Response In Mouse Model Research Articles

Ginsenoside compound K restrains hepatic fibrotic response by dual-inhibition of GLS1 and LDHA

BackgroundLiver fibrosis is a dynamic process marked by the accumulation of extracellular matrix due to hepatic stellate cells (HSCs) activation. Ginsenoside compound K (CK), a rare derivative of its parent ginsenosides, is known to significantly ameliorate metabolic disorders. PurposeThe aim of this study was to elucidate the protective effects of CK against liver fibrosis with a focus on metabolic regulation. MethodsWe established liver fibrosis models in mice using carbon tetrachloride (CCl4) challenge, bile duct ligation, or a methionine-choline deficient diet, with continuous oral administration of CK at specified doses and intervals. Simultaneously, we examined the impact of CK on metabolic regulation in cultured HSCs and investigated the associated mechanisms. ResultsCK was found to alleviate liver injury and curb fibrotic responses in mouse models, as well as decrease elevated levels of liver enzyme. Metabolomic analysis in vitro highlighted the crucial roles of pyruvate and glutamine metabolism in metabolic remodeling. Immunohistochemical staining indicated significantly elevated expressions of lactate dehydrogenase A (LDHA) (p = 0.014) and glutaminase 1 (GLS1) (p = 0.024) in liver cirrhosis patients. Comparable alterations were noted in the liver of model mice and in cultured HSCs. Molecular docking and bio-layer interferometry demonstrated that CK interacts with and inhibits the activities of LDHA and GLS1. As expected, CK attenuated glycolysis and glutaminolysis, reducing HSC growth dependently on lactate and α-ketoglutarate (α-KG). Upon HSC activation, metabolism is reprogrammed with Myc as a key regulator, transcriptionally controlling LDHA, GLS1, and glutamine transporters SLC1A5 and SLC38A5. CK inhibited Myc induction, integrating glycolysis and glutaminolysis regulation to counteract the fibrotic response. ConclusionCK inhibited LDHA and GLS1 activities, thereby inhibiting hepatic fibrosis. These findings offer new insights into the role of ginsenosides in liver protection, especially regarding metabolic disorders.

Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1.

Mechanistic understanding of the immune checkpoint receptor PD1 is largely based on mouse models, but human and mouse PD1 orthologs exhibit only 59.6% identity in amino acid sequences. Here we show that human PD1 is more inhibitory than mouse PD1 due to stronger interactions with the ligands PDL1 and PDL2 and with the effector phosphatase Shp2. A novel motif highly conserved among PD1 orthologs in vertebrates except in rodents is primarily responsible for the differential Shp2 recruitment. Evolutionary analysis suggested that rodent PD1 orthologs uniquely underwent functional relaxation, particularly during the K-Pg boundary. Humanization of the PD1 intracellular domain disrupted the anti-tumor activity of mouse T cells while increasing the magnitude of anti-PD1 response. Together, our study uncovers species-specific features of the PD1 pathway, with implications to PD1 evolution and differential anti-PD(L)1 responses in mouse models and human patients.

Mixed-charge hyperbranched polymer nanoparticles with selective antibacterial action for fighting antimicrobial resistance

