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2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Chih-Hao Lu ◽  
Wei-Min Chung ◽  
Chun-Hao Tsai ◽  
Ju-Chien Cheng ◽  
Kai-Cheng Hsu ◽  
...  

AbstractTargeting the programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) axis with monoclonal antibodies (mAbs) represents a crucial breakthrough in anticancer therapy, but mAbs are limited by their poor oral bioavailability, adverse events in multiple organ systems, and primary, adaptive, and acquired resistance, amongst other issues. More recently, the advent of small molecule inhibitors that target the PD-1/PD-L1 axis have shown promising cellular inhibitory activity and the potential to counteract the disadvantages of mAbs. In this study, structure-based virtual screening identified small molecule inhibitors that effectively inhibited the PD-1/PD-L1 interaction. Six of those small molecule inhibitors were applied to cell-based experiments targeting PD-1: CH-1, CH-2, CH-3, CH-4, CH-5, and CH-6. Of all 6, CH-4 displayed the lowest cytotoxicity and strongest inhibitory activity towards the PD-1/PD-L1 interaction. The experiments revealed that CH-4 inhibited the interaction of soluble form PD-L1 (sPD-L1) with PD-1 surface protein expressed by KG-1 cells. Investigations into CH-4 analogs revealed that CH-4.7 effectively blocked the PD-1/sPD-L1 interaction, but sustained the secretion of interleukin-2 and interferon-γ by Jurkat cells. Our experiments revealed a novel small molecule inhibitor that blocks the interaction of PD-1/sPD-L1 and potentially offers an alternative PD-1 target for immune checkpoint therapy.


Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 64
Author(s):  
Antonella Capozzi ◽  
Daniela Caissutti ◽  
Vincenzo Mattei ◽  
Francesca Gado ◽  
Stefano Martellucci ◽  
...  

The endocannabinoid system (ECS) exerts immunosuppressive effects, which are mostly mediated by cannabinoid receptor 2 (CBR2), whose expression on leukocytes is higher than CBR1, mainly localized in the brain. Targeted CBR2 activation could limit inflammation, avoiding CBR1-related psychoactive effects. Herein, we evaluated in vitro the biological activity of a novel, selective and high-affinity CBR2 agonist, called JT11, studying its potential CBR2-mediated anti-inflammatory effect. Trypan Blue and MTT assays were used to test the cytotoxic and anti-proliferative effect of JT11 in Jurkat cells. Its pro-apoptotic activity was investigated analyzing both cell cycle and poly PARP cleavage. Finally, we evaluated its impact on LPS-induced ERK1/2 and NF-kB-p65 activation, TNF-α, IL-1β, IL-6 and IL-8 release in peripheral blood mononuclear cells (PBMCs) from healthy donors. Selective CB2R antagonist SR144528 and CBR2 knockdown were used to further verify the selectivity of JT11. We confirmed selective CBR2 activation by JT11. JT11 regulated cell viability and proliferation through a CBR2-dependent mechanism in Jurkat cells, exhibiting a mild pro-apoptotic activity. Finally, it reduced LPS-induced ERK1/2 and NF-kB-p65 phosphorylation and pro-inflammatory cytokines release in human PBMCs, proving to possess in vitro anti-inflammatory properties. JT11 as CBR2 ligands could enhance ECS immunoregulatory activity and our results support the view that therapeutic strategies targeting CBR2 signaling could be promising for the treatment of chronic inflammatory diseases.


2021 ◽  
Author(s):  
James M Heather ◽  
Matthew J Spindler ◽  
Marta Herrero Alonso ◽  
Yifang Ivana Shui ◽  
David G Millar ◽  
...  

