Lytic Function Research Articles

Inhibitors identify an auxiliary role for mTOR signalling in necroptosis execution downstream of MLKL activation.

Necroptosis is a lytic and pro-inflammatory form of programmed cell death executed by the terminal effector, the MLKL (mixed lineage kinase domain-like) pseudokinase. Downstream of death and Toll-like receptor stimulation, MLKL is trafficked to the plasma membrane via the Golgi-, actin- and microtubule-machinery, where activated MLKL accumulates until a critical lytic threshold is exceeded and cell death ensues. Mechanistically, MLKL's lytic function relies on disengagement of the N-terminal membrane-permeabilising four-helix bundle domain from the central autoinhibitory brace helix: a process that can be experimentally mimicked by introducing the R30E MLKL mutation to induce stimulus-independent cell death. Here, we screened a library of 429 kinase inhibitors for their capacity to block R30E MLKL-mediated cell death, to identify co-effectors in the terminal steps of necroptotic signalling. We identified 13 compounds - ABT-578, AR-A014418, AZD1480, AZD5363, Idelalisib, Ipatasertib, LJI308, PHA-793887, Rapamycin, Ridaforolimus, SMI-4a, Temsirolimus and Tideglusib - each of which inhibits mammalian target of rapamycin (mTOR) signalling or regulators thereof, and blocked constitutive cell death executed by R30E MLKL. Our study implicates mTOR signalling as an auxiliary factor in promoting the transport of activated MLKL oligomers to the plasma membrane, where they accumulate into hotspots that permeabilise the lipid bilayer to cause cell death.

Proteomic and phenotypic characteristics of memory-like natural killer cells for cancer immunotherapy

BackgroundHuman and mouse natural killer (NK) cells have been shown to develop memory-like function after short-term exposure to the cocktail of IL-12/15/18 or to overnight co-culture with some tumor cell...

Abstract 1096: Enhancing effector function in T cell adoptive cell therapy in murine models of pediatric sarcomas

Abstract New treatment approaches for high-risk pediatric sarcomas are desperately needed. Immunotherapy advances with adoptive cell therapy in hematologic malignancies have not yet translated to pediatric solid tumors including sarcomas. Hallmarks of the immune microenvironment in pediatric sarcomas include the presence of immunosuppressive cytokines and immune cells and a lack of potent T cell activation. We are seeking ways to overcome these barriers to effective adoptive cell therapy in pediatric sarcoma. We are exploring introduction of a novel synthetic co-receptor, CD8a:MyD88, that can augment tumor-specific T cell effector function and potentially improve outcomes in murine models of sarcoma. Tumor-antigen specific T cells or tumor-infiltrating T cell populations are transduced ex vivo to express the CD8a:MyD88 synthetic co-receptor resulting in MyD88 signaling when T cells encounter their cognate MHC I/epitope complex. We have studied the effects of CD8a:MyD88 expression on cytokine production and lytic activity against murine sarcoma cell lines in vitro. Initial experiments utilized the OT1 T cell model (specific to the model antigen chicken ovalbumin) which we introduced into our murine sarcoma cell lines (KPSarc pleomorphic sarcoma and M3-9M rhabdomyosarcoma). Using GFP as a marker of T cell transduction, the production of effector cytokine interferon gamma was measured following T cell activation compared with GFP-negative non-transduced T cells. CD8a:MyD88 expressing T cells expressed higher levels of interferon gamma at basal levels and following antigen-specific activation when co-cultured with Ova-expressing sarcoma cells. Improved tumor-specific lytic function in CD8a:MyD88 expressing T cells was also observed in co-culture experiments compared to untransduced or control vector- transduced T cells. We further hypothesized that skewing T cell differentiation toward an effector phenotype with the addition of interleukin-12 during T cell expansion would increase the anti-tumor effect of the CD8a:MyD88 expressing T cells. Co-culture with our murine sarcoma cell lines showed increased tumor-lytic function in T cells primed with IL-12. We have ongoing experiments studying the in vivo effects of the CD8a:MyD88 expressing T cells in model antigen-expressing murine sarcoma models. In addition, the ability of the CD8a:MyD88 co-receptor to augment the effector function of polyclonal sarcoma infiltrating T cells is being explored. These results will further define the obstacles and potential solutions to successful adoptive T cell therapy for pediatric sarcomas. Citation Format: Jessica P. Sanchez, Eduardo Davila, Brian H. Ladle. Enhancing effector function in T cell adoptive cell therapy in murine models of pediatric sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1096.