The escalating menace of antimicrobial resistance (AMR) presents a profound global threat to life and assets. However, the incapacity of metal ions/reactive oxygen species (ROS) or the indiscriminate intrinsic interaction of cationic groups to distinguish between bacteria and mammalian cells undermines the essential selectivity required in these nanomaterials for an ideal antimicrobial agent. Hence, we devised and synthesized a range of biocompatible mixed-charge hyperbranched polymer nanoparticles (MCHPNs) incorporating cationic, anionic, and neutral alkyl groups to effectively combat multidrug-resistant bacteria and mitigate AMR. This outcome stemmed from the structural, antibacterial activity, and biocompatibility analysis of seven MCHPNs, among which MCHPN7, with a ratio of cationic groups, anionic groups, and long alkyl chains at 27:59:14, emerged as the lead candidate. Importantly, owing to inherent differences in membrane potential among diverse species, alongside its nano-size (6 - 15 nm) and high hydrophilicity (Kow = 0.04), MCHPN7 exhibited exceptional selective bactericidal effects over mammalian cells (selectivity index > 564) in vitro and in vivo. By inducing physical membrane disruption, MCHPN7 effectively eradicated antibiotic-resistant bacteria and significantly delayed the emergence of bacterial resistance. Utilized as a coating, MCHPN7 endowed initially inert surfaces with the ability to impede biofilm formation and mitigate infection-related immune responses in mouse models. This research heralds the advent of biocompatible polymer nanoparticles and harbors significant implications in our ongoing combat against AMR. Statement of SignificanceThe escalating prevalence of antimicrobial resistance (AMR) has been acknowledged as one of the most significant threats to global health.Therefore, a series of mixed-charge hyperbranched polymer nanoparticles (MCHPNs) with selective antibacterial action were designed and synthesized. Owing to inherent differences in membrane potential among diverse species and high hydrophilicity (Kow = 0.04), the optimal nanoparticles exhibited exceptional selective bactericidal effects over mammalian cells (selectivity index >564) and significantly delayed the emergence of bacterial resistance. Importantly, they endowed surfaces with the ability to impede biofilm formation and mitigate infection-related immune responses.Furthermore, the above findings focus on addressing the problem of AMR in Post-Pandemic, which will for sure attract attention from both academic and industry research.

Recombinant outer membrane protein Lipl41 from Leptospira interrogans robust immune responses in mice model.

Leptospirosis is an infectious zoonotic disease that can result in severe complications. It is widespread, especially in hot and humid climates such as the northern region of Iran. The immune responses to leptospirosis are multifaceted. Lipl41 is an outer membrane protein that is expressed during infection and is highly conserved among pathogenic species. This makes it a good candidate for diagnosis and induction of specific immune responses. The aim of the present study was to evaluate immune responses against recombinant Lipl41 in mice. After immunizing of different groups of mice with recombinant Lipl41 (rLipl41), the levels of specific antibodies and cytokine profiles interferon-gamma/interleukin-4 (IFN-γ/IL-4) were measured. The results revealed that rLipl41 showed a significant increase in antibody levels compared with the control groups (P< 0.05). Although the level of IL-4 in the groups that received Lipl41 was similar to that in the other control groups, the IFN-γ levels showed a significant increase (P<0.05). It has been concluded that recombinant Lipl41 protein could strongly stimulate specific immune responses and be considered a potential candidate for vaccine development and diagnostic research.

Immunopeptidomics Mapping of Listeria monocytogenes T Cell Epitopes in Mice

Listeria monocytogenes is a foodborne intracellular bacterial model pathogen. Protective immunity against Listeria depends on an effective CD8+ T cell response, but very few T cell epitopes are known in mice as a common animal infection model for listeriosis. To identify epitopes, we screened for Listeria immunopeptides presented in the spleen of infected mice by mass spectrometry-based immunopeptidomics. We mapped more than 6000mouse self-peptides presented on MHC class I molecules, including 12 high confident Listeria peptides from 12 different bacterial proteins. Bacterial immunopeptides with confirmed fragmentation spectra were further tested for their potential to activate CD8+ T cells, revealing VTYNYINI from the putative cell wall surface anchor family protein LMON_0576 as a novel bona fide peptide epitope. The epitope showed high biological potency in a prime boost model and can be used as a research tool to probe CD8+ T cell responses in the mouse models of Listeria infection. Together, our results demonstrate the power of immunopeptidomics for bacterial antigen identification.

Influence of Diet on Reproducible Corticosterone Levels in a Mouse Model of Maternal Separation with Early Weaning.

Maternal separation with early weaning (MSEW) is a popular early life stress (ELS) model in rodents, which emulates childhood neglect through scheduled mother-offspring separation. Although variations of ELS models, including maternal separation and MSEW, have been published for the mouse species, the reported results are inconsistent. Corticosterone is considered the main stress hormone involved in regulating stress responses in rodents-yet generating a robust and reproducible corticosterone response in mouse models of ELS has been elusive. Considering the current lack of standardization for MSEW protocols, these inconsistent results may be attributed to variations in model methodologies. Here, we compared the effects of select early wean diet sources-which are the non-milk diets used to complete early weaning in MSEW pups-on the immediate stress phenotype of C57BL/6J mice at postnatal day 21. Non-aversive handling was an integral component of our modified MSEW model. The evaluation of body weight and serum corticosterone revealed the early wean diet to be a key variable in the resulting stress phenotype. Interestingly, select non-milk diets facilitated a stress phenotype in which low body weight was accompanied by significant corticosterone elevation. Our data indicate that dietary considerations are critical in MSEW-based studies and provide insight into improving the reproducibility of key stress-associated outcomes as a function of this widely used ELS paradigm.

