Target Cells Research Articles

Chitosan-modified manganese oxide-conjugated methotrexate nanoparticles delivering 5-aminolevulinic acid as a dual-modal T1-T2* MRI contrast agent in U87MG cell detection.

Glioblastoma multiforme is a highly aggressive form of brain cancer, and early diagnosis plays a pivotal role in improving patient survival rates. In this regard, molecular magnetic resonance imaging has emerged as a promising imaging modality due to its exceptional sensitivity to minute tissue changes and the ability to penetrate deep into the brain. This study aimed to assess the efficacy of a novel contrast agent in detecting gliomas during MRI scans. The contrast agent utilized modified chitosan coating on manganese oxide nanoparticles. The modification included adding methotrexate and 5-aminolevulinic acid (MnO2/CS@5-ALA-MTX) to target cells with overexpressed folate receptors and breaking down excess hydrogen peroxide in tumor tissue, resulting in enhanced signal intensity in T1-weighted MR images but diminished signal intensity in T2*-weighted MR images. The nanosystem was characterized and evaluated in MR imaging, safety, and ability to target cells both in vivo and in vitro. MTX-free nanoparticles (MnO2/CS@5-ALA NPs) had no obvious cytotoxicity on cell lines U87MG and NIH3T3 after 24/48 h at a concentration of up to 160 µgr/mL (cell viability more than 80%). In this system, methotrexate enables tumor targeting and the MnO2/5-ALA improves T1-T2*-weighted MRI. In addition, MRI scans of mice with M109 carcinoma indicated significant tumor uptake and NP capacity to improve the positive contrast effect. This developed MnO2/CS@5-ALA-MTX nanoparticle system may exhibit great potential in the accurate diagnosis of folate receptor over-expressing cancers such as glioblastoma.

Engineering Dimeric EGFR-directed IgA Antibodies Reveals a Central Role of CD147 during Neutrophil-mediated Tumor Cell Killing of Head and Neck Squamous Cancer Cells.

Human IgA Abs engage neutrophils for cancer immunotherapy more effectively than IgG Abs. Previous studies demonstrated that engineering approaches improved biochemical and functional properties. In this study, we report a novel, to our knowledge, IgA2 Ab against the epidermal growth factor receptor generated by protein engineering and polymerization. The resulting molecule demonstrated a covalent linkage of L and H chains and an effective polymerization by the joining chain. The engineered dimer outperformed its monomeric variant in functional experiments on Fab-mediated modes of action and binding to the Fc receptor. The capacity to engage neutrophils for Ab-dependent cell-mediated cytotoxicity (ADCC) of adherent growing target cancer cells was cell line dependent. Although the engineered dimer displayed a long-term efficacy against the vulva carcinoma cell line A431, there was a notable in-efficacy against human papillomavirus (HPV)- head and neck squamous cell carcinoma (HNSCC) cell lines. However, the highly engineered IgA Abs triggered a neutrophil-mediated cytotoxicity against HPV+ HNSCC cell lines. Short-term ADCC efficacy correlated with the target cells' epidermal growth factor receptor expression and the ability of cancer cell-conditioned media to enhance the CD147 surface level on neutrophils. Notably, the HPV+ HNSCC cell lines demonstrated a significant increment in releasing soluble CD147 and a reduced induction of membranous CD147 on neutrophils compared with HPV- cells. Although membranous CD147 on neutrophils may impair proper IgA-Fc receptor binding, soluble CD147 enhanced the IgA-neutrophil-mediated ADCC in a dose-dependent manner. Thus, engineering IgA Abs and impedance-based ADCC assays provided valuable information regarding the target-effector cell interaction and identified CD147 as a putative critical parameter for neutrophil-mediated cytotoxicity.

Mechanisms of neuromodulatory volume transmission.

