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Cellular & Molecular Immunology Jun 2024CD28 and 4-1BB costimulatory endodomains included in chimeric antigen receptor (CAR) molecules play a critical role in promoting sustained antitumor activity of CAR-T...
CD28 and 4-1BB costimulatory endodomains included in chimeric antigen receptor (CAR) molecules play a critical role in promoting sustained antitumor activity of CAR-T cells. However, the molecular events associated with the ectopic and constitutive display of either CD28 or 4-1BB in CAR-T cells have been only partially explored. In the current study, we demonstrated that 4-1BB incorporated within the CAR leads to cell cluster formation and cell death in the forms of both apoptosis and necroptosis in the absence of CAR tonic signaling. Mechanistic studies illustrate that 4-1BB sequesters A20 to the cell membrane in a TRAF-dependent manner causing A20 functional deficiency that in turn leads to NF-κB hyperactivity, cell aggregation via ICAM-1 overexpression, and cell death including necroptosis via RIPK1/RIPK3/MLKL pathway. Genetic modulations obtained by either overexpressing A20 or releasing A20 from 4-1BB by deleting the TRAF-binding motifs of 4-1BB rescue cell cluster formation and cell death and enhance the antitumor ability of 4-1BB-costimulated CAR-T cells.
PubMed: 38937625
DOI: 10.1038/s41423-024-01198-y -
Scientific Reports Jun 2024Both hypoxia and the complement lectin pathway (CLP) are involved in atherosclerosis and atherosclerosis-related stroke and acute myocardial infarction (AMI). We have...
Both hypoxia and the complement lectin pathway (CLP) are involved in atherosclerosis and atherosclerosis-related stroke and acute myocardial infarction (AMI). We have previously shown that mannose-binding lectin-associated serine protease-1 (MASP-1), the most abundant enzyme of CLP, induces an inflammatory phenotype of endothelial cells (ECs) by cleaving protease activated receptors (PARs). In the absence of data, we aimed to investigate whether hypoxia and MASP-1 interact at the level of ECs, to better understand their role in atherosclerosis-related diseases. Hypoxia attenuated the wound healing ability of ECs, increased ICAM-1 and decreased ICAM-2 expression and upregulated PAR2 gene expression. Hypoxia and MASP-1 increased GROα and IL-8 production, and endothelial permeability without potentiating each other's effects, whereas they cooperatively disrupted vascular network integrity, activated the Ca, CREB and NFκB signaling pathways, and upregulated the expression of E-selectin, a crucial adhesion molecule in neutrophil homing. VCAM-1 expression was not influenced either by hypoxia, or by MASP-1. In summary, hypoxia potentiates the effect of MASP-1 on ECs, at least partially by increasing PAR expression, resulting in interaction at several levels, which may altogether exacerbate stroke and AMI progression. Our findings suggest that MASP-1 is a potential drug target in the acute phase of atherosclerosis-related diseases.
Topics: Humans; Mannose-Binding Protein-Associated Serine Proteases; Atherosclerosis; Endothelial Cells; Signal Transduction; Cell Hypoxia; NF-kappa B; Receptor, PAR-2; Human Umbilical Vein Endothelial Cells; Intercellular Adhesion Molecule-1; E-Selectin; Interleukin-8
PubMed: 38937560
DOI: 10.1038/s41598-024-64479-6 -
Nature Communications Jun 2024Peptidoglycan (PG) sacculi surround the cytoplasmic membrane, maintaining cell integrity by withstanding internal turgor pressure. During cell growth, PG endopeptidases...