Spider minor ampullate silk protein nanoparticles: an effective protein delivery system capable of enhancing systemic immune responses.

Spider silk proteins (spidroins) are particularly attractive due to their excellent biocompatibility. Spider can produce up to seven different types of spidroins, each with unique properties and functions. Spider minor ampullate silk protein (MiSp) might be particularly interesting for biomedical applications, as the constituent silk is mechanically strong and does not super-contract in water, attributed to its amino acid composition. In this study, we evaluate the potential of recombinant nanoparticles derived from Araneus ventricosus MiSp as a protein delivery carrier. The MiSp-based nanoparticles were able to serve as an effective delivery system, achieving nearly 100% efficiency in loading the model protein lysozyme, and displayed a sustained release profile at physiological pH. These nanoparticles could significantly improve the delivery efficacy of the model proteins through different administration routes. Furthermore, nanoparticles loaded with model protein antigen lysozyme after subcutaneous or intramuscular administration could enhance antigen-specific immune responses in mouse models, through a mechanism involving antigen-depot effects at the injection site, long-term antigen persistence, and efficient uptake by dendritic cells as well as internalization by lymph nodes. These findings highlight the transnational potential of MiSp-based nanoparticle system for protein drug and vaccine delivery.

Phase and Defect Engineering of MoSe2 Nanosheets for Enhanced NIR-II Photothermal Immunotherapy.

Inducing immunogenic cell death (ICD) during photothermal therapy (PTT) has the potential to effectively trigger photothermal immunotherapy (PTI). However, ICD induced by PTT alone is often limited by inefficient PTT, low immunogenicity of tumor cells, and a dysregulated redox microenvironment. Herein, we develop MoSe2 nanosheets with high-percentage metallic 1T phase and rich exposed active Mo centers through phase and defect engineering of MoSe2 as an effective nanoagent for PTI. The metallic 1T phase in MoSe2 nanosheets endows them with strong PTT performance, and the abundant exposed active Mo centers endow them with high activity for glutathione (GSH) depletion. The MoSe2-mediated high-performance PTT synergizing with efficient GSH depletion facilitates the release of tumor-associated antigens to induce robust ICD, thus significantly enhancing checkpoint blockade immunotherapy and activating systemic immune response in mouse models of colorectal cancer and triple-negative metastatic breast cancer.

Intravesical local administration of pembrolizumab for treatment of bladder cancer: A novel strategy to minimize toxicity.

e14687 Background: A major hallmark of bladder cancer (BCa) is a higher treatment success rate than prostate cancer following stimulation of innate anti-tumor immune response either by intravesical BCG or to FDA approved intravenous anti-PD1 checkpoint antibody (pembrolizumab) approved for BCG resistant BCa since 2021. However, injected pembrolizumab is characterized by a markedly higher incidence (66%) of serious adverse events than the median duration of complete response of ~40% for non-muscle invasive BCa (NMIBC) and 15-20% for invasive BCa. Since the toxicity (fatigue, Pneumonitis and diarrhea) of injected pembrolizumab is linked to systemic breakdown of T-cell mediated immune surveillance we enquired whether analogous to BCG, pembrolizumab can be administered intravesically to safely induce a localized anti-tumor response in mouse model of BCa. Methods: Six weeks old female mice of B6D2F1 strain (n=10) were ad libitum fed carcinogen, N-butyl-N-4-hydroxybutyl nitrosamine (BBN) in drinking water (0.05% w/v) for 12 weeks. The presence of BBN evoked tumor was ascertained by unenhanced T2 weighted MRI at week 11 prior to single intravesical dosing by 24G transurethral catheter under isoflurane anesthesia of 0.1mL vehicle (sterile water) with or without 0.3mg of Pembrolizumab (Lipella Pharmaceuticals). 6 weeks later, T2 weighted MRI was repeated together with T1 mapping by ICE-MRI prior to organ harvest. Results: BBN feeding evoked multiclonal tumors (red*) of comparable size on T2 weighted MRI at week 11 but repeat MRI 7 weeks after treatment revealed that tumor progression and perivesical invasion noted in vehicle group was arrested in pembrolizumab group. Longest dimension of BBN tumor in vehicle treated mice was ≥ 1mm longer (p&lt;0.05) than in pembrolizumab treated mice. T1 mapping revealed thickened bladder wall missed by T2 MRI. Conclusions: These findings on single intravesical dose of pembrolizumab (10mg/kg) in mice are consistent with human study on multiple intravesical dosing of BCG plus pembrolizumab (1-2mg/kg) in BCG resistant NMIBC patients. Intravesical pembrolizumab mirrors the limited systemic uptake of radiolabelled antibodies together with substantially higher ingress into tumor foci. The tumor selective ingress for BCa immunotherapy without any toxicity will be enhanced with Lipella Pharmaceutical’s liposomal delivery of Pembrolizumab.