A wealth of neuromodulatory transmitters regulate synaptic circuits in the brain. Their mode of signaling, often called volume transmission, differs from classical synaptic transmission in important ways. In synaptic transmission, vesicles rapidly fuse in response to action potentials and release their transmitter content. The transmitters are then sensed by nearby receptors on select target cells with minimal delay. Signal transmission is restricted to synaptic contacts and typically occurs within ~1 ms. Volume transmission doesn't rely on synaptic contact sites and is the main mode of monoamines and neuropeptides, important neuromodulators in the brain. It is less precise than synaptic transmission, and the underlying molecular mechanisms and spatiotemporal scales are often not well understood. Here, we review literature on mechanisms of volume transmission and raise scientific questions that should be addressed in the years ahead. We define five domains by which volume transmission systems can differ from synaptic transmission and from one another. These domains are (1) innervation patterns and firing properties, (2) transmitter synthesis and loading into different types of vesicles, (3) architecture and distribution of release sites, (4) transmitter diffusion, degradation, and reuptake, and (5) receptor types and their positioning on target cells. We discuss these five domains for dopamine, a well-studied monoamine, and then compare the literature on dopamine with that on norepinephrine and serotonin. We include assessments of neuropeptide signaling and of central acetylcholine transmission. Through this review, we provide a molecular and cellular framework for volume transmission. This mechanistic knowledge is essential to define how neuromodulatory systems control behavior in health and disease and to understand how they are modulated by medical treatments and by drugs of abuse.

#2416 Bicc1 and PKD: linking RNA metabolism to kidney disease

Abstract Background and Aims Mutations in PKD1 or PKD2 lead to autosomal dominant polycystic kidney disease (PKD) by allowing urine flow-sensing primary cilia to activate tissue growth signals. However, the responsible cilia-associated factors and a strategy to block them and cure PKD are elusive. Cilia also specify organ laterality. In this process, flow-stimulated cilia activate the cytosolic protein Bicaudal-C (Bicc1) to curb the translation and/or the stability of specific target mRNAs. To identify candidate regulatory factors, we investigated if Bicc1 ribonucleoprotein particle (RNP) formation involves known proteins linked to cilia and cystic kidney diseases. Method To address this question, we here used AlphaFold structure modeling of protein complexes, combined with in vitro reconstitution assays and live imaging of Bicc1 RNP assembly. To begin to validate these interactions, we tested by genetic approaches if specific interacting factors also regulate Bicc1 targets in cells and mice. Results We found that multivalent interactions with structured domains of the ankyrin repeat and SAM domain containing (ANKS) 3 and their allosteric regulation by the related ANKS6 protein inhibit or restore Bicc1 access to target mRNAs, respectively. Concomitantly, ANKS3 alone increased the solubility and fluidity of Bicc1, whereas co-recruitment of ANKS6 had the opposite effect, liberating Bicc1 to self-polymerize and to induce phase-transitioning of target mRNAs in gel-like meshworks. Conclusion Perturbations in Bicc1-ANKS3-ANKS6 RNP dynamics and altered translation of Bicc1 target mRNAs emerge as likely culprits regulating cyst formation downstream of ciliary defects. ANKS6 can be sequestered in cilia by inversin, and mutations in these proteins provoke kidney cysts in nephronophthisis patients. Therefore, besides systematically profiling Bicc1 RNPs, future studies should test if ANKS6 mediates their dynamic regulation by flow-stimulated cilia.

#2207 CD3(+) T cell depletion does not predict short term outcomes in kidney transplant patients

