Murine Model Research Articles

The small inhibitor WM-1119 effectively targets KAT6A-rearranged AML, but not KMT2A-rearranged AML, despite shared KAT6 genetic dependency

BackgroundThe epigenetic factors KAT6A (MOZ/MYST3) and KMT2A (MLL/MLL1) interact in normal hematopoiesis to regulate progenitors’ self-renewal. Both proteins are recurrently translocated in AML, leading to impairment of critical differentiation pathways in these malignant cells. We evaluated the potential of different KAT6A therapeutic targeting strategies to alter the growth of KAT6A and KMT2A rearranged AMLs.MethodsWe investigated the action and potential mechanisms of the first-in-class KAT6A inhibitor, WM-1119 in KAT6A and KMT2A rearranged (KAT6Ar and KMT2Ar) AML using cellular (flow cytometry, colony assays, cell growth) and molecular (shRNA knock-down, CRISPR knock-out, bulk and single-cell RNA-seq, ChIP-seq) assays. We also used two novel genetic murine KAT6A models combined with the most common KMT2Ar AML, KMT2A::MLLT3 AML. In these murine models, the catalytic activity of KAT6A, or the whole protein, can be conditionally abrogated or deleted. These models allowed us to compare the effects of specific KAT6A KAT activity inhibition with the complete deletion of the whole protein. Finally, we also tested these therapeutic approaches on human AML cell lines and primary patient AMLs.ResultsWe found that WM-1119 completely abrogated the proliferative and clonogenic potential of KAT6Ar cells in vitro. WM-1119 treatment was associated with a dramatic increase in myeloid differentiation program. The treatment also decreased stemness and leukemia pathways at the transcriptome level and led to loss of binding of the fusion protein at critical regulators of these pathways. In contrast, our pharmacologic and genetic results indicate that the catalytic activity of KAT6A plays a more limited role in KMT2Ar leukemogenicity, while targeting the whole KAT6A protein dramatically affects leukemic potential in murine KMT2A::MLLT3 AML.ConclusionOur study indicates that inhibiting KAT6A KAT activity holds compelling promise for KAT6Ar AML patients. In contrast, targeted degradation of KAT6A, and not just its catalytic activity, may represent a more appropriate therapeutic approach for KMT2Ar AMLs.

A modified BPaL regimen for tuberculosis treatment replaces linezolid with inhaled spectinamides.

The Nix-TB clinical trial evaluated a new 6 month regimen containing three oral drugs; bedaquiline (B), pretomanid (Pa), and linezolid (L) (BPaL regimen) for the treatment of tuberculosis (TB). This regimen achieved remarkable results as almost 90% of the multidrug-resistant or extensively drug-resistant TB participants were cured but many patients also developed severe adverse events (AEs). The AEs were associated with the long-term administration of the protein synthesis inhibitor linezolid. Spectinamide 1599 is also a protein synthesis inhibitor of Mycobacterium tuberculosis with an excellent safety profile, but it lacks oral bioavailability. Here, we propose to replace L in the BPaL regimen with spectinamide (S) administered via inhalation and we demonstrate that inhaled spectinamide 1599, combined with BPa --BPaS regimen--has similar efficacy to that of the BPaL regimen while simultaneously avoiding the L-associated AEs. The BPaL and BPaS regimens were compared in the BALB/c and C3HeB/FeJ murine chronic TB efficacy models. After 4-weeks of treatment, both regimens promoted equivalent bactericidal effects in both TB murine models. However, treatment with BPaL resulted in significant weight loss and the complete blood count suggested the development of anemia. These effects were not similarly observed in mice treated with BPaS. BPaL and BPa, but not the BPaS treatment, also decreased myeloid to erythroid ratio suggesting the S in the BPaS regimen was able to recover this effect. Moreover, the BPaL also increased concentration of proinflammatory cytokines in bone marrow compared to mice receiving BPaS regimen. These combined data suggest that inhaled spectinamide 1599 combined with BPa is an effective TB regimen without L-associated AEs.

Calcitonin gene-related peptide promotes epithelial reparative and anti-colitic functions of IL-4 educated human macrophages.