3-Fucosyllactose-mediated modulation of immune response against virus infection

Viral pathogens, particularly influenza and SARS-CoV-2, pose a significant global health challenge. Given the immunomodulatory properties of human milk oligosaccharides, in particular 2′-fucosyllactose and 3-fucosyllactose (3-FL), we investigated their dietary supplementation effects on antiviral responses in mouse models. This study revealed distinct immune modulations induced by 3-FL. RNA-sequencing data showed that 3-FL increased the expression of interferon receptors, such as Interferon Alpha and Beta Receptor (IFNAR) and Interferon Gamma Receptor (IFNGR), while simultaneously downregulating interferons and interferon-stimulated genes, an effect not observed with 2′-fucosyllactose supplementation. Such modulation enhanced antiviral responses in both cell culture and animal models while attenuating pre-emptive inflammatory responses. Nitric oxide concentrations in 3-FL-supplemented A549 cells and mouse lung tissues were elevated exclusively upon infection, reaching 5.8- and 1.9-fold increases over control groups, respectively. In addition, 3-FL promoted leukocyte infiltration into the site of infection upon viral challenge. 3-FL supplementation provided protective efficacy against lethal influenza challenge in mice. The demonstrated antiviral efficacy spanned multiple influenza strains and extended to SARS-CoV-2. In conclusion, 3-FL is a unique immunomodulator that helps protect the host from viral infection while suppressing inflammation prior to infection.

Helminth-derived proteins as immune system regulators: a systematic review of their promise in alleviating colitis

Helminth-derived proteins have immunomodulatory properties, influencing the host’s immune response as an adaptive strategy for helminth survival. Helminth-derived proteins modulate the immune response by inducing anti-inflammatory cytokines, promoting regulatory T-cell development, and ultimately favouring a Th2-biased immune response. This systematic review focused on helminth-derived proteins and explored their impact on reducing inflammatory responses in mouse models of colitis. A systematic search across Medline, EMBASE, Web of Science, and Cochrane Library identified fourteen relevant studies. These studies reported immunomodulatory changes, including increased production of anti-inflammatory cells and cytokines. In mouse models of colitis treated with on helminth-derived proteins, significant improvements in pathological parameters such as body weight, colon length, and microscopic inflammatory scores were observed compared to control groups. Moreover, helminth-derived proteins can enhance the function of Tregs and alleviate the severity of inflammatory conditions. The findings underscore the pivotal role of helminth-derived proteins in immunomodulation, specifically in the axis of cytokine secretion and immune cell polarization. The findings offer new opportunities for treating chronic inflammatory conditions such Crohn’s disease.