Abstract Background and Aims Anti-thymocyte globulin (ATG) is the most frequently agent used for immunosuppression induction therapy after kidney transplantation. The efficacy of the ATG treatment is monitored by peripheral blood CD3(+) T cell counts. ATG dosing can be tailored to reach and keep the target commonly accepted as below 50/μL within the week of transplant. In this study, we investigated the significance of reaching CD3(+) T cell target regarding short term adverse outcomes of biopsy-proved rejection, BK viremia and infection related hospitalization in living donor kidney transplant recipients. Method This retrospective observational study was conducted on adult patients who received living donor kidney transplants from January 1, 2015 to October 1, 2022. Demographic, clinical and laboratory information were collected using electronical medical records. The average CD3(+) T cell counts of postoperative first 3 days were calculated. Patients with CD3(+) T cell counts ≤ 50/μL were classified as “Optimally Treated”, while the patients with CD3(+) T cell counts >50/μL were classified as “Suboptimally Treated”. The incidence of biopsy-proven rejection, BK viremia and infection related hospitalization in 6 months following the transplant surgery were compared in these two groups. Results Sixty one patients were included in the study. Twenty eight were women and the average age was 39 ± 12.7 years. Average HLA mismatch count was 3.6 ± 1.7. Ten patients were desensitized before kidney transplant. Average ATG dose was 6.8 ± 2.3 mg/kg. Mean average CD3(+) T cell count of postoperative first 3 days were 93.9 ± 87.8/μL. During 6 months of follow up, 34.4% (n = 21) had at least one biopsy proved rejection episode, 21.3% (n = 13) had at least one infection related hospitalization and 11.4% (n = 7) had BK viremia. According to average CD3(+) T cell counts of postoperative first 3 days, 16 patients were in “Optimally Treated” group, whereas 45 patients were in “Suboptimally Treated” group. The patients in both groups had similar mean age, gender distribution, HLA mismatch, pretransplant desensitization status and total ATG doses. In 6 months of follow-up, the patients in “Optimally Treated” and “Suboptimally Treated” groups had no statistically difference in incidence of biopsy-proved rejection, BK viremia and infection related hospitalization. Conclusion In our study, we could not find any evidence to confirm the significance of reaching CD3(+) T cell target of 50/μL in first 3 days after transplant surgery regarding short term adverse outcomes of biopsy-proved rejection, BK viremia and infection related hospitalization. The value of CD3(+) T cell monitoring to adjust ATG therapy after kidney transplant surgery is questionable. Thus, in the light of our findings, we do not support the use of CD3(+) T cell counts in optimization of induction therapy with ATG after renal transplantation.

Transposon DNA sequences facilitate the tissue-specific gene transfer of circulating tumor DNA between human cells.

The exchange of genes between cells is known to play an important physiological and pathological role in many organisms. We show that circulating tumor DNA (ctDNA) facilitates cell-specific gene transfer between human cancer cells and explain part of the mechanisms behind this phenomenon. As ctDNA migrates into the nucleus, genetic information is transferred. Cell targeting and ctDNA integration require ERVL, SINEor LINE DNA sequences. Chemically manufactured AluSp and MER11C sequences replicated multiple myeloma (MM) ctDNA cell targeting and integration. Additionally, we found that ctDNA may alter the treatment response of MM and pancreatic cancer models. This study shows that retrotransposon DNA sequences promote cancer gene transfer. However, because cell-free DNA has been detected in physiological and other pathological conditions, our findings have a broader impact than just cancer. Furthermore, the discovery that transposon DNA sequences mediate tissue-specific targeting will open up a new avenue for the delivery of genes and therapies.

Diabetes and cancer: two epidemic diseases requiring an opposite therapeutic approach to target cells

Diabetes and cancer are two chronic metabolic diseases with ever-increasing incidence rates worldwide. These disorders can often occur together, as diabetes presents an important risk factor for cancer and some cancers could in turn lead to diabetes. In this perspective article, many more commonalities between diabetes and cancer are highlighted, including the role of lifestyle and environmental factors in the pathogenesis, the presence of a rather long latency period before clinical diagnosis of invasive disease, as well as the ultimate progression to diabetic complications or malignant metastases. Moreover, both of these devastating disorders still lack curative treatment options, whereas several currently approved antidiabetic and anticancer drugs have been originally derived from different natural sources. However, while in the case of diabetes, the main therapeutic goal is to maintain the pancreatic islet mass by preserving β-cells survival, the major purpose of cancer therapy is to kill malignant cells and reduce the neoplastic mass of solid tumors. It is expected that both diabetes and cancer, two systemic diseases with epidemic proportions, would be managed more effectively through an integral approach, considering many different aspects related to their pathogenesis, including also lifestyle changes and dietary modifications.