Whole-Cell Electron Tomography Analysis of Vacuole Biogenesis.

Vacuoles in plant cells are the most prominent organelles that harbor distinctive features, including lytic function, storage of proteins and sugars, balance of cell volume, and defense responses. Despite their dominant size and functional versatility, the nature and biogenesis of vacuoles in plants per se remain elusive and several models have been proposed. Recently, we used the whole-cell 3D electron tomography (ET) technique to study vacuole formation and distribution at nanometer resolution and demonstrated that small vacuoles are derived from multivesicular body maturation and fusion. Good sample preparation is a critical step to get high-quality electron tomography images. In this chapter, we provide detailed sample preparation methods for high-resolution ET in Arabidopsis thaliana root cells, including high-pressure freezing, subsequent freeze-substitution fixation, embedding, and serial sectioning.

Prevalence of anti-lymphocyte IgM autoantibodies driving complement activation in COVID-19 patients.

COVID-19 patients can develop autoantibodies against a variety of secreted and membrane proteins, including some expressed on lymphocytes. However, it is unclear what proportion of patients might develop anti-lymphocyte antibodies (ALAb) and what functional relevance they might have. We evaluated the presence and lytic function of ALAb in the sera of a cohort of 85 COVID-19 patients (68 unvaccinated and 17 vaccinated) assigned to mild (N=63), or moderate/severe disease (N=22) groups. Thirty-seven patients were followed-up after recovery. We also analyzed in vivo complement deposition on COVID-19 patients' lymphocytes and examined its correlation with lymphocyte numbers during acute disease. Compared with healthy donors (HD), patients had an increased prevalence of IgM ALAb, which was significantly higher in moderate/severe disease patients and persisted after recovery. Sera from IgM ALAb+ patients exhibited complement-dependent cytotoxicity (CDC) against HD lymphocytes. Complement protein C3b deposition on patients' CD4 T cells was inversely correlated with CD4 T cell numbers. This correlation was stronger in moderate/severe disease patients. IgM ALAb and complement activation against lymphocytes may contribute to the acute lymphopenia observed in COVID-19 patients.

A GH19 lysozyme and peptidase from Myoviridae cyanophages lacking the typical holin-endolysin system exhibit lytic activity

Most of the dsDNA cyanophages employ holin-endolysin lysis systems to damage the host cells. This study aimed to elucidate the lytic activity of ORF91 and ORF117 in the cyanophage MaMV-DH01, which lacked a conventional cholinesterase system. These two proteins contained Lyz-like superfamily domains and were annotated as a member of GH family 19 (named DHGH19) and peptidase (named DHpeptidase), respectively. Overexpression of DHGH19 in E. coli over a 5 h course demonstrated potent bactericidal activity, evident from significant growth inhibition, membrane damage, and leakage of intracellular enzymes of E. coli cells. However, the lytic activity of DHpeptidase was relatively weaker, exhibiting a bacteriostatic effect. It was important to highlight that the specific mutation of enzyme-catalyzed residues in DHGH19 (E122 and E131) showed that these were the essential amino acids for DHGH19 to exert its bactericidal activity. Furthermore, the lytic function of DHGH19 and DHpeptidase on cyanobacteria cells was confirmed by their overexpression in the cyanobacterium Synechocystis sp. PCC6803. Overall, this study provides novel insights into the lytic mechanism of Myoviridae cyanophage, offering potential alternatives for the development of GH19 and peptidase as new antibacterial agents in the future.

Rapid assessment of changes in phage bioactivity using dynamic light scattering.

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. In this study, we use dynamic light scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-y-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web application (Phage-Estimator of Lytic Function) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and nondestructive tool for quality control of phage preparations in academic and commercial settings.