Abstract 4236: PDX-derived organoids (PDXO) are valuable tools to unveil the shortcomings of new anti-cancer drug candidates

Abstract Introduction: Tumor organoids are increasingly being used as predictive preclinical cancer models, as they reflect the original tumor features and patient drug responses. However, systematic comparisons between organoids and traditional 2D cell lines or 3D spheroids are lacking. Method: We established over 30 PDX-derived organoid (PDXO) models with KRAS mutations and tested three inhibitors targeting the KRAS pathway: VS-6766(RAF/MEK inhibitor), Trametinib (MEK inhibitor), and MRTX1133 (a Phase II drug targeting KRASG12D) against these models. Results: IC50 results indicated that most models responded to treatment. For some cell lines we detected differential drug responses in 2D vs. 3D testing with 3Dspheroids being more sensitive to inhibitors than 2D cell lines (on average ~10 fold). PDXO models exhibited greater individual differences in drug response, with a subset of models demonstrating no sensitivity to MRTX1133. This is consistent with published results showing that some KRASG12D-mutant PDX models were less sensitive to MRTX11331. Similarly, the clinical trial data for the KRASG12C inhibitor Adagrasib revealed distinctions in the response of patients2. Further analysis with WES (whole exome sequencing) and RNA-sequencing identified potential genotypic mutations and differential expressions of signaling pathways related to drug resistance. Subsequent drug combination testing showed that targeting these pathways could enhance the MRTX1133 efficacy in the resistant PDXO models. Summary: Our research confirms that drug testing on organoids predicts responses in mouse models and clinical trials. In combination with multi-omics and bioinformatics analysis, the platform can be used for evaluating drug resistances, identifying new targets, developing new drug combination therapies, and ultimately improving the creation of new cancer therapeutics. Citation Format: Yuhui Wang, Lin Feng, Han Liu, Junwen Zhang, Kaiqiang Hu, Pengwei Pan, Fang He. PDX-derived organoids (PDXO) are valuable tools to unveil the shortcomings of new anti-cancer drug candidates [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 4236.

Identification of mouse CD4+ T cell epitopes in SARS-CoV-2 BA.1 spike and nucleocapsid for use in peptide:MHCII tetramers.

Understanding adaptive immunity against SARS-CoV-2 is a major requisite for the development of effective vaccines and treatments for COVID-19. CD4+ T cells play an integral role in this process primarily by generating antiviral cytokines and providing help to antibody-producing B cells. To empower detailed studies of SARS-CoV-2-specific CD4+ T cell responses in mouse models, we comprehensively mapped I-Ab-restricted epitopes for the spike and nucleocapsid proteins of the BA.1 variant of concern via IFNγ ELISpot assay. This was followed by the generation of corresponding peptide:MHCII tetramer reagents to directly stain epitope-specific T cells. Using this rigorous validation strategy, we identified 6 immunogenic epitopes in spike and 3 in nucleocapsid, all of which are conserved in the ancestral Wuhan strain. We also validated a previously identified epitope from Wuhan that is absent in BA.1. These epitopes and tetramers will be invaluable tools for SARS-CoV-2 antigen-specific CD4+ T cell studies in mice.

Advancing the Boundaries of Immunotherapy in Lung Adenocarcinoma with Idiopathic Pulmonary Fibrosis by a Biomimetic Proteinoid Enabling Selective Endocytosis.

The coexistence of lung adenocarcinoma (LUAD) with idiopathic pulmonary fibrosis (IPF), which has been extensively documented as a prominent risk factor for checkpoint inhibitor-related pneumonitis (CIP) in patients undergoing immunotherapy, has long been considered a restricted domain for the use of immune checkpoint inhibitors (ICIs). To overcome it, an approach was employed herein to specifically target PD-L1 within the cellular interior, surpassing the conventional focus solely on the cytomembrane, thereby facilitating the development of ICIs capable of distinguishing between LUAD cells and noncancerous cells based on their distinctive endocytic propensities. By exploiting the aurophilicity-driven self-assembly of a PD-L1 binding peptide (PDBP) and subsequently encapsulating it within erythrocyte membranes (EM), the resulting biomimetic ICIs protein EMS-PDBP exhibited extraordinary selectivity in internalizing LUAD cells, effectively targeting PD-L1 within cancer cells while hindering its membrane translocation. The EMS-PDBP treatment not only reactivated the antitumor immune response in the LUAD orthotopic allograft mouse model but also demonstrated a favorable safety profile by effectively eliminating any immune-related adverse events (irAEs). Most significantly, EMS-PDBP successfully and safely restored the antitumor immune response in a mouse model of LUAD with coexistent IPF, thus shattering the confines of ICIs immunotherapy. The reported EMS-PDBP collectively offers a potential strategy for immune reactivation to overcome the limitations of immunotherapy in LUAD coexisting with IPF.