The study and manipulation of T cell receptors (TCRs) is central to multiple fields across basic and translational immunology research. Produced by V(D)J recombination, TCRs are often only recorded in the literature and data repositories as a combination of their V and J gene symbols, plus their hypervariable CDR3 amino acid sequence. However, numerous applications require full-length coding nucleotide sequences. Here we present Stitchr, a software tool developed to specifically address this limitation. Given minimal V/J/CDR3 information, Stitchr produces complete coding sequences representing a fully spliced TCR cDNA. Due to its modular design, Stitchr can be used for TCR engineering using either published germline or novel/modified variable and constant region sequences. Sequences produced by Stitchr were validated by synthesizing and transducing TCR sequences into Jurkat cells, recapitulating the expected antigen specificity of the parental TCR. Using a companion script, Thimble, we demonstrate that Stitchr can process a million TCRs in under ten minutes using a standard desktop personal computer. By systemizing the production and modification of TCR sequences, we propose that Stitchr will increase the speed, repeatability, and reproducibility of TCR research. Stitchr is available on GitHub.


Pharmaceutics ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2173
Author(s):  
Martin Kello ◽  
Tomas Kuruc ◽  
Klaudia Petrova ◽  
Michal Goga ◽  
Zuzana Michalova ◽  
...  

Acute lymphoblastic leukemia (ALL) is the most frequently diagnosed type of leukemia among children. Although chemotherapy is a common treatment for cancer, it has a wide range of serious side effects, including myelo- and immunosuppression, hepatotoxicity and neurotoxicity. Combination therapies using natural substances are widely recommended to attenuate the adverse effects of chemotherapy. The aim of the present study was to investigate the anti-leukemic potential of extract from the lichen Pseudevernia furfuracea (L.) Zopf (PSE) and isolated physodic acid (Phy) in an in vitro ALL model. A screening assay, flow cytometry and Western blotting were used to analyze apoptosis occurrence, oxidative stress, DNA damage and stress/survival/apoptotic pathway modulation induced by the tested substances in Jurkat cells. We demonstrate for the first time that PSE and Phy treatment-induced intrinsic caspase-dependent cell death was associated with increased oxidative stress, DNA damage and cell cycle arrest with the activation of cell cycle checkpoint proteins p53, p21 and p27 and stress/survival kinases p38 MAPK, JNK and PI3K/Akt. Moreover, using peripheral T lymphocytes, we confirmed that PSE and Phy treatment caused minimal cytotoxicity in normal cells, and therefore, these naturally occurring lichen secondary metabolites could be promising substances for ALL therapy.


2021 ◽  
Vol 21 (4) ◽  
pp. 1924-37
Author(s):  
Joy Nkechinyere Adeniyi ◽  
Manimbulu Nlooto ◽  
Mlungisi Ngcobo ◽  
Roshila Moodley ◽  
Exnevia Gomo

Background: Three decoctions, namely Emelia M (EMB), Mshikazi and Delosma H are used by traditional health practitioners in KwaZulu-Natal, South Africa to treat and manage leukaemia and related conditionsObjectives: This study evaluated the in vitro antioxidant activity and phytochemical profile of the aqueous extracts of Emelia M (EMB), Mshikazi and Delosma H decoctions.Methods: Antioxidant activity of the extracts was evaluated using1-diphenyl-2-picrylhydrazyl (DPPH), glutathione (GSH), phosphomolybdate and thiobarbituric acid reactive substance (TBARS) assays. Phytochemical screening was used to determinethe presence of compounds.Results: The DPPH radical scavenging activity was similar to ascorbic acid for EMB and Delosma H, but not for Mshikazi. At 24 h, EMB increased GSH in both THP-1 and Jurkat cells similar to Delosma H while Mshikazi demonstrated the lowest activity. At 48 h, EMB and Delosma H revealed increased GSH in THP-1 cells with no significant decrease in GSH levels in Jurkat cells. However, EMB showed the lowest lipid peroxidation activity compared to Delosma H and Mshikazi after 24 h treatment of both cells. Phenols, flavonoids, terpenoids, saponins were present in all extracts.Conclusion: Extracts of the three decoctions possess both antioxidant and prooxidant properties through high scavenging activity and increased in lipid peroxidation. Keywords: Antioxidants; herbal medicines; Emelia M; Mshikazi; Delosma H.