SIV infection and ARV treatment reshape the transcriptional and epigenetic profile of naïve and memory T cells in vivo.

Human and simian immunodeficiency viruses (HIV and SIV) are lentiviruses that reverse transcribe their RNA genome with subsequent integration into the genome of the target cell. How progressive infection and administration of antiretrovirals (ARVs) longitudinally influence the transcriptomic and epigenetic landscape of particular T cell subsets, and how these may influence the genetic location of integration are unclear. Here, we use RNAseq and ATACseq to study the transcriptomics and epigenetic landscape of longitudinally sampled naïve and memory CD4+ and CD8+ T cells in two species of non-human primates prior to SIV infection, during chronic SIV infection, and after administration of ARVs. We find that SIV infection leads to significant alteration to the transcriptomic profile of all T cell subsets that are only partially reversed by administration of ARVs. Epigenetic changes were more apparent in animals with longer periods of untreated SIV infection and correlated well with changes in corresponding gene expression. Known SIV integration sites did not vary due to SIV status but did contain more open chromatin in rhesus macaque memory T cells, and the expression of proteasome-related genes at the pre-SIV timepoint correlated with subsequent viremia.IMPORTANCEChronic inflammation during progressive human and simian immunodeficiency virus (HIV and SIV) infections leads to significant co-morbidities in infected individuals with significant consequences. Antiretroviral (ARV)-treated individuals also manifest increased levels of inflammation which are associated with increased mortalities. These data will help guide rational development of modalities to reduce inflammation observed in people living with HIV and suggest mechanisms underlying lentiviral integration site preferences.

The effect of bone marrow mesenchymal stromal cell exosomes on acute myeloid leukemia’s biological functions: a focus on the potential role of LncRNAs

Acute myeloid leukemia represents a group of malignant blood disorders that originate from clonal over-proliferation and the differentiation failure of hematopoietic precursors, resulting in the accumulation of blasts in the bone marrow. Mesenchymal stromal cells (MSCs) have been shown to exert diverse effects on tumor cells through direct and indirect interaction. Exosomes, as one of the means of indirect intercellular communication, are released from different types of cells, including MSCs, and their various contents, such as lncRNAs, enable them to exert significant impacts on target cells. Our study aims to investigate the effects of BM-MSC exosomes on the cellular and molecular characterization of HL-60 AML cells, particularly detecting the alterations in the expression of lncRNAs involved in AML leukemogenesis, cell growth, drug resistance, and poor prognosis. BM-MSCs were cultured with serum-free culture media to isolate exosomes from their supernatants. The validation of exosomes was performed in three stages: morphological analysis using TEM, size evaluation using DLS, and CD marker identification using flow cytometry. Subsequently, the HL-60 AML cells were treated with isolated BM-MSC exosomes to determine the impact of their contents on leukemic cells. Cell metabolic activity was evaluated by the MTT assay, while cell cycle progression, apoptosis, ROS levels, and proliferation were assessed by flow cytometry. Furthermore, RT-qPCR was conducted to determine the expression levels of lncRNAs and apoptosis-, ROS-, and cell cycle-related genes. MTT assay and flow cytometry analysis revealed that BM-MSC exosomes considerably suppressed cell metabolic activity, proliferation, and cell cycle progression. Also, these exosomes could effectively increase apoptosis and ROS levels in HL-60 cells. The expression levels of p53, p21, BAX, and FOXO4 were increased, while the BCL2 and c-Myc levels decreased. MALAT1, HOTAIR, and H19 expression levels were also significantly decreased in treated HL-60 cells compared to their untreated counterparts. BM-MSC exosomes suppress cell cycle progression, proliferation, and metabolic activity while simultaneously elevating the ROS index and apoptosis ratio in HL-60 cells, likely by reducing the expression levels of MALAT1, HOTAIR, and H19. These findings suggest that BM-MSC exosomes might serve as potential supportive therapies for leukemia.