Peptidoglycan (PG) sacculi surround the cytoplasmic membrane, maintaining cell integrity by withstanding internal turgor pressure. During cell growth, PG endopeptidases cleave the crosslinks of the fully closed sacculi, allowing for the incorporation of new glycan strands and expansion of the peptidoglycan mesh. Outer-membrane-anchored NlpI associates with hydrolases and synthases near PG synthesis complexes, facilitating spatially close PG hydrolysis. Here, we present the structure of adaptor NlpI in complex with the endopeptidase MepS, revealing atomic details of how NlpI recruits multiple MepS molecules and subsequently influences PG expansion. NlpI binding elicits a disorder-to-order transition in the intrinsically disordered N-terminal of MepS, concomitantly promoting the dimerization of monomeric MepS. This results in the alignment of two asymmetric MepS dimers respectively located on the two opposite sides of the dimerization interface of NlpI, thus enhancing MepS activity in PG hydrolysis. Notably, the protein level of MepS is primarily modulated by the tail-specific protease Prc, which is known to interact with NlpI. The structure of the Prc-NlpI-MepS complex demonstrates that NlpI brings together MepS and Prc, leading to the efficient MepS degradation by Prc. Collectively, our results provide structural insights into the NlpI-enabled avidity effect of cellular endopeptidases and NlpI-directed MepS degradation by Prc.
Topics: Peptidoglycan; Endopeptidases; Lipoproteins; Protein Binding; Protein Multimerization; Bacterial Proteins; Models, Molecular; Crystallography, X-Ray; Hydrolysis; Escherichia coli
PubMed: 38937433
DOI: 10.1038/s41467-024-49552-y -
The Journal of Pharmacology and... Jun 2024Through its pathological and genetic association to Parkinson's Disease (PD), α-synuclein (α-syn) remains a favorable therapeutic target that is being investigated...
Through its pathological and genetic association to Parkinson's Disease (PD), α-synuclein (α-syn) remains a favorable therapeutic target that is being investigated using various modalities, including many passive immunotherapy approaches clinically targeting different forms of α-syn and epitopes. Whereas published studies from some immunotherapy trials have demonstrated engagement in plasma, none have shown direct drug-antigen interactions in the disease-relevant compartment, the central nervous system (CNS). Cinpanemab (BIIB054) selectively targets pathological aggregated α-syn with low affinity binding to monomeric forms. The avidity-driven binding, low drug concentration, and the very low α-syn levels plus its heterogeneous nature in cerebrospinal fluid (CSF) made it not possible to measure drug-target interactions by conventional assays. Here we overcame these challenges by using zero-length crosslinking to stabilize the BIIB054-α-syn complexes and then quantified the crosslinked complexes using a Meso Scale Discovery (MSD) electrochemiluminescence assay. CSF samples from healthy volunteers (HV, n=46) and individuals with PD (PD, n=18) from study 228HV101 (Phase I clinical trial of BIIB054), demonstrated dose- and time- dependent binding of cinpanemab to α-syn with measurable complexes detected at doses {greater than or equal to}15 mg/kg. Complex formation displayed a direct positive correlation to drug concentration (Spearman rank correlation = 0.8295 (HV), 0.8032 (PD) p < 0.0001 (HV, PD)). The observed binding of cinpanemab to α-syn in CSF is consistent with its low intrinsic affinity for α-syn monomer and provides evidence that the drug is behaving with expected binding dynamics in the central nervous system compartment. A zero-length cross-linking method with MSD detection was developed to enable quantification of cinpanemab-α-syn complexes in Phase 1 clinical CSF samples by preventing signal loss caused by their rapid dissociation. Observed dose- and time-dependent binding were consistent with cinpanemab's affinity for α-syn and provided confidence that the drug had engaged its target at the desired site of action. This is the first demonstration of α-syn binding by an antibody in clinical samples from the CNS.
PubMed: 38936981
DOI: 10.1124/jpet.124.002199 -
In Vivo (Athens, Greece) 2024The RNA binding protein quaking (QKI) is associated with the development and progression of tumor suppressors in various cancers. However, the clinical implications of...
