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Chimeric antigen receptor-expressing NK (CAR-NK) cells represent an advancing frontier in cancer immunotherapy, building upon decades of natural killer cell research and recent breakthroughs in CAR technology. While early CAR-NK manufacturing protocols have demonstrated feasibility, existing manufacturing methods, whether utilizing cord blood or peripheral blood sources, often require extended culture periods and intensive labor, creating bottlenecks for widespread therapeutic application. To address these manufacturing hurdles, we have developed an optimized protocol for ex vivo CAR-NK cell production from human peripheral blood that incorporates lessons learned from previous methodologies while introducing novel efficiency improvements. This protocol offers a practical solution for scalable CAR-NK cell manufacturing that can be readily adapted across different production facilities, potentially accelerating the clinical development of CAR-NK therapies.

Suppression of human megakaryocytes by dengue virus (DENV) infection significantly reduces the platelet count that eventually leads to thrombocytopenia, severe dengue and death. To understand DENV interactions with megakaryocytes, we investigated the cell cycle in leukemic human megakaryocytic in vitro cell line (MEG-01 cells). Megakaryocytes are known for complex endomitotic cell cycle leading to their polyploidy state. Our study shows that DENV uses these polyploid cells for its replication. Understanding the modulation of DENV-mediated cell cycle regulation in megakaryocytes is therefore highly important. We show that DENV2 (serotype 2) infection significantly modulates cell cycle signaling. Our protein profile microarray data showed significant upregulation of several cell cycle regulatory proteins including CDK4, CDK1, Cyclin B1 and others or downregulation of Chk1, GSK3-beta, CUL-3, and E2F-3. Quantitative real-time PCR and immunoblotting analyses further confirmed the upregulation of CDK4, CDK1, and Cyclin B1 upon DENV2 infection. Gene silencing of CDK4, CDK1 and Cyclin B1 showed significant reduction in DENV2 loads. Immunoprecipitation analysis further revealed an enhanced interaction between Cyclin B1 and CDK1 upon DENV2 infection that perhaps suggest the substantial changes noted in cell cycle regulation. Overall, our study suggests that DENV2 modulates cell cycle signaling in megakaryocytes and interferes with the critical regulatory proteins that may eventually lead to changes in endomitosis process. In conclusion, we report an important molecular insight regarding DENV2-mediated cell cycle modulation in human megakaryocytes.

Our previous study showed that cells from medically important arthropods, such as ticks, secrete extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Understanding the molecular determinants and mechanism(s) of arthropod-borne flavivirus transmission via exosome biogenesis is very important. In this current study, we showed that in the presence of tick-borne Langat Virus (LGTV; a member of tick-borne encephalitis virus complex), the expression of arthropod SMase, a sphingomyelinase D (SMase D) that catalyzes the hydrolytic cleavage of substrates like sphingomyelin (SM) lipids, was significantly reduced in both ticks ( ) and in tick cells ( ). The SMase reduced levels correlated with down-regulation of its activity upon LGTV replication in tick cells. Our data show that LGTV-mediated suppression of SMase allowed accumulation of SM lipid levels that supported membrane-associated viral replication and exosome biogenesis. Inhibition of viral loads and SM lipid built up upon GW4869 inhibitor treatment reversed the SMase levels and restored its activity. Our results suggest an important role for this spider venomous ortholog SMase in regulating viral replication associated with membrane-bound SM lipids in ticks. In summary, our study not only suggests a novel role for arthropod SMase in tick-LGTV interactions but also provides new insights into its important function in vector defense mechanism(s) against tick-borne virus infection and in anti-viral pathway(s).

IntroductionCAR-T cell therapy is associated with life-threatening inflammatory toxicities, partly due to the activation and secretion of inflammatory cytokines by bystander myeloid cells (BMCs). However, due to limited clinical data, it is unclear whether CAR-NK cells cause similar toxicities.MethodsWe characterized the soluble factors (SFs) released by activated human CAR-T and CAR-NK cells and assessed their role in BMC activation (BMCA).ResultsWe found that SFs from both activated, peripheral blood-derived CAR-T (PB-CAR-T) and CAR-NK (PB-CAR-NK) cells induced BMCA; however, PB-CAR-NK cells caused significantly lower BMCA compared to PB-CAR-T cells. Interestingly, SFs from cord-blood-derived (CB) NK cells caused little to no BMCA, consistent with previous clinical studies showing minimal inflammatory toxicity with CB-CAR-NK cells. Comparative analysis of SFs released by PB-NK and PB-CAR-NK cells following CAR-dependent and CAR-independent activation revealed several candidate factors with the potential to cause BMCA. Antibody-mediated neutralization studies identified a combination of four factors that contribute to PB-CAR-NK cell-mediated BMCA. siRNA-mediated knockdown studies confirmed that inactivating these four factors in PB-CAR-NK cells significantly reduces BMCA. Importantly, neutralization or knockdown of these four factors did not affect CAR-NK cell potency.DiscussionThese data suggest that specific SFs released by PB-CAR-NK cells activate BMCs and have the potential to contribute to inflammatory toxicities. Furthermore, inactivation of these four factors in PB-CAR-NK cells could reduce inflammatory toxicities and improve safety of PB-CAR-NK cell therapy without compromising potency.