Tools for Cre-mediated conditional deletion of floxed alleles from developing cerebellar Purkinje cells.

The Cre-lox system is an indispensable tool in neuroscience research for targeting gene deletions to specific cellular populations. Here we assess the utility of several transgenic Cre lines, along with a viral approach, for targeting cerebellar Purkinje cells in mice. Using a combination of a fluorescent reporter line (Ai14) to indicate Cre-mediated recombination and a floxed Dystroglycan line (Dag1flox ), we show that reporter expression does not always align precisely with loss of protein. The commonly used Pcp2Cre line exhibits a gradual mosaic pattern of Cre recombination in Purkinje cells from P7-P14, while loss of Dag1 protein is not complete until P30. Ptf1aCre drives recombination in precursor cells that give rise to GABAergic neurons in the embryonic cerebellum, including Purkinje cells and molecular layer interneurons. However, due to its transient expression in precursors, Ptf1aCre results in stochastic loss of Dag1 protein in these neurons. NestinCre , which is often described as a "pan-neuronal" Cre line for the central nervous system, does not drive Cre-mediated recombination in Purkinje cells. We identify a Calb1Cre line that drives efficient and complete recombination in embryonic Purkinje cells, resulting in loss of Dag1 protein before the period of synaptogenesis. AAV8-mediated delivery of Cre at P0 results in gradual transduction of Purkinje cells during the second postnatal week, with loss of Dag1 protein not reaching appreciable levels until P35. These results characterize several tools for targeting conditional deletions in cerebellar Purkinje cells at different developmental stages and illustrate the importance of validating the loss of protein following recombination.Significance Statement The development of Cre lines for targeting gene deletions to defined cellular populations has led to important discoveries in neuroscience. As with any tool, there are inherent limitations that must be carefully considered. Here we describe several Cre lines available for targeting of cerebellar Purkinje cells at various developmental stages. We use the combination of a Cre-dependent fluorescent reporter line and conditional deletion of the synaptic scaffolding molecule Dystroglycan as an example to highlight the potential disconnect between the presence of a fluorescent reporter and the loss of protein.

Comparison of the Differences between Two-Photon Excitation, Upconversion, and Conventional Photodynamic Therapy on Cancers in In Vitro and In Vivo Studies

Photodynamic therapy (PDT) is a minimally invasive treatment for several diseases. It combines light energy with a photosensitizer (PS) to destroy the targeted cells or tissues. A PS itself is a non-toxic substance, but it becomes toxic to the target cells through the activation of light at a specific wavelength. There are some limitations of PDT, although it has been used in clinical studies for a long time. Two-photon excitation (TPE) and upconversion (UC) for PDT have been recently developed. A TPE nanoparticle-based PS combines the advantages of TPE and nanotechnology that has emerged as an attractive therapeutic agent for near-infrared red (NIR) light-excited PDT, whilst UC is also used for the NIR light-triggered drug release, activation of ‘caged’ imaging, or therapeutic molecules during PDT process for the diagnosis, imaging, and treatment of cancers. Methods: Nine electronic databases were searched, including WanFang Data, PubMed, Science Direct, Scopus, Web of Science, Springer Link, SciFinder, and China National Knowledge Infrastructure (CNKI), without any language constraints. TPE and UCNP were evaluated to determine if they had different effects from PDT on cancers. All eligible studies were analyzed and summarized in this review. Results: TPE-PDT and UCNP-PDT have a high cell or tissue penetration ability through the excitation of NIR light to activate PS molecules. This is much better than the conventional PDT induced by visible or ultraviolet (UV) light. These studies showed a greater PDT efficacy, which was determined by enhanced generation of reactive oxygen species (ROS) and reduced cell viability, as well as inhibited abnormal cell growth for the treatment of cancers. Conclusions: Conventional PDT involves Type I and Type II reactions for the generation of ROS in the treatment of cancer cells, but there are some limitations. Recently, TPE-PDT and UCNP-PDT have been developed to overcome these problems with the help of nanotechnology in in vitro and in vivo studies.