Abstract IA022: Uracil DNA glycosylase activity limits deoxyuridine contamination in genomic DNA and is essential for the type-1 interferon response in cells treated with ATR kinase inhibitors

Abstract Most chemotherapies target DNA replication, and their efficacy depends on the DNA damage response that integrates DNA repair with the cell cycle. Widely used standard-of-care antimetabolites inhibit thymidylate synthase and increase the incorporation of deoxyuridine (dU) into DNA by polymerases. Contamination of dU in DNA is limited by the DNA damage response kinase ATR that induces cell cycle checkpoints and reduces the rate of DNA replication. ATR inhibitors (ATRi) induce origin firing across active replicons and cause ribonuclease reductase degradation in otherwise unperturbed cells. This increases both the amount of DNA replication and the amount of free dUTP in cells. Thus, ATRi increase the incorporation of dU into DNA by polymerases. Since ATRi also inhibit cell cycle checkpoints, ATRi induce more dU contamination than antimetabolites. ATRi-induced dU contamination in DNA is associated with an innate immune response. We showed this with the simple observation that ATRi-induced dU contamination and IFN-α/β expression is reversed by low doses of thymidine. Here we show that ATRi-induced IFN-α/β response is dependent on uracil DNA glycosylase (UNG) which removes dU from DNA. Our data are consistent with a model in which UNG-dependent base excision repair (BER) removes dU from DNA, ultimately generating cytoplasmic dsDNA that induces IFN-α/β. We propose that ATRi-induced dU contamination contributes to dose-limiting leukocytopenia and inflammation in the clinic and CD8+ T cell dependent anti-tumor responses in mouse models of cancer treated with radiation therapy. Citation Format: Pinakin Pandya, Frank P. Vendetti, Joseph A. Ghoubaira, Sudipta Pathak, Joshua J. Deppas, Reyna E. Jones, Yunqi Zhang, Daniel Ivanov, Raquel Buj, Katherine M. Aird, Jan H. Beumer, Robert W. Sobol, Christopher J. Bakkenist. Uracil DNA glycosylase activity limits deoxyuridine contamination in genomic DNA and is essential for the type-1 interferon response in cells treated with ATR kinase inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA022.

Safety and Efficacy of Azacitidine and Chidamide Maintenance after Allogeneic Hematopoietic Stem Cell Transplantation for Patients with High-Risk Acute Myeloid Leukemia: Interim Analysis of a Prospective, Multicenter, Single-Arm, Phase Ⅱ Clinical Trial