Background The genus Swertia is reported to contain potent bitter compounds like iridoids, xanthones and c-glucoflavones that are known to heal many human disorders. In contrast to high ethnomedicinally valued Swertia chirayita , its other species have not been studied extensively, in spite of their common use in traditional medicinal system in Nepalese communities. So, the present study attempts to investigate the content of total polyphenols, flavonoids, antioxidant activity and estimate the rough content of amarogentin, swertiamarin and mangiferin from different species of Swertia from Nepalese Himalayas. Methods Whole plant parts of S. chirayita (SCH), S. angustifolia (SAN), S. paniculata (SPA), S. racemosa (SRA), S. nervosa (SNE), S. ciliata (SCI) and S. dilatata (SDI) were collected; total phenolic and flavonoid contents were quantified spectrophotometrically and in vitro DPPH free radical scavenging assay was measured. Thin layer chromatography was performed on TLC aluminium plates pre-coated with silica gel for identification of swertiamarin, amarogentin and mangiferin from those species and semi quantitative estimation was done using GelQuant.NET software using their standard compounds. Results The phenolic content was highest in the methanol extract of SCH (67.49 ± 0.5 mg GAE/g) followed by SDI, SRA, SNE, SCI, SPA and SAN. The contents of flavonoids were found in the order of SCH, SPA, SRA, SNE, SDI, SCI and SAN. Promising concentration of phenolics and flavonoids produced promising DPPH free radical scavenging values. The IC50 values for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test was lowest in SCH (23.35 ± 0.6 μg/ml), even lower than the standard ascorbic acid among the seven studied species. A significant correlation of 0.977 was observed between the polyphenol content and antioxidant values. The TLC profile showed the presence of all three major phytochemicals; amarogentin, swertiamarin and mangiferin in all of the plant samples. Conclusion Among the seven studied species, SCH showed anticipating results in total phenol content, flavonoid content and DPPH radical scavenging test. The less considered species of Swertia can be a potential source of bioactive amarogentin, and other useful therapeutic compounds in the alarming status of Swertia chirayita as shown by the phytochemical analysis.

Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis infects neutrophils and other cells from hematopoietic origin. Using human megakaryocytic cell line, MEG-01, we show that expression of cell cycle genes in these cells are altered upon A. phagocytophilum infection. Expression of several cell cycle genes in MEG-01 cells was significantly up regulated at early and then down regulated at later stages of A. phagocytophilum infection. Lactate dehydrogenase (LDH) assays revealed reduced cellular cytotoxicity in MEG-01 cells upon A. phagocytophilum infection. The levels of both PI3KCA (p110 alpha, catalytic subunit) and PI3KR1 (p85, regulatory subunit) of Class I PI3 kinases and phosphorylated protein kinase B (Akt/PKB) and inhibitory kappa B (IκB) were elevated at both early and late stages of A. phagocytophilum infection. Inhibition of PI3 kinases with LY294002 treatment resulted in significant reduction in the expression of tested cell cycle genes, A. phagocytophilum burden and phosphorylated Akt levels in these MEG-01 cells. Collectively, these results suggest a role for PI3K-Akt-NF-κB signaling pathway in the modulation of megakaryocyte cell cycle genes upon A. phagocytophilum infection.