Enhancing Effector Jurkat Cell Activity and Increasing Cytotoxicity against A549 Cells Using Nivolumab as an Anti-PD-1 Agent Loaded on Gelatin Nanoparticles

The current research investigated the use of gelatin nanoparticles (GNPs) for enhancing the cytotoxic effects of nivolumab, an immune checkpoint inhibitor. The unique feature of GNPs is their biocompatibility and functionalization potential, improving the delivery and the efficacy of immunotherapeutic drugs with fewer side effects compared to traditional treatments. This exploration of GNPs represents an innovative direction in the advancement of nanomedicine in oncology. Nivolumab-loaded GNPs were prepared and characterized. The optimum formulation had a particle size of 191.9 ± 0.67 nm, a polydispersity index of 0.027 ± 0.02, and drug entrapment of 54.67 ± 3.51%. A co-culture experiment involving A549 target cells and effector Jurkat cells treated with free nivolumab solution, and nivolumab-loaded GNPs, demonstrated that the latter had significant improvements in inhibition rate by scoring 87.88 ± 2.47% for drug-loaded GNPs against 60.53 ± 3.96% for the free nivolumab solution. The nivolumab-loaded GNPs had a lower IC50 value, of 0.41 ± 0.01 µM, compared to free nivolumab solution (1.22 ± 0.37 µM) at 72 h. The results indicate that administering nivolumab-loaded GNPs augmented the cytotoxicity against A549 cells by enhancing effector Jurkat cell activity compared to nivolumab solution treatment.

Novel Cholesterol-Based Dihydropyrimidinone Derivatives with Potential Biological Activities: An In-silico Investigation

Steroids play essential physiological roles in the living systems, including their role as sex hormones, glucocorticoids, and mineralocorticoids. Usually, they engage in interactions with steroid receptors. Target cell nuclei, cytosol and plasma membranes all have steroid hormone receptors. Cytoplasmic or nuclear, intracellular receptors start the signal transduction process for steroid hormones and cause gene expression alterations over the course of several hours to days. Certain steroid hormones bind to cell surface receptors, including ion channels, G protein-coupled receptors and several nuclear receptors. Dihydropyrimidinones are also significant moieties with important physiological roles. The hybrid molecules can offer several potential biological activities. In this work, we have checked the in silico interaction of some novel cholesterol–dihydropyrimidinones interactions with the sex hormones, mineralocorticoid and glucocorticoid receptors to screen their potential use in the regulation of the sexual cycle or the carbohydrate metabolism or the mineral imbalance.

A simple protocol to establish a conditionally immortalized mouse podocyte cell line

Podocytes are specialized terminally differentiated cells in the glomerulus that are the primary target cells in many glomerular diseases. However, the current podocyte cell lines suffer from prolonged in vitro differentiation and limited survival time, which impede research progress. Therefore, it is necessary to establish a cell line that exhibits superior performance and characteristics. We propose a simple protocol to obtain an immortalized mouse podocyte cell (MPC) line from suckling mouse kidneys. Primary podocytes were cultured in vitro and infected with the SV40 tsA58 gene to obtain immortalized MPCs. The podocytes were characterized using Western blotting and quantitative real-time PCR. Podocyte injury was examined using the Cell Counting Kit-8 assay and flow cytometry. First, we successfully isolated an MPC line and identified 39 °C as the optimal differentiation temperature. Compared to undifferentiated MPCs, the expression of WT1 and synaptopodin was upregulated in differentiated MPCs. Second, the MPCs ceased proliferating at a nonpermissive temperature after day 4, and podocyte-specific proteins were expressed normally after at least 15 passages. Finally, podocyte injury models were induced to simulate podocyte injury in vitro. In summary, we provide a simple and popularized protocol to establish a conditionally immortalized MPC, which is a powerful tool for the study of podocytes.