Prevalence and complement activation of anti-lymphocyte IgM antibodies in hospitalized COVID-19 patients

Abstract Recent studies have shown that a proportion of COVID-19 patients develop auto-antibodies against a wide range of secreted and membrane proteins, some of which are expressed by lymphocytes. It is unclear, however, if and what proportion of patients might develop anti-lymphocyte antibodies (ALAb) and whether they might have cytotoxic function. We systematically characterized the presence, isotype and lytic function of ALAb in a cohort of 85 COVID-19 patients during hospital admission from May 2020 to June 2022. As controls we analyzed 20 healthy donors (HD) and seven Flu infected volunteers. Patients were assigned into mild (N=65), or moderate/severe (N=20) groups, based on oxygen requirements, and 37 patients were followed up after recovery. We found that 27% of COVID-19 patients had IgM ALAb (binding CD4, CD8 and/or B cells), compared to 15% of the control populations. IgM ALAb prevalence was independent of disease severity and persisted during early convalescence. Sera from some of the IgM ALAb positive COVID-19 patients, and not from the controls, induced complement dependent cytotoxicity (CDC) of HD PBMCs, mostly targeting B cells. Complement activation was corroborated by detecting complement deposition ex vivo on lymphocytes from 70% of COVID-19 acutely infected patients. The level of C3b deposition on CD4 T cells inversely correlated with CD4 numbers in blood of moderate/severe patients. Our findings indicate that the antibody mediated auto-immune response found in COVID-19 patients includes targeting of lymphocytes with potential contribution to the observed lymphopenia. This work was supported by the Divisions of Intramural Research of NIAID and NIBIB of the NIH

Exploitation of a Bacterium-Encoded Lytic Transglycosylase by a Human Oral Lytic Phage To Facilitate Infection.

Bacteriophages (phages) are an integral part of the human oral microbiome. Their roles in modulating bacterial physiology and shaping microbial communities have been discussed but remain understudied due to limited isolation and characterization of oral phage. Here, we report the isolation of LC001, a lytic phage targeting human oral Schaalia odontolytica (formerly known as Actinomyces odontolyticus) strain XH001. We showed that LC001 attached to and infected surface-grown, but not planktonic, XH001 cells, and it displayed remarkable host specificity at the strain level. Whole-genome sequencing of spontaneous LC001-resistant, surface-grown XH001 mutants revealed that the majority of the mutants carry nonsense or frameshift mutations in XH001 gene APY09_05145 (renamed ltg-1), which encodes a putative lytic transglycosylase (LT). The mutants are defective in LC001 binding, as revealed by direct visualization of the significantly reduced attachment of phage particles to the XH001 spontaneous mutants compared that to the wild type. Meanwhile, targeted deletion of ltg-1 produced a mutant that is defective in LC001 binding and resistant to LC001 infection even as surface-grown cells, while complementation of ltg-1 in the mutant background restored the LC001-sensitive phenotype. Intriguingly, similar expression levels of ltg-1 were observed in surface-grown and planktonic XH001, which displayed LC001-binding and nonbinding phenotypes, respectively. Furthermore, the overexpression of ltg-1 failed to confer an LC001-binding and -sensitive phenotype to planktonic XH001. Thus, our data suggested that rather than directly serving as a phage receptor, ltg-1-encoded LT may increase the accessibility of phage receptor, possibly via its enzymatic activity, by cleaving the peptidoglycan structure for better receptor exposure during peptidoglycan remodeling, a function that can be exploited by LC001 to facilitate infection. IMPORTANCE The evidence for the presence of a diverse and abundant phage population in the host-associated oral microbiome came largely from metagenomic analysis or the observation of virus-like particles within saliva/plaque samples, while the isolation of oral phage and investigation of their interaction with bacterial hosts are limited. Here, we report the isolation of LC001, the first lytic phage targeting oral Schaalia odontolytica. Our study suggested that LC001 may exploit the host bacterium-encoded lytic transglycosylase function to gain access to the receptor, thus facilitating its infection.