Ixodes scapularis is a medically important tick that transmits several microbes to humans, including rickettsial pathogen Anaplasma phagocytophilum. In nature, these ticks encounter several abiotic factors including changes in temperature, humidity, and light. Many organisms use endogenously generated circadian pathways to encounter abiotic factors. In this study, we provide evidence for the first time to show that A. phagocytophilum modulates the arthropod circadian gene for its transmission to the vertebrate host. We noted a circadian oscillation in the expression of arthropod clock, bmal1, period and timeless genes when ticks or tick cells were exposed to alternate 12 h light: 12 h dark conditions. Moreover, A. phagocytophilum significantly modulates the oscillation pattern of expression of these genes. In addition, increased levels of clock and bmal1 and decreased expression of Toll and JAK/STAT pathway immune genes such as pelle and jak, respectively, were noted during A. phagocytophilum transmission from ticks to the vertebrate host. RNAi-mediated knockdown of clock gene expression in ticks resulted in the reduced expression of jak and pelle that increased bacterial transmission from ticks to the murine host. Furthermore, clock-deficient ticks fed late and had less engorgement weights. These results indicate an important role for circadian modulation of tick gene expression that is critical for arthropod blood feeding and transmission of pathogens from vector to the vertebrate host.

Ixodes scapularis ticks transmit several pathogens to humans including rickettsial bacterium, Anaplasma phagocytophilum . Here, we report that A . phagocytophilum uses tick transcriptional activator protein-1 (AP-1) as a molecular switch in the regulation of arthropod antifreeze gene, iafgp . RNAi-mediated silencing of ap-1 expression significantly affected iafgp gene expression and A . phagocytophilum burden in ticks upon acquisition from the murine host. Gel shift assays provide evidence that both the bacterium and AP-1 influences iafgp promoter and expression. The luciferase assays revealed that a region of approximately 700 bp upstream of the antifreeze gene is sufficient for AP-1 binding to promote iafgp gene expression. Furthermore, survival assays revealed that AP-1-deficient ticks were more susceptible to cold in comparison to the mock controls. In addition, this study also indicates arthropod AP-1 as a global regulator for some of the tick genes critical for A . phagocytophilum survival in the vector. In summary, our study defines a novel mode of arthropod signaling for the survival of both rickettsial pathogen and its medically important vector in the cold.

Vector-borne diseases (VBDs) are illnesses transmitted to humans and other animals by arthropods such as ticks, mosquitoes, and fleas. These arthropod vectors transmit infectious pathogens such as viruses, bacteria, and protozoa, to humans during blood-feeding. We have very few control strategies to treat or control these diseases. Human anaplasmosis, caused by the bacterium Anaplasma phagocytophilum, is the second most common tick-borne disease in the United States. This work defines three studies elucidating Anaplasma phagocytophilum-mediated modulation of cell signaling in mammalian cells and arthropod vector Ixodes scapularis ticks. The first study focused on mammalian PI3 kinases signaling in regulating cell cycle gene expression during A. phagocytophilum infection. Using the human megakaryocytic cell line, MEG-01, we observed a differential expression of cell cycle genes in these cells upon A. phagocytophilum infection. Both PI3KCA (p110 alpha, catalytic subunit) and PI3KR1 (p85, regulatory subunit) of Class I PI3 kinases and phosphorylated protein kinase B (Akt/PKB) and IκB were higher at early and late stages of A. phagocytophilum infection. Inhibition of PI3 kinases with LY294002 treatment resulted in a significant reduction in the bacterial load and the expression of cell cycle gene expression. These results suggest a role for PI3K-Akt-NF-κB signaling in the modulation of megakaryocyte cell cycle genes upon A. phagocytophilum infection. The second study showed that A. phagocytophilum uses tick transcriptional activator protein-1 (AP-1) as a molecular switch in the regulation of the arthropod antifreeze gene, iafgp. RNAi-mediated silencing of ap-1 significantly affected iafgp gene expression and bacterial burden in ticks during acquisition from the murine host. The electrophoretic mobility shift assays (EMSAs) revealed that both the bacterium and AP-1 protein influence iafgp promoter and expression. The luciferase assays demonstrated that a 700bp upstream region of the antifreeze gene is sufficient for AP-1 binding to drive iafgp gene expression. Furthermore, survival assays revealed that ap-1 deficient ticks were more susceptible to cold than the mock control ticks. These data show that AP-1 acts as an upstream transcriptional activator to drive the iafgp expression that is critical for A. phagocytophilum survival in I. scapularis ticks. The third study identified and characterized the circadian components in I. scapularis. The identification of the core clock genes in ticks was made using bioinformatic analysis from the Ixodes scapularis genome. Core clock genes like clock1 and bmal1 were upregulated upon tick feeding on the murine host. RNAi-mediated knockdown of the arthropod clock1 gene resulted in an increased bacterial transmission from ticks to the murine host. These results indicate that arthropod clock-mediated signaling is essential for transmitting A. phagocytophilum from tick to the vertebrate host. Taken together, these studies highlight several undefined mechanisms that A. phagocytophilum modulates for its survival in mammalian cells and ticks.