Adverse events associated with chimeric antigen receptor T-Cell therapy in ophthalmology: a narrative review

Chimeric antigen receptors are synthetically produced receptors engineered to engage with target cells with high specificity. These cells are created by inserting an artificial T-cell receptor into an immunoglobulin’s antigen-binding region, allowing the cells to combine and target specific antigens. The use of chimeric antigen receptor (CAR) T-cell therapy has been a remarkable achievement in the field of immunotherapy, particularly in the treatment of ophthalmic tumors like retinoblastoma and uveal melanoma. However, there are some documented side effects, such as cytokine release syndrome (CRS) and immunological effector cell-associated neurotoxicity syndrome (ICANS). Additionally, ocular side effects such as blurred vision, vision impairment, and intraocular infections are also concerning and require further evaluation. This review highlights the advances made in chimeric antigen receptor (CAR) immunotherapy, including its structure and manufacture, as well as relevant clinical discoveries and associated adverse effects. By identifying the gaps in current research, this analysis provides insights into potential strategies and solutions for addressing some of the most severe side effects.

S309-CAR-NK cells bind the Omicron variants in vitro and reduce SARS-CoV-2 viral loads in humanized ACE2-NSG mice.

Chimeric antigen receptor (CAR)-NK cells can be "off-the-shelf" products that treat various diseases, including cancer, infections, and autoimmune diseases. In this study, we engineered natural killer (NK) cells to express S309 single-chain fragment variable (scFv), to target the Spike protein of SARS-CoV-2, hereinafter S309-CAR-NK cells. Our study shows that S309-CAR-NK cells are effective against different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants. The S309-CAR-NK cells can (i) directly bind to SARS-CoV-2 pseudotyped virus (PV), (ii) competitively bind to SARS-CoV-2 PV with 293T cells expressing the human angiotensin-converting enzyme 2 (hACE2) receptor (293T-hACE2 cells), (iii) specifically target and lyse A549 cells expressing the spike protein, and (iv) significantly reduce the viral loads of SARS-CoV-2 wild-type (strain USA/WA1/2020) in the lungs of NOD/SCID gamma (NSG) mice expressing hACE2 (hACE2-NSG mice). Altogether, the current study demonstrates the potential use of S309-CAR-NK immunotherapy as an alternative treatment for COVID-19 patients.

NK cell receptor modulation in viral infections.

Natural killer (NK) cells play a crucial role in controlling viral infections. The ability to kill infected cells without prior immunization, yet being tolerant to self, healthy cells, depends on the balance of germ-line encoded surface receptors. NK cell receptors are divided to either activating, leading to activation of NK cell and its cytotoxic and pro-inflammatory activity, or inhibitory, providing tolerance for a target cell. The signals from inhibitory receptors dominate and NK cell activation requires stimulation of activating receptors. In viral infections, NK cell interaction with infected cell can result in activation, memory-like NK cell differentiation or NK cell exhaustion, which constitutes one of the viral immune evasion mechanisms. All of these states are associated with the modulation of NK cell receptor expression. In this review, we summarize the current knowledge of NK cell receptors and their role in viral infection control, as well as the alterations of their expression observed in acute or chronic infections. We present recently discovered SARS-CoV-2-mediated modulation of NK cell receptor expression and compare them with other human viral infections. Finally, since modulation of NK cell receptor activation gives promising addition to currently used antiviral therapies, we briefly discuss the clinical significance and future perspective of application of agonists or antagonists of activating and inhibitory receptors, respectively. In sum, our review shows that although much is known about NK cell receptor biology, a deeper understanding of NK cell receptors role in viral infections is still needed.