Background Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the potentially curative therapy for AML, the relapse rate after HSCT is high with 30-60% for patients with high-risk AML. Maintenance therapy with efficacy and low toxicity is needed to mitigate relapse. It has been reported that histone deacetylases inhibitor chidamide exhibits anti-tumor activity against AML cells, and is able to elicit anti-tumor immune response in mouse models. However, efficacy and safety of chidamide as maintenance therapy following allo-HSCT for patients with AML have not been reported. Therefore, we designed a multicenter, single arm, phase II clinical trial for high-risk AML patients to use azacitidine combined with chidamide as maintenance therapy post allo-HSCT to prevent relapse. Methods In this multicenter, single arm, phase II clinical trial, patients aged 18-60 years with high-risk AML, defined as the European LeukemiaNet (ELN) 2017 adverse risk group, refractory, relapsed or secondary AML were given AZA and chidamide as maintenance therapy after 60 days post transplantation (NCT05270200). AZA was given at 100mg/day (days 1-5) for 6 cycles and chidamide 5mg daily (days 1-28) with a cycle of 28 days for up to 2 years. The dose of AZA was amended at 50mg/m 2/day (days 1-3) from Dec 23,2022 due to occurrence of thrombocytopenia. If PLT was below 50×10 9/L or grade 3/4 non-hematological toxicity occurs, maintenance should be interrupted or delayed until PLT count was greater than 75×10 9/L or the non-hematological toxicity resolved to ≦ grade 1. Primary objectives were safety and one year cumulative incidence of relapse. Secondary objections were relapse-free survival (RFS), overall survival (OS) and incidence of severity of acute and chronic graft-versus-host disease (GVHD). Results From February 12,2022 to June 30,2023, seven patients were enrolled, with a median age of 46 years（28-60), four patients (57%) were female. One patient (14%) received matched sibling donor transplantation, six patients (86%) received haploidentical donor transplantation. Five patients (71%) received myeloablative and 2/7 (29%) reduced intensity conditioning regimen. Six (86%) were in ELN 2017 adverse risk with three patients harboring KMT2A rearrangement, one patient with TP53 mutation, one patient with -7, one patient with RUNX1 mutation, while one patient (14%) was diagnosed as secondary AML with broad extramedullary infiltration. All but one patients entered transplant in CR, while five patients (83%) were MRD negative and one patient (17%) were positive. The baseline patient characteristics are summarized in Table1. Outcome data were updated as of June 30,2023 (Figure1). The median time to start maintenance therapy after transplantation was 77 days (range 64-80). The median follow-up time was 9.9 months, only one patient relapsed and the accumulative incidence of relapse of one year was 16.7%（95%CI,0.4%-64.1%). The relapsed patient diagnosed with MLL-AF10 rearrangement had extramedullary infiltration of left tonsil, gum and neck lymph nodes at 197 days post transplantation while bone marrow remained in MRD negative remission. AZA/chidamide were well tolerated, with no severe adverse event was recorded. Two grade 3/4 non-hematologic adverse events were observed, with one increased bilirubin due to cGVHD and one pneumonia. Two patients (29%) experienced grade4 thrombocytopenia requiring platelet transfusion. After amendment of AZA dose, no grade4 thrombocytopenia was recorded. No one discontinued treatment due to toxicity. During follow-up, none of patients had aGVHD. Three patients (43%) experienced mild or moderate cGVHD, no severe cGVHD was observed. The median OS was not reached, with six months and one year OS both of 100%. The median RFS is not reached, with six months and one year RFS of 100% and 83.3%(95%CI,35.9%-99.6%). Conclusions Preliminary results suggest that maintenance of AZA combined with chidamide post allo-HSCT in patients with high-risk AML exhibits promising efficacy and well-tolerated. Recruitment of patients with high-risk AML post allo-HSCT for this trial is ongoing. Disclosures No relevant conflicts of interest to declare.

Tumor-specific GPX4 degradation enhances ferroptosis-initiated antitumor immune response in mouse models of pancreatic cancer

Lipid peroxidation–dependent ferroptosis has become an emerging strategy for tumor therapy. However, current strategies not only selectively induce ferroptosis in malignant cells but also trigger ferroptosis in immune cells simultaneously, which can compromise anti-tumor immunity. Here, we used In-Cell Western assays combined with an unbiased drug screening to identify the compound N6F11 as a ferroptosis inducer that triggered the degradation of glutathione peroxidase 4 (GPX4), a key ferroptosis repressor, specifically in cancer cells. N6F11 did not cause the degradation of GPX4 in immune cells, including dendritic, T, natural killer, and neutrophil cells. Mechanistically, N6F11 bound to the RING domain of E3 ubiquitin ligase tripartite motif containing 25 (TRIM25) in cancer cells to trigger TRIM25-mediated K48-linked ubiquitination of GPX4, resulting in its proteasomal degradation. Functionally, N6F11 treatment caused ferroptotic cancer cell death that initiated HMGB1-dependent antitumor immunity mediated by CD8 + T cells. N6F11 also sensitized immune checkpoint blockade that targeted CD274/PD-L1 in advanced cancer models, including genetically engineered mouse models of pancreatic cancer driven by KRAS and TP53 mutations. These findings may establish a safe and efficient strategy to boost ferroptosis-driven antitumor immunity.

Whole genome sequencing characteristics of Chlamydia psittaci caprine AMK-16 strain, a promising killed whole cell veterinary vaccine candidate against chlamydia infection.