Gene expression profile of human colorectal cancer identified NKTR as a biomarker for liver metastasis.

Objective: Liver metastasis is one of the prognostic factors of colorectal cancer (CRC). The aim of this study is to identify biomarkers that facilitate easier detection of liver metastasis.Methods: Significance Analysis of Microarrays (SAM) and Array Data Analyzer (ADA) were applied used for the analysis of differentially differently expressed mRNAs. mRNA expression was verified by quantitative real-timer reverse transcriptiontase polymerase chain reaction (qRT-PCR). Immunohistochemistry were was used to show natural killer-tumor recognition (NKTR) expression in CRC. NKTR-knockdown CRC cells were constructed obtained by using short hairpin RNA (shRNA). Followed by CCK-8 assay, plate colony formation test, and transwell assay were used to evaluate the influence of NKTR on cell proliferation, migration, and invasion in vitro.Results: SAM yielded showed 256 up-regulated and 224 down-regulated differentially differently expressed genes. Seven genes were identified by using ADA, tools and four genes were verified by using qRT-PCR. Three genes (metastasis associated lung adenocarcinoma transcript 1 (MALAT1), nuclear factor I/B (NKTR), and nuclear factor I/B (NFIB)) showed a statistically significant considerabley difference between CRC with and liver metastasis and CRC without liver metastasis. Immunohistochemical analysis showed that NKTR expression was much lower in primary CRC with liver metastasis than that in primary CRC without liver metastasis. The NKTR protein plays a role in the lytic function of natural killer (NK) cells and it has been rarely studied in the CRC. The down-regulation of NKTR by shRNA interference in CRC cells increased cell proliferation, migration, and invasion in vitro.

Characterization and Genomic Analysis of a Novel Jumbo Bacteriophage vB_StaM_SA1 Infecting Staphylococcus aureus With Two Lysins.

The development of new antimicrobial agents is critically needed due to the alarming increase in antibiotic resistance in bacterial pathogens. Phages have been widely considered as effective alternatives to antibiotics. A novel phage vB_StaM_SA1 (hereinafter as SA1) that can infect multiple Staphylococcus strains was isolated from untreated sewage of a pig farm, which belonged to Myoviridae family. At MOI of 0.1, the latent period of phage SA1 was 55 min, and the final titer reached about 109 PFU/mL. The genome of phage SA1 was 260,727 bp, indicating that it can be classified as a jumbo phage. The genome of SA1 had 258 ORFs and a serine tRNA, while only 53 ORFs were annotated with functions. Phage SA1 contained a group of core genes that was characterized by multiple RNA polymerase subunits and also found in phiKZ-related jumbo phages. The phylogenetic tree showed that phage SA1 was a phiKZ-related phage and was closer to jumbo phages compared with Staphylococcus phages with small genome. Three proteins (lys4, lys210, and lys211) were predicted to be associated with lysins, and two proteins with lytic function were verified by recombinant expression and bacterial survival test. Both lys210 and lys211 possessed efficient bactericidal ability, and lys210 could lyse all test strains. The results show that phage SA1 and lys210/lys211 could be potentially used as antibiotic agents to treat Staphylococcus infection.

Proteome Analysis of Vacuoles Isolated from Fig (Ficus carica L.) Flesh during Fruit Development.

Fruit flesh cell vacuoles play a pivotal role in fruit growth and quality formation. In the present study, intact vacuoles were carefully released and collected from protoplasts isolated from flesh cells at five sampling times along fig fruit development. Label-free quantification and vacuole proteomic analysis identified 1,251 proteins, 1,137 of which were recruited as differentially abundant proteins (DAPs) by fold change ≥ 1.5, P < 0.05. DAPs were assigned to 10 functional categories; among them, 238, 186, 109, 93 and 90 were annotated as metabolism, transport proteins, membrane fusion or vesicle trafficking, protein fate and stress response proteins, respectively. Decreased numbers of DAPs were uncovered along fruit development. The overall changing pattern of DAPs revealed two major proteome landscape conversions in fig flesh cell vacuoles: the first occurred when fruit developed from late-stage I to mid-stage II, and the second occurred when the fruit started ripening. Metabolic proteins related to glycosidase, lipid and extracellular proteins contributing to carbohydrate storage and vacuole expansion, and protein-degrading proteins determining vacuolar lytic function were revealed. Key tonoplast proteins contributing to vacuole expansion, cell growth and fruit quality formation were also identified. The revealed comprehensive changes in the vacuole proteome during flesh development were compared with our previously published vacuole proteome of grape berry. The information expands our knowledge of the vacuolar proteome and the protein basis of vacuole functional evolution during fruit development and quality formation.