BACKGROUND/AIM
The RNA binding protein quaking (QKI) is associated with the development and progression of tumor suppressors in various cancers. However, the clinical implications of QKI expression have not yet been fully elucidated. In this study, we aimed to investigate the clinicopathological and prognostic significance of QKI expression in hepatocellular carcinoma (HCC).
MATERIALS AND METHODS
We performed QKI, Zinc finger E-box-binding homeobox 1 (ZEB1), E-cadherin, and glutathione peroxidase 4 (GPX4) immunohistochemical staining on 166 HCC patient tissue samples. X-tile bioinformatics software was used to set the cut-off value for high QKI expression. Correlations between QKI expression and various clinicopathological parameters were assessed.
RESULTS
The best cut-off value for high QKI expression was 12.5. High QKI expression was observed in 28 of 166 patients (16.9%) and was an independent prognostic factor for inferior recurrence-free survival (RFS). In addition, high ZEB1 and GPX4 expression correlated with high QKI expression, but not with the loss of E-cadherin expression.
CONCLUSION
High QKI expression was identified in HCCs and associated with poor RFS. QKI might be a prognostic biomarker of HCCs associated with epithelial-to-mesenchymal transition and a potential candidate therapeutic target.
Topics: Humans; Carcinoma, Hepatocellular; Liver Neoplasms; Male; Female; Prognosis; Middle Aged; Biomarkers, Tumor; RNA-Binding Proteins; Zinc Finger E-box-Binding Homeobox 1; Aged; Gene Expression Regulation, Neoplastic; Adult; Cadherins; Phospholipid Hydroperoxide Glutathione Peroxidase; Immunohistochemistry; Epithelial-Mesenchymal Transition
PubMed: 38936929
DOI: 10.21873/invivo.13665 -
In Vivo (Athens, Greece) 2024Hyperthermia represents an adjuvant local anticancer strategy which relies on the increase of temperature beyond the physiological level. In this study, we investigated...
BACKGROUND/AIM
Hyperthermia represents an adjuvant local anticancer strategy which relies on the increase of temperature beyond the physiological level. In this study, we investigated the anticancer potential of FeO and FeO Au nanoparticles as hyperthermic agents in terms of cytotoxicity and studied the expression of cellular markers of proliferation (changes in mRNA levels via real-time polymerase chain reaction).
MATERIALS AND METHODS
The human breast cancer cell line SK-BR-1 was incubated with either FeO or FeO Au nanoparticles stabilized with tryptophan, prior to hyperthermia treatment. The normal HEK293 cell line was used as a control. Toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay to estimate possible toxic effects of the tested nanoparticles. After RNA extraction and cDNA synthesis, mRNA expression of three indicators of proliferation, namely marker of proliferation Ki-67, DNA topoisomerase II alpha (TOP2A) and TPX2 microtubule nucleation factor (TPX2), was investigated.
RESULTS
At each concentration tested, FeO Au nanoparticles showed greater toxicity compared to FeO, while SK-BR-3 cells were more susceptible to their cytotoxic effects compared to the HEK293 cell line. The expression of Ki-67, TOP2A and TPX2 was reduced in SK-BR-3 cells by both FeO or FeO Au nanoparticles compared to untreated cells, while the only observed change in HEK293 cells was the up-regulation of TOP2A.
CONCLUSION
Both FeO Au and FeO NPs exhibit increased cytotoxicity to the cancer cell line tested (SK-BR-3) compared to HEK293 cells. The down-regulation in SK-BR-3 cells of the three proliferative markers studied, Ki-67, TOP2A and TPX2, after incubation with NPs suggests that cells that survived thermal destruction were not actively proliferating.
Topics: Humans; Breast Neoplasms; DNA Topoisomerases, Type II; Cell Proliferation; Hyperthermia, Induced; Ki-67 Antigen; Cell Line, Tumor; Microtubule-Associated Proteins; Female; Cell Cycle Proteins; Poly-ADP-Ribose Binding Proteins; HEK293 Cells; Gene Expression Regulation, Neoplastic; Biomarkers, Tumor
PubMed: 38936909
DOI: 10.21873/invivo.13616 -
Journal of Immunological Methods Jun 2024Antibody-dependent cellular phagocytosis (ADCP) is a cellular process by which antibody-opsonized targets (pathogens or cells) activate the Fc receptors on the surface...