Interleukin-4 activated human macrophages (M(IL4) promote epithelial wound healing and exert an anti-colitic effect in a murine model. Blood monocyte-derived M(IL4)s from healthy donors and individuals with Crohn's disease had increased mRNA expression of the calcitonin gene-related peptide (CGRP) receptor chain, RAMP1, raising the issue of neural modulation of the M(IL4)s reparative function. Thus, human (MIL4)s were treated with CGRP and the cells phagocytotic, epithelial wound repair and anti-colitic functions were assessed. Initial studies confirmed up-regulation of expression of the CGRP receptor, which was localized to the cell surface and was functional as determined by CGRP-evoked increases in cAMP. M(IL4,CGRP)s had increased mannose receptor (CD206) and FcgRIIa (CD32a) mRNA expression, a subtle, but significant increase in phagocytosis, and decreased chemokine production following exposure to E. coli. When delivered systemically (106 cells, ip.) to oxazolone-treated rag1-/- mice, M(IL4,CGRP) had an anti-colitic effect superior to M(IL4)s from the same blood donor. Conditioned medium (CM) from M(IL4,CGRP) had increased amounts of TGFb and increased wound-healing capacity compared to matched M(IL4)-CM in the human CaCo2 epithelial cell line in vitro wounding assay. Moreover, M(IL4,CGRP)s displayed increased cyclooxygenase (COX)-1 and prostaglandin D2, and CM from M(IL4,CGRP)s treated with indomethacin or SC-560 to inhibit COX1 activity failed to promote repair of wounded CaCo2 cell monolayers. These data confirm the human M(IL4)s' anti-colitic effect that was enhanced by CGRP, and may be partially dependent on macrophage COX1/PDG2 activity. Thus, input from neurone-derived molecules is a local modifier capable of boosting the anti-colitic effect of autologous M(IL4) transfer.

Novel Peptides LFLLP and DFFL from Jack Bean Protein Hydrolysates Suppress the Inflammatory Response in Lipopolysaccharide-Stimulated RAW 264.7 Cells.

The production of inflammatory cytokines such as tumor necrosis factor (TNF)-α by activated macrophage cells plays an important role in the development of intestinal inflammation. The present study investigated the anti-inflammatory effect of the protein hydrolysates prepared from the jack bean (JBPHs), Canavalia ensiformis (L.) DC, using the enzyme Alcalase, in a murine macrophage model, RAW 264.7 cells, which were stimulated by lipopolysaccharides. JBPHs reduced the TNF-α expression at the protein and mRNA levels through the downregulation of cellular signaling pathways involved in nuclear factor kappa B (NF-κB), extracellular signal-regulated kinase (ERK), and p38. A combination of mass spectrometry and in silico approaches identified 10 potential anti-inflammatory peptides in the JBPHs, including LFLLP and DFFL. Interestingly, while LFLLP targeted the NF-κB pathway, DFFL targeted p38 and ERK to suppress the TNF-α production in the RAW 264.7 cells. In addition, LFLLP and DFFL were localized in the cytosol of the cells. These results demonstrated that LFLLP and DFFL were incorporated by RAW 264.7 cells and, at least in part, contributed to the reduction in TNF-α by JBPHs. These peptides isolated from JBPHs may well be utilized as new alternatives to alleviate intestinal inflammation.

Human Amniotic Epithelial Stem Cells Promote Colonic Recovery in Experimental Colitis via Exosomal MiR-23a-TNFR1-NF-κB Signaling.

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, manifests as chronic intestinal inflammation with debilitating symptoms, posing a significant burden on global healthcare. Moreover, current therapies primarily targeting inflammation can lead to immunosuppression-related complications. Human amniotic epithelial stem cells (hAESCs), which exhibit low immunogenicity and ethical acceptability, have gained attention as potential therapeutics. In this study, it is demonstrated that their encapsulation in a hydrogel and administration via anal injection enhanced the colonic mucosal barrier repair in a murine colitis model induced by dextran sodium sulfate during the recovery phase. The underlying mechanism involved the release of exosomes from hAESCs enriched with microRNA-23a-3p, which post-transcriptionally reduced tumor necrosis factor receptor 1 expression, suppressing the nuclear factor-κB pathway in colonic epithelial cells, thus played a key role in inflammation. The novel approach shows potential for IBD treatment by restoring intestinal epithelial homeostasis without the immunosuppressive therapy-associated risks. Furthermore, the approach provides an alternative strategy to target the key molecular pathways involved in inflammation and promotes intestinal barrier function using hAESCs and their secreted exosomes. Overall, this study provides key insights to effectively treat IBD, addresses the unmet needs of patients, and reduces related healthcare burden.