Inflammasome and gasdermin signaling in neutrophils.

Inflammasomes and gasdermins mount potent host defense pathways against invading microbial pathogens, however, dysregulation in these pathways can drive a variety of inflammatory disorders. Neutrophils, historically regarded as effector phagocytes that drive host defense via microbial killing, are now emerging as critical drivers of immunity in vivo. Here, we summarize, the latest advancement in inflammasome, gasdermin, and cell death signaling in neutrophils. We discuss the mechanisms by which neutrophils resist caspase-1-dependent pyroptosis, the lytic function of gasdermin D and E during NETosis and Yersinia infection, and the contribution of neutrophil inflammasomes to inflammatory disorders.

Anti-prion activity of cellulose ether is impaired in mice lacking pre T-cell antigen receptor α, T-cell receptor δ, or lytic granule function

The anti-prion activity of cellulose ether (CE) has been reported in rodents, but the mechanism of action is not well understood. As defects in early T-cell development have been reported in Tga20 mice which show only a slight effect of CE administration, we investigated the involvement of immune functions in the CE action. We confirmed an insertion of the prion protein transgene into the pre T-cell antigen receptor α gene of Tga20 mice, and its impaired expression in the thymus and other tissues. The influence of immune suppression on the CE effect was then examined in high CE-responder mice treated with immunosuppressive agents or neonatal thymectomy. As neonatal thymectomy significantly reduced the CE effect, we compared the influence of various T-cell defects in mice with similar genetic backgrounds. The CE effect was increased or unchanged in mice with defects in the αβ T-cell lineage, whereas it was abolished in T-cell receptor δ deficient mice. Further, when other immune defects were examined, the CE effect was reduced in mice with lysosomal trafficking dysfunction, but was unchanged in mice deficient in B-cell differentiation or toll-like receptor 4 signaling. These findings collectively suggest that the mechanism of CE action may involve γδ T cells and lytic granule function, as well as immune factors like natural killer T cells which are lacking in pre T-cell antigen receptor α deficient mice and neonatally thymectomized mice.

Membrane permeabilization is mediated by distinct epitopes in mouse and human orthologs of the necroptosis effector, MLKL.

Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL's N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.

Hemophagocytic Lymphohistiocytosis Gene Variants in Multisystem Inflammatory Syndrome in Children.

Simple SummaryChildren with a COVID-19 infection are at risk of developing a novel syndrome called multisystem inflammatory syndrome in children (MIS-C). This disease state is characterized by a high level of inflammation. It is unclear why only some children infected with SARS-CoV-2 later develop MIS-C. There may be genetic risk factors for MIS-C development, but none have previously been reported. We report genetic findings in a group of children with MIS-C.Multisystem inflammatory syndrome in children (MIS-C) affects few children previously infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In 2020, 45 children admitted to our hospital for MIS-C underwent genetic screening with a commercial 109-immune-gene panel. Thirty-nine children were diagnosed with MIS-C, and 25.4% of the 39 MIS-C patients harbored rare heterozygous missense mutations either in primary hemophagocytic lymphohistiocytosis (pHLH) genes (LYST, STXBP2, PRF1, UNC13D, AP3B1) or the HLH-associated gene DOCK8 (four variants). We demonstrate that foamy virus introduction of cDNA for the four DOCK8 variants into human NK-92 natural killer (NK) cells led to decreased CD107a expression (degranulation) and decreased NK cell lytic function in vitro for each variant. Heterozygous carriers of missense mutations in pHLH genes and DOCK8 may serve as risk factors for development of MIS-C among children previously infected with SARS-CoV-2.