Chlamydia psittaci is a primary zoonotic pathogen with a broad host range causing severe respiratory and reproductive system infection in animals and humans. To reduce the global burden of C. psittaci-associated diseases on animal welfare and health and to control the pathogen spread in husbandry, effective vaccines based on promising vaccine candidate(s) are required. Recently, the caprine C. psittaci AMK-16 strain (AMK-16) demonstrated a high level of protection (up to 80-100%) in outbred mice and pregnant rabbits immunized with these formaldehyde-inactivated bacteria against experimental chlamydial wild-type infection. This study investigated the molecular characteristics of AMK-16 by whole-genome sequencing followed by molecular typing, phylogenetic analysis and detection of main immunodominant protein(s) eliciting the immune response in mouse model. Similarly to other C. psittaci, AMK-16 harbored an extrachromosomal plasmid. The whole-genome phylogenetic analysis proved that AMK-16 strain belonging to ST28 clustered with only C. psittaci but not with Chlamydia abortus strains. However, AMK-16 possessed the insert which resulted from the recombination event as the additional single chromosome region of a 23,100 bp size with higher homology to C. abortus (98.38-99.94%) rather than to C. psittaci (92.06-92.55%). At least six of 16 CDSs were absent in AMK-16 plasticity zone and 41 CDSs in other loci compared with the reference C. psittaci 6BC strain. Two SNPs identified in the AMK-16 ompA sequence resulted in MOMP polymorphism followed by the formation of a novel genotype/subtype including three other C. psittaci strains else. AMK-16 MOMP provided marked specific cellular and humoral immune response in 100% of mice immunized with the inactivated AMK-16 bacteria. Both DnaK and GrpE encoded by the recombination region genes were less immunoreactive, inducing only a negligible T-cell murine immune response, while homologous antibodies could be detected in 50% and 30% of immunized mice, respectively. Thus, AMK-16 could be a promising vaccine candidate for the development of a killed whole cell vaccine against chlamydiosis in livestock.

Quantitative Threshold Determination of Auditory Brainstem Responses in Mouse Models.

The auditory brainstem response (ABR) is a scalp recording of potentials produced by sound stimulation, and is commonly used as an indicator of auditory function. However, the ABR threshold, which is the lowest audible sound pressure, cannot be objectively determined since it is determined visually using a measurer, and this has been a problem for several decades. Although various algorithms have been developed to objectively determine ABR thresholds, they remain lacking in terms of accuracy, efficiency, and convenience. Accordingly, we proposed an improved algorithm based on the mutual covariance at adjacent sound pressure levels. An ideal ABR waveform with clearly defined waves I-V was created; moreover, using this waveform as a standard template, the experimentally obtained ABR waveform was inspected for disturbances based on mutual covariance. The ABR testing was repeated if the value was below the established cross-covariance reference value. Our proposed method allowed more efficient objective determination of ABR thresholds and a smaller burden on experimental animals.

Self-Adjuvanting Calcium-Phosphate-Coated Microcrystal-Based Vaccines Induce Pyroptosis in Human and Livestock Immune Cells.

Successful vaccines require adjuvants able to activate the innate immune system, eliciting antigen-specific immune responses and B-cell-mediated antibody production. However, unwanted secondary effects and the lack of effectiveness of traditional adjuvants has prompted investigation into novel adjuvants in recent years. Protein-coated microcrystals modified with calcium phosphate (CaP-PCMCs) in which vaccine antigens are co-immobilised within amino acid crystals represent one of these promising self-adjuvanting vaccine delivery systems. CaP-PCMCs has been shown to enhance antigen-specific IgG responses in mouse models; however, the exact mechanism of action of these microcrystals is currently unclear. Here, we set out to investigate this mechanism by studying the interaction between CaP-PCMCs and mammalian immune cells in an in vitro system. Incubation of cells with CaP-PCMCs induced rapid pyroptosis of peripheral blood mononuclear cells and monocyte-derived dendritic cells from cattle, sheep and humans, which was accompanied by the release of interleukin-1β and the activation of Caspase-1. We show that this pyroptotic event was cell-CaP-PCMCs contact dependent, and neither soluble calcium nor microcrystals without CaP (soluble PCMCs) induced pyroptosis. Our results corroborate CaP-PCMCs as a promising delivery system for vaccine antigens, showing great potential for subunit vaccines where the enhancement or find tuning of adaptive immunity is required.