Segment-Based Peptide Design Reveals the Importance of N-Terminal High Cationicity for Antimicrobial Activity Against Gram-Negative Pathogens.

Host defense antimicrobial peptides (AMPs) are recognized candidates to develop a new generation of peptide antibiotics. While high hydrophobicity can be deployed in peptides for eliminating Gram-positive bacteria, high cationicity is usually observed in AMPs against Gram-negative pathogen. This study investigates how the sequence distribution of basic amino acids affects peptide activity. For this purpose, we utilized human cathelicidin LL-37 as a template and designed four highly selective ultrashort peptides with similar length, net charge, and hydrophobic content. LL-10 + , RK-9 + , KR-8 + , and RIK-10 + showed similar activity against methicillin-resistant Staphylococcus aureus in vitro and comparable antibiofilm efficacy in a murine wound model. However, these peptides showed clear activity differences against Gram-negative pathogens with RIK-10 + (i.e., LL-37mini2) being the strongest and LL-10 + the weakest. To understand this activity difference, we characterized peptide toxicity; the effects of salts, pH, and serum on peptide activity; and the mechanism of action and determined the membrane-bound helical structure for RIK-10 + by two-dimensional NMR spectroscopy. By writing an R program, we generated charge density plots for these peptides and uncovered the importance of the N-terminal high-density basic charges for antimicrobial potency. To validate this finding, we reversed the sequences of two peptides. Interestingly, sequence reversal weakened the activity of RIK-10 + but increased the activity of LL-10 + especially against Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Those more active peptides with high cationicity at the N-terminus are also more hydrophobic based on HPLC retention times. A database search found numerous natural sequences that arrange basic amino acids primarily at the N-terminus. Combined, this study not only obtained novel peptide leads but also discovered one useful strategy for designing novel antimicrobials to control drug-resistant Gram-negative pathogens.

Zidovudine in synergistic combination with nitrofurantoin or omadacycline: in vitro and in murine urinary tract or lung infection evaluation against multidrug-resistant Klebsiella pneumoniae.

Limited treatment options and multidrug-resistant (MDR) Klebsiella pneumoniae present a significant therapeutic challenge, underscoring the need for novel approaches. Drug repurposing is a promising tool for augmenting the activity of many antibiotics. This study aimed to identify novel synergistic drug combinations against K. pneumoniae based on drug repurposing. We used the clinically isolated GN 172867 MDR strain of K. pneumoniae to determine the reversal resistance activity of zidovudine (AZT). The combined effects of AZT and various antibiotics, including nitrofurantoin (NIT) and omadacycline (OMC), were examined using the checkerboard method, growth curves, and crystal violet assays to assess biofilms. An in vitro combination activity testing was carried out in 12 isolates of K. pneumoniae. In vivo murine urinary tract and lung infection models were used to evaluate the therapeutic effects of AZT + NIT and AZT + OMC, respectively. The fractional inhibitory concentration index and growth curve demonstrated that AZT synergized with NIT or OMC against K. pneumoniae strains. In addition, AZT + NIT inhibited biofilm formation and cleared mature biofilms. In vivo, compared with untreated GN 172867-infected mice, AZT + NIT and AZT + OMC treatment decreased colony counts in multiple tissues (P < 0.05) and pathological scores in the bladder and kidneys (P < 0.05) and increased the survival rate by 60% (P < 0.05). This study evaluated the combination of AZT and antibiotics to treat drug-resistant K. pneumoniae infections and found novel drug combinations for the treatment of acute urinary tract infections. These findings suggest that AZT may exert significant anti-resistance activity.

Lactoferrin modulates oxidative stress and inflammatory cytokines in a murine model of dysbiosis induced by clindamycin.

Antibiotics, specifically clindamycin, cause intestinal dysbiosis, reducing the microbiota with anti-inflammatory properties. Furthermore, clindamycin can induce alterations in the immune responses and oxidative stress. Lactoferrin, among other activities, participates in the maintenance of intestinal homeostasis and reduces dysbiosis induced by antibiotic treatment. The aim of this study was to analyze the effect of native and iron-saturated bovine LF in a murine model of dysbiosis induced by clindamycin. Six groups of male C57BL/6 mice were treated with saline (control), clindamycin (Clin), native lactoferrin (nLF), iron-saturated lactoferrin (sLF), nLF/Clin or sLF/Clin. Oxidation caused in the intestinal cells of the ileum of animals subjected to different treatments was analyzed, focusing on lipid peroxidation and protein carbonyl content. The expression of inflammatory mediators was determined by qRT-PCR. Treatment with clindamycin did not modify lipid peroxidation, but significantly increased protein carbonyl levels up to almost 5-fold respect to the control, an effect that was reversed by orally administering sLF to mice. Furthermore, clindamycin increased the expression of interleukin-6 and TNF-α by 1- and 2-fold change, respectively. This effect was reversed by treatment with nLF and sLF, decreasing the expression to basal levels. In conclusion, this study indicates that lactoferrin can prevent some of the effects of clindamycin on intestinal cells and their associated immune system.