Novel Efficient Lipid-Based Delivery Systems Enable a Delayed Uptake and Sustained Expression of mRNA in Human Cells and Mouse Tissues.

Over the past decade, mRNA-based therapy has displayed significant promise in a wide range of clinical applications. The most striking example of the leap in the development of mRNA technologies was the mass vaccination against COVID-19 during the pandemic. The emergence of large-scale technology and positive experience of mRNA immunization sparked the development of antiviral and anti-cancer mRNA vaccines as well as therapeutic mRNA agents for genetic and other diseases. To facilitate mRNA delivery, lipid nanoparticles (LNPs) have been successfully employed. However, the diverse use of mRNA therapeutic approaches requires the development of adaptable LNP delivery systems that can control the kinetics of mRNA uptake and expression in target cells. Here, we report effective mRNA delivery into cultured mammalian cells (HEK293T, HeLa, DC2.4) and living mouse muscle tissues by liposomes containing either 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) or the newly applied 1,30-bis(cholest-5-en-3β-yloxycarbonylamino)-9,13,18,22-tetraaza-3,6,25,28-tetraoxatriacontane tetrahydrochloride (2X7) cationic lipids. Using end-point and real-time monitoring of Fluc mRNA expression, we showed that these LNPs exhibited an unusually delayed (of over 10 h in the case of the 2X7-based system) but had highly efficient and prolonged reporter activity in cells. Accordingly, both LNP formulations decorated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000) provided efficient luciferase production in mice, peaking on day 3 after intramuscular injection. Notably, the bioluminescence was observed only at the site of injection in caudal thigh muscles, thereby demonstrating local expression of the model gene of interest. The developed mRNA delivery systems hold promise for prophylactic applications, where sustained synthesis of defensive proteins is required, and open doors to new possibilities in mRNA-based therapies.

Enhancing in vivo cell and tissue targeting by modulation of polymer nanoparticles and macrophage decoys.

The in vivo efficacy of polymeric nanoparticles (NPs) is dependent on their pharmacokinetics, including time in circulation and tissue tropism. Here we explore the structure-function relationships guiding physiological fate of a library of poly(amine-co-ester) (PACE) NPs with different compositions and surface properties. We find that circulation half-life as well as tissue and cell-type tropism is dependent on polymer chemistry, vehicle characteristics, dosing, and strategic co-administration of distribution modifiers, suggesting that physiological fate can be optimized by adjusting these parameters. Our high-throughput quantitative microscopy-based platform to measure the concentration of nanomedicines in the blood combined with detailed biodistribution assessments and pharmacokinetic modeling provides valuable insight into the dynamic in vivo behavior of these polymer NPs. Our results suggest that PACE NPs-and perhaps other NPs-can be designed with tunable properties to achieve desired tissue tropism for the in vivo delivery of nucleic acid therapeutics. These findings can guide the rational design of more effective nucleic acid delivery vehicles for in vivo applications.

Light‐Driven Micronavigators for Directional Migration of Cells

AbstractCell migration is an essential physiological process in the life cycle of cells, playing a crucial role in cancer metastasis, neural development, and cellular immune response. However, achieving precise control of cell migration at single‐cell level is challenging due to the intricate and diverse microenvironments of cells. Here, an optical technique is presented that utilizes light‐actuated micronavigators to guide the directional migration of individual cells both in vitro and in vivo. Employing high‐speed scanning optical tweezers, micronavigators near target cells are trapped and rotated at a rotation speed of up to 12 000 rpm, which, to the best of knowledge, represents the fastest rotation of light‐driven micromotors in a biological environment to date. The micronavigators generate a powerful fluid shear force (up to 40 pN) which can guide the migration of immune and nerve cells in a predetermined direction. Furthermore, micronavigators are employed to guide cell migration in various biological systems, including lab‐on‐a‐chip devices and blood vessels within living animals. This technique offers new opportunities for controlling cell migration, enabling precise immune activation, and neuron repair at the single‐cell level.