Antibody-dependent cellular phagocytosis (ADCP) is a cellular process by which antibody-opsonized targets (pathogens or cells) activate the Fc receptors on the surface of phagocytes to induce phagocytosis, resulting in internalization and degradation of pathogens or target cells through phagosome acidification. Besides NK cells-mediated antibody-dependent cellular cytotoxicity (ADCC), tumor-infiltrated monocytes and macrophages can directly kill tumor cells in the presence of tumor antigen-specific antibodies through ADCP, representing another attractive strategy for cancer immunotherapy. Even though several methods have been developed to measure ADCP, an automated and high-throughput quantitative assay should offer highly desirable advantages for drug discovery. In this study we established a new ADCP assay to identify therapeutical monoclonal antibodies (mAbs) that facilitate macrophages phagocytosis of live target cells. We used Incucyte, an imaging system for live cell analysis. By labeling the live target cells with a pH sensitive dye (pHrodo), we successfully monitored the ADCP in real time. We demonstrated that our image-based assay is robust and quantitative, suitable for screening and characterization of therapeutical mAbs that directly kill target cells through ADCP. Furthermore, we found different subtypes of macrophages have distinct ADCP activities using both mouse and human primary macrophages differentiated in vitro. By studying various mAbs with mutations in their Fc regions using our assay, we showed that the variants with increased binding to Fc gamma receptors (FcγRs) have enhanced ADCP activities.
PubMed: 38936465
DOI: 10.1016/j.jim.2024.113715 -
International Immunopharmacology Jun 2024Hypoxia is a hallmark of solid tumors. Cancer-associated fibroblasts (CAFs) are an important component of the tumor microenvironment, and CAF-derived exosomes are...
Hypoxia is a hallmark of solid tumors. Cancer-associated fibroblasts (CAFs) are an important component of the tumor microenvironment, and CAF-derived exosomes are involved in cancer genesis and progression. Here, this work investigated the role and mechanism of exosomal circHIF1A derived from hypoxia-induced CAFs in hepatocellular carcinoma (HCC) tumorigenesis. CAFs isolated from fresh HCC tissues were incubated in normoxia or hypoxia condition (N/CAFs or H/CAFs), and then the exosomes from N/CAFs or H/CAFs were isolated for functional analysis. Cell proliferation, migration and invasion were analyzed by cell counting kit-8, colony formation, and transwell assays. Immune evasion was evaluated by measuring the cytotoxicity and viability of CD8T cells. qRT-PCR and western blotting analyses were used for the level measurement of genes and proteins. The binding between Hu antigen R (HuR) and circHIF1A or Programmed death ligand 1 (PD-L1) was analyzed by RNA immunoprecipitation assay. Functionally, we found that CAFs, especially CAFs under hypoxic stress (H/CAFs), promoted the proliferation, migration, invasion and EMT progression in HCC cells, as well as induced immune escape by suppressing CD8T cell cytotoxicity and activity in an exosome-dependent manner. H/CAFs-derived exosomes showed highly expressed circHIF1A, and could secrete circHIF1A into HCC cells via exosomes. The oncogenic effects of H/CAFs-secreted exosomes were abolished by circHIF1A knockdown. Mechanistically, circHIF1A interacted with HuR to stabilize PD-L1 expression in HCC cells. Meanwhile, circHIF1A silencing suppressed HCC cell proliferation, mobility and immune escape by regulating PD-L1 expression. In all, exosomal circHIF1A derived from hypoxic-induced CAFs promoted the proliferation, migration, invasion, EMT progression and immune escape in HCC cells by up-regulating PD-L1 expression in a HuR-dependent manner.