A WICB 50th Favorite: Rapid lytic granule convergence to the MTOC in natural killer cells is dependent on dynein but not cytolytic commitment.

Molecular Biology of the CellVol. 33, No. 2 ASCB WICB 50th Anniversary FavoriteFree AccessA WICB 50th Favorite: Rapid lytic granule convergence to the MTOC in natural killer cells is dependent on dynein but not cytolytic commitmentAbigail E. Reed and Emily M. MaceAbigail E. ReedDepartment of Pediatrics, Columbia University Irving Medical Center, New York, NY 10032Search for more papers by this author and Emily M. Mace*Address correspondence to: Emily Mace (E-mail Address: [email protected]).Department of Pediatrics, Columbia University Irving Medical Center, New York, NY 10032Search for more papers by this authorErin D Goley, Monitoring EditorPublished Online:18 Jan 2022https://doi.org/10.1091/mbc.E21-04-0218AboutSectionsView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail To celebrate the 50th anniversary of the American Society for Cell Biology's Women in Cell Biology Committee (WICB), members of WICB and the MBoC Editorial Board invited a diverse group of scientists to highlight MBoC papers by women that have had a scientific or personal impact on the authors of the highlight.Cytotoxic immune cells, including NK cells and T-cells, kill target cells through the mobilization of specialized secretory lysosomes containing perforin and granzymes, termed lytic granules (LG), that undergo directed secretion at the immunological synapse (IS). While polarization of LG had been previously described in concert with polarization of the microtubule- organizing center (MTOC) to the IS, the kinetic mechanisms of LG trafficking to the synapse were poorly understood.We have chosen this article, which has been cited more than 140 times since publication, for the WICB 50 collection to highlight the contribution of women to this study. Namely, three of the four authors were women. In addition, the first author, who was a graduate student and a member of a historically underrepresented group in science, benefited from a training environment in the senior authors’ labs that has continuously supported and promoted women and other underrepresented minority trainees.Using quantitative live cell confocal microscopy and mass spectrometry, the authors demonstrated that LG in human NK cells converged rapidly to the MTOC before its polarization to the IS. Further, this convergence was dependent on minus-ended transport of LG by dynein along microtubules but occurred independently of NK cell commitment to killing (Mentlik et al., 2010). Colocalization of dynein and LG throughout the convergence process, and disruption of this interaction, definitively demonstrated the requirement of dynein for the rapid movement of LG during convergence. This finding, made by a diverse and collaborative investigative team, defined LG convergence as a fundamental priming step for cytotoxicity that increases the efficiency of lytic function by promoting directed secretion.FOOTNOTESDOI:10.1091/mbc.E21-04-0218REFERENCEMentlik AN, Sanborn KB, Holzbaur EL, Orange JS (2010). Rapid lytic granule convergence to the MTOC in natural killer cells is dependent on dynein but not cytolytic commitment. Mol Biol Cell 21, 2241–2256. Link, Google ScholarFiguresReferencesRelatedDetails Vol. 33, No. 2 February 01, 2022 Metrics Downloads & Citations Downloads: 275 History Submitted: 5 November 2021 Revised: 12 November 2021 Accepted: 19 November 2021 Information© 2022 Mace and Reed. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial-Share Alike 4.0 International Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/4.0).PDF download

Polymer-Mediated Cryopreservation of Bacteriophages.