Memory-enhancing activity of verapamil in murine models of stress

Purpose: To evaluate the benefit of verapamil on stress-induced memory impairment in mice. Methods: Forty-eight (48) mice were used in this study. They were divided into two equal groups based on the two models of stress used in this study (sleep deprivation and hypoxia). Each model was further divided into 4 groups of six animals each. The mice in the sleep deprivation model were suspended on a platform above water while in the hypoxic model, mice were locked in an airless container for 20 min daily throughout the experiment. As for the intervention, 25 and 50 mg/kg verapamil were preadministered via the oral route to study groups, except the normal control group and negative control group in both models. After seven-day stress and intervention, the mice were subjected to behavioural tests (Y-maze and object recognition tests), biochemical assays (for acetylcholinesterase activity) and histochemical analysis. Results: Stress caused a significant (p &lt; 0.05) impairment in the consolidation and retrieval of working and recognition memories. Also, acetylcholinesterase activity was significantly (p &lt; 0.05) enhanced in the stressed groups when compared to control groups. Similarly, the histological analysis revealed a significant decline in population (p &lt; 0.05), distribution and density of viable neurons in specific areas of the hippocampus and prefrontal cortex. These alterations were significantly (p &lt; 0.05) attenuated in verapamil-treated groups almost in a dose-dependent pattern. Conclusion: Verapamil displays significant memory-enhancing effects in two (2) murine models of stress. Antihypertensives should therefore be considered a viable prospect in the management of stress-related memory disorders after additional studies have been done to establish the mechanisms of action.

Integrated metabolomics and gut microbiota analysis to explore potential mechanism of Qi-Huo-Yi-Fei formula against chronic obstructive pulmonary disease

Metabolic disorders and gut microbiota dysbiosis contribute to the complicated pathology of chronic obstructive pulmonary disease (COPD). Qi-Huo-Yi-Fei formula (QHYFF) is a Chinese medicine prescription for COPD treatment and has showed beneficial clinical effects, but the underlying mechanism remains elusive. This study integrated metabolomics and gut microbiota analysis to explore potential mechanism of QHYFF against COPD. The therapeutic effects of QHYFF were evaluated using a murine model of COPD induced by cigarette smoke and lipopolysaccharide. QHYFF effectively improved pulmonary function, suppressed inflammation, and relieved lung pathological changes. Serum and urine metabolomics analysis identified 19 differential metabolites, such as L-tyrosine, epinephrine, dopamine, hypotaurine, citric acid, L-tryptophan and indoleacrylic acid, involving tyrosine metabolism, taurine and hypotaurine metabolism, citrate cycle and tryptophan metabolism. QHYFF also enriched Bifidobacterium, Blautia, Faecalibaculum and Parasutterella. Moreover, Spearman’s correlation analysis showed that discriminative metabolites and bacteria were closely correlated with efficacy indices. The findings indicated that QHYFF could be an effective therapeutic measure against COPD by regulating metabolism and gut microbiota.

NINJ1-mediated Macrophage Plasma Membrane Rupture and Neutrophil Extracellular Trap Formation Contribute to Oxalate Nephropathy.