PubMed: 38936058
DOI: 10.1016/j.intimp.2024.112282 -
Angewandte Chemie (International Ed. in... Jun 2024Pancreatic cancer is one of the deadliest cancers worldwide, mainly due to late diagnosis. Therefore, there is an urgent need for novel diagnostic approaches to identify...
Pancreatic cancer is one of the deadliest cancers worldwide, mainly due to late diagnosis. Therefore, there is an urgent need for novel diagnostic approaches to identify the disease as early as possible. We have developed a diagnostic assay for pancreatic cancer based on the detection of naturally occurring tumor associated autoantibodies against Mucin-1 (MUC1) using engineered glycopeptides on nanoparticle probes. We used a structure-guided approach to develop unnatural glycopeptides as model antigens for tumor-associated MUC1. We designed a collection of 13 glycopeptides to bind either SM3 or 5E5, two monoclonal antibodies with distinct epitopes known to recognize tumor associated MUC1. Glycopeptide binding to SM3 or 5E5 was confirmed by surface plasmon resonance and rationalized by molecular dynamics simulations. These model antigens were conjugated to gold nanoparticles and used in a dot-blot assay to detect autoantibodies in serum samples from pancreatic cancer patients and healthy volunteers. Nanoparticle probes with glycopeptides displaying the SM3 epitope did not have diagnostic potential. Instead, nanoparticle probes displaying glycopeptides with high affinity for 5E5 could discriminate between cancer patients and healthy controls. Remarkably, the best-discriminating probes show significantly better true and false positive rates than the current clinical biomarkers CA19-9 and carcinoembryonic antigen (CEA).
PubMed: 38935849
DOI: 10.1002/anie.202407131 -
Neuromolecular Medicine Jun 2024Glioma is the most common primary intracranial tumor with high mortality and poor prognosis. The purpose of this study was to investigate how single-nucleotide...
Glioma is the most common primary intracranial tumor with high mortality and poor prognosis. The purpose of this study was to investigate how single-nucleotide polymorphisms (SNPs) of the NID2 gene affect glioma risk and prognosis. Four candidate SNPs of NID2 in 529 glioma patients and 478 healthy controls were successfully genotyped by Agena MassARRAY mass spectrometer. Logistic regression was utilized to assess the associations between NID2 SNPs and glioma risk under different genetic models. Furthermore, the relationship between risk-related SNPs in NID2 and the prognosis of glioma patients was explored through Kaplan-Meier (KM) survival curve and Cox proportional hazard regression analysis. The results showed that rs11846847 (OR 1.24, p = 0.017) and rs1874569 (OR 1.22, p = 0.026) were significantly associated with an increased risk of glioma, and rs11846847 also had a risk-increasing effect on glioma in participants ≤ 40 years old. The interaction model of rs11846847 and rs1874569 could be more suitable for forecasting glioma risk. We also discovered a significant association between rs1874569 and poor prognosis in glioma patients (HR 1.32, p = 0.039) and especially CC genotype was relevant to shorter overall survival (OS) and progression-free survival (PFS) in patients with high-grade glioma. Additionally, the study demonstrated that gross total resection or chemotherapy improve glioma prognosis in the Chinese Han population. This study is the first to provide evidence for the association of NID2 SNPs with glioma risk and prognosis, suggesting that NID2 variants might be potential factors for glioma.
Topics: Humans; Polymorphism, Single Nucleotide; Glioma; Female; Male; Brain Neoplasms; Prognosis; Adult; Middle Aged; Asian People; Genetic Predisposition to Disease; Calcium-Binding Proteins; China; Case-Control Studies; Kaplan-Meier Estimate; Genotype; Proportional Hazards Models; Risk Factors; East Asian People; Cell Adhesion Molecules
PubMed: 38935278
DOI: 10.1007/s12017-024-08795-0