Bacteriophages (phages, bacteria-specific viruses) have biotechnological and therapeutic potential. To apply phages as pure or heterogeneous mixtures, it is essential to have a robust mechanism for transport and storage, with different phages having very different stability profiles across storage conditions. For many biologics, cryopreservation is employed for long-term storage and cryoprotectants are essential to mitigate cold-induced damage. Here, we report that poly(ethylene glycol) can be used to protect phages from cold damage, functioning at just 10 mg·mL–1 (∼1 wt %) and outperforms glycerol in many cases, which is a currently used cryoprotectant. Protection is afforded at both −20 and −80 °C, the two most common temperatures for frozen storage in laboratory settings. Crucially, the concentration of the polymer required leads to frozen solutions at −20 °C, unlike 50% glycerol (which results in liquid solutions). Post-thaw recoveries close to 100% plaque-forming units were achieved even after 2 weeks of storage with this method and kill assays against their bacterial host confirmed the lytic function of the phages. Initial experiments with other hydrophilic polymers also showed cryoprotection, but at this stage, the exact mechanism of this protection cannot be concluded but does show that water-soluble polymers offer an alternative tool for phage storage. Ice recrystallization inhibiting polymers (poly(vinyl alcohol)) were found to provide no additional protection, in contrast to their ability to protect proteins and microorganisms which are damaged by recrystallization. PEG’s low cost, solubility, well-established low toxicity/immunogenicity, and that it is fit for human consumption at the concentrations used make it ideal to help translate new approaches for phage therapy.

C-Met-Specific Chimeric Antigen Receptor T Cells Demonstrate Anti-Tumor Effect in c-Met Positive Gastric Cancer.

Simple Summaryc-Met is known to be overexpressed in gastric cancers. Here, we developed anti-c-Met CAR T cell and measured its anti-tumor efficacy in vitro and in vivo. Our anti c-Met CAR T cells have shown selective killing of c-Met overexpressed gastric cancer cells. Based on our results, we suggest that anti-c-Met CAR T cell therapy could be effective for gastric cancer patients.Chimeric antigen receptor (CAR) technology has been highlighted in recent years as a new therapeutic approach for cancer treatment. Although the impressive efficacy of CAR-based T cell adoptive immunotherapy has been observed in hematologic cancers, limited effect has been reported on solid tumors. Approximately 20% of gastric cancer (GC) patients exhibit a high expression of c-Met. We have generated an anti c-Met CAR construct that is composed of a single-chain variable fragment (scFv) of c-Met antibody and signaling domains consisting of CD28 and CD3ζ. To test the CAR construct, we used two cell lines: the Jurkat and KHYG-1 cell lines. These are convenient cell lines, compared to primary T cells, to culture and to test CAR constructs. We transduced CAR constructs into Jurkat cells by electroporation. c-Met CAR Jurkat cells secreted interleukin-2 (IL-2) only when incubated with c-Met positive GC cells. To confirm the lytic function of CAR, the CAR construct was transduced into KHYG-1, a NK/T cell line, using lentiviral particles. c-Met CAR KHYG-1 showed cytotoxic effect on c-Met positive GC cells, while c-Met negative GC cell lines were not eradicated by c-Met CAR KHYG-1. Based on these data, we created c-Met CAR T cells from primary T cells, which showed high IL-2 and IFN-γ secretion when incubated with the c-Met positive cancer cell line. In an in vivo xenograft assay with NSG bearing MKN-45, a c-Met positive GC cell line, c-Met CAR T cells effectively inhibited the tumor growth of MKN-45. Our results show that the c-Met CAR T cell therapy can be effective on GC.

The role of NK cells in rheumatoid arthritis.

Natural killer (NK) cells are part of the innate immune system which not only provides a primary response to pathogenic conditions but can also play an important regulatory role in immune responses. Furthermore, these cells can influence immune responses by affecting other involved cells. Human NK cells can be classified as CD56dim and CD56bright; the former demonstrates mostly cytotoxic effects, while the latter comprises mostly tolerant or regulatory NK cells. These cells participate in the immunopathogenesis of rheumatoid arthritis (RA) and their role remains still unclear. We searched PubMed/MEDLINE and Scopus databases to review and analyze relevant literature on the impact of NK cells in the pathogenesis of RA. Although the percentage of NK cells increases in peripheral blood of RA patients compared to healthy individuals, the cytotoxic function of these cells is impaired. It is demonstrated by reduced "perforin+ NK cells" and decreased per-cell lytic function. These cytotoxic NK cells may control the pathogenic bone absorptive function of osteoclasts by directly targeting these cells. Collectively, the evidence collected in the current review emphasizes the possible protective role of CD56dim NK cells in the pathogenesis of RA.