Pharmaceutics ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2097
Author(s):  
Hiroki Tanaka ◽  
Ryo Miyama ◽  
Yu Sakurai ◽  
Shinya Tamagawa ◽  
Yuta Nakai ◽  
...  

(1) Background: T cells are important target cells, since they exert direct cytotoxic effects on infected/malignant cells, and affect the regulatory functions of other immune cells in a target antigen-specific manner. One of the current approaches for modifying the function of T cells is gene transfection by viral vectors. However, the insertion of the exogenous DNA molecules into the genome is attended by the risk of mutagenesis, especially when a transposon-based gene cassette is used. Based on this scenario, the transient expression of proteins by an in vitro-transcribed messenger RNA (IVT-mRNA) has become a subject of interest. The use of lipid nanoparticles (LNPs) for the transfection of IVT-mRNA is one of the more promising strategies for introducing exogenous genes. In this study, we report on the development of LNPs with transfection efficiencies that are comparable to that for electroporation in a T cell line (Jurkat cells). (2) Methods: Transfection efficiency was improved by optimizing the phospholipids and polyethylene glycol (PEG)-conjugated lipid components. (3) Results: Modification of the lipid composition resulted in the 221-fold increase in luciferase activity compared to a previously optimized formulation. Such a high transfection activity was due to the efficient uptake by clathrin/dynamin-dependent endocytosis and the relatively efficient escape into the cytoplasm at an early stage of endocytosis.


2021 ◽  
Vol 22 (23) ◽  
pp. 12905
Author(s):  
Irina Yermak ◽  
Stanislav Anastyuk ◽  
Anna Kravchenko ◽  
William Helbert ◽  
Valery Glazunov ◽  
...  

New insights into the structure of the hybrid κ/β-carrageenan (κ/β-CRG) of the red alga Tichocarpus crinitus have been obtained. Carrageenan oligosaccharides were prepared through the chemical and enzymatic depolymerization of κ/β-CRG with κ-carrageenase and its the enzyme-resistant fraction. The composition and distribution of the repetition units of κ/β- CRG were investigated by using the negative ion tandem MALDI-TOFMS and ESIMS method, which made it possible to prove and characterize the hybrid structure of this polysaccharide. An analysis revealed the blockwise distribution of the long β-blocks along the polysaccharide chain, with the inclusion of κ/β, μ/ν-blocks and some ι-blocks. Furthermore, the desulfated κ/β-CRG was shown to contain of –G–D– repeating units up to 3.5 kDa. Previous studies have demonstrated that CRGs suppress the replication of several viruses. Here, we established that κ/β-CRG and its oligosaccharides significantly inhibit the transduction efficiency of replication-defective lentiviral particles pseudotyped with the envelope proteins of three different viruses. We found that the polysaccharide and its oligosaccharides strongly reduced the transduction efficiency of lentiviral particles pseudotyped with GP160—the envelope protein of the human immunodeficiency virus HIV-1—when added to T-lymphocyte Jurkat cells. The CRG oligosaccharides displayed significantly higher antiviral activity.


2021 ◽  
Author(s):  
Morgan J. Smith ◽  
Lucia Pastor ◽  
Jeremy R.B. Newman ◽  
Patrick Concannon

