because of their non-controversial nutritional, health and safety benefits as compared with chemically synthesized commercially rivalry antioxidants. However, none of natural de-novo biosynthesized phyto-compounds has been commercially available and used in customary food business and processing. In this study, efficacy of sesame seed extracts (SSEs) in stabilizing sunflower oil during storage has been studied. Fine powder of sesame seed was extracted in different solvents. The results showed that significant differences in extractability of different solvents and maximum extraction yield (29.48%) were achieved with methanol. The antioxidant components and capability of different extracts were further investigated and evaluated via total phenolic contents, DPPH radical scavenging activity and ß-carotene/linoleic acid calorimetric assays respectively. Being highest in yield and antioxidant potential, methanolic extract was used; three different concentrations of SSE (500, 750, and 1000 µL) were added in 100 mL of sunflower oil to further evaluate its oxidative stability. Sensory and oxidative analysis of baked product from these groups was also evaluated.