<b>Signal regulatory protein SIRPγ (CD172G) is expressed on the surface of lymphocytes where it acts by engaging its ligand, CD47. <i>SIRPG,</i> which encodes SIRPγ, contains a non-synonymous coding variant, rs6043409, which is significantly associated with risk for type 1 diabetes. <i>SIRPG</i> produces multiple transcript isoforms via alternative splicing, all encoding potentially functional proteins. We show that rs6043409 alters a predicted exonic splicing enhancer, resulting in significant shifts in the distribution of <i>SIRPG</i> transcript isoforms. All of these transcript isoforms produced protein upon transient expression <i>in vitro</i>. However, CRISPR targeting of one of the alternatively spliced exons in <i>SIRPG</i> eliminated all SIRPγ expression in Jurkat T cells. These targeted cells formed fewer cell-cell conjugates with each other than with wild type Jurkat cells, expressed reduced levels of genes associated with CD47 signaling and had significantly increased levels of cell surface CD47. In primary CD4<sup>+</sup> and CD8<sup>+</sup> T cells cell surface SIRPγ levels in response to anti-CD3 stimulation varied quantitatively by rs6043409 genotype. Our results suggest that <i>SIRPG</i> is the most likely causative gene for type 1 diabetes risk in the 20p13 region and highlight the role of alternative splicing in lymphocytes in mediating the genetic risk for autoimmunity.</b>


2021 ◽  
Author(s):  
Morgan J. Smith ◽  
Lucia Pastor ◽  
Jeremy R.B. Newman ◽  
Patrick Concannon

<b>Signal regulatory protein SIRPγ (CD172G) is expressed on the surface of lymphocytes where it acts by engaging its ligand, CD47. <i>SIRPG,</i> which encodes SIRPγ, contains a non-synonymous coding variant, rs6043409, which is significantly associated with risk for type 1 diabetes. <i>SIRPG</i> produces multiple transcript isoforms via alternative splicing, all encoding potentially functional proteins. We show that rs6043409 alters a predicted exonic splicing enhancer, resulting in significant shifts in the distribution of <i>SIRPG</i> transcript isoforms. All of these transcript isoforms produced protein upon transient expression <i>in vitro</i>. However, CRISPR targeting of one of the alternatively spliced exons in <i>SIRPG</i> eliminated all SIRPγ expression in Jurkat T cells. These targeted cells formed fewer cell-cell conjugates with each other than with wild type Jurkat cells, expressed reduced levels of genes associated with CD47 signaling and had significantly increased levels of cell surface CD47. In primary CD4<sup>+</sup> and CD8<sup>+</sup> T cells cell surface SIRPγ levels in response to anti-CD3 stimulation varied quantitatively by rs6043409 genotype. Our results suggest that <i>SIRPG</i> is the most likely causative gene for type 1 diabetes risk in the 20p13 region and highlight the role of alternative splicing in lymphocytes in mediating the genetic risk for autoimmunity.</b>


Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 7028
Author(s):  
Chandrasekar Balachandran ◽  
Kenta Yokoi ◽  
Kana Naito ◽  
Jebiti Haribabu ◽  
Yuichi Tamura ◽  
...  

In our previous paper, we reported that amphiphilic Ir complex–peptide hybrids (IPHs) containing basic peptides such as KK(K)GG (K: lysine, G: glycine) (e.g., ASb-2) exhibited potent anticancer activity against Jurkat cells, with the dead cells showing a strong green emission. Our initial mechanistic studies of this cell death suggest that IPHs would bind to the calcium (Ca2+)–calmodulin (CaM) complex and induce an overload of intracellular Ca2+, resulting in the induction of non-apoptotic programmed cell death. In this work, we conduct a detailed mechanistic study of cell death induced by ASb-2, a typical example of IPHs, and describe how ASb-2 induces paraptotic programmed cell death in a manner similar to that of celastrol, a naturally occurring triterpenoid that is known to function as a paraptosis inducer in cancer cells. It is suggested that ASb-2 (50 µM) induces ER stress and decreases the mitochondrial membrane potential (ΔΨm), thus triggering intracellular signaling pathways and resulting in cytoplasmic vacuolization in Jurkat cells (which is a typical phenomenon of paraptosis), while the change in ΔΨm values is negligibly induced by celastrol and curcumin. Other experimental data imply that both ASb-2 and celastrol induce paraptotic cell death in Jurkat cells, but this induction occurs via different signaling pathways.


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