Oxalate nephropathy is characterized by calcium oxalate crystals deposition, which triggers necrosis of renal tubular epithelial cells, initiates an inflammatory cascade characterized by neutrophil and macrophage activation within the renal microenvironment. Despite the close association of immune cells with acute oxalate nephropathy, the underlying mechanisms still remain unclear. Nerve injury-induced protein 1 (NINJ1) plays an essential role in the induction of plasma membrane rupture (PMR), leading to damage-associated molecular patterns (DAMPs) release and triggering inflammation. We hypothesize that NINJ1-mediated high mobility group box 1 (HMGB1) release from macrophage PMR and neutrophil extracellular traps (NETs) formation synergistically contribute to the progression of acute oxalate nephropathy. Using a murine model of acute oxalate nephropathy, myeloid cell-specific deletion of Ninj1 mice (Ninj1fl/flvavcre) and their wild-type littermate control mice (Ninj1wt/wtvavcre) were administered intraperitoneal injection of 100 mg/kg sodium oxalate followed by drinking water with 3% sodium oxalate. Evaluation was conducted on tubular injury and inflammatory cell infiltration. In vitro studies involved isolation and culture of renal proximal tubular epithelial cells (RTECs), bone marrow-derived macrophages, and neutrophils to investigate NETs formation and HMGB1 release. Targeted deletion of Ninj1 in myeloid cells significantly mitigated oxalate-induced acute kidney injury by suppressing both HMGB1 release and NETs formation in vivo. In vitro investigations demonstrated that HMGB1 release from macrophage PMR and NETs formation in neutrophils mediated by NINJ1 oligomerization, which consequently coordinated to enhance renal tubular epithelial cell death. Our findings elucidate the pivotal role of NINJ1-dependent macrophage PMR and NETs formation in the progression of acute oxalate nephropathy, providing novel insights for its prevention and therapeutic targets.

Single-cell RNA sequencing reveals the pro-inflammatory roles of liver-resident Th1-like cells in primary biliary cholangitis

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease characterized by multilineage immune dysregulation, which subsequently causes inflammation, fibrosis, and even cirrhosis of liver. Due to the limitation of traditional assays, the local hepatic immunopathogenesis of PBC has not been fully characterized. Here, we utilize single-cell RNA sequencing technology to depict the immune cell landscape and decipher the molecular mechanisms of PBC patients. We reveal that cholangiocytes and hepatic stellate cells are involved in liver inflammation and fibrosis. Moreover, Kupffer cells show increased levels of inflammatory factors and decreased scavenger function related genes, while T cells exhibit enhanced levels of inflammatory factors and reduced cytotoxicity related genes. Interestingly, we identify a liver-resident Th1-like population with JAK-STAT activation in the livers of both PBC patients and murine PBC model. Finally, blocking the JAK-STAT pathway alleviates the liver inflammation and eliminates the liver-resident Th1-like cells in the murine PBC model. In conclusion, our comprehensive single-cell transcriptome profiling expands the understanding of pathological mechanisms of PBC and provides potential targets for the treatment of PBC in patients.

LHPP deficiency aggravates liver fibrosis through TGF-β/Smad3 signaling.

Liver fibrosis is characterized by a wound-healing response and may progress to liver cirrhosis and even hepatocellular carcinoma. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a tumor suppressor that participates in malignant diseases. However, the role of LHPP in liver fibrosis has not been determined. Herein, the function and regulatory network of LHPP were explored in liver fibrosis. The expression of LHPP in human and murine fibrotic liver tissues was assessed via immunohistochemistry and Western blot analysis. In addition, liver fibrosis was induced in wild-type (WT) and LHPP-/- (KO) mice after carbon tetrachloride (CCl4) or thioacetamide (TAA) treatment. The effect of LHPP was systematically assessed by using specimens acquired from the above murine models. The functional role of LHPP was further explored by detecting the pathway activity of TGF-β/Smad3 and apoptosis after interfering with LHPP invitro. To explore whether the function of LHPP depended on the TGF-β/Smad3 pathway invivo, an inhibitor of the TGF-β/Smad3 pathway was used in CCl4-induced WT and KO mice. LHPP expression was downregulated in liver tissue samples from fibrosis patients and fibrotic mice. LHPP deficiency aggravated CCl4- and TAA-induced liver fibrosis. Moreover, through immunoblot analysis, we identified the TGF-β/Smad3 pathway as a key downstream pathway of LHPP invivo and invitro. The effect of LHPP deficiency was reversed by inhibiting the TGF-β/Smad3 pathway in liver fibrosis. These results revealed that LHPP deficiency exacerbates liver fibrosis through the TGF-β/Smad3 pathway. LHPP may be a potential therapeutic target in hepatic fibrosis.

Cysteinyl Leukotrienes in Allergic Inflammation.

The cysteinyl leukotrienes (CysLTs), LTC4, LTD4, and LTE4, are potent lipid mediators derived from arachidonic acid through the 5-lipoxygenase pathway. These mediators produce both inflammation and bronchoconstriction through three distinct G protein-coupled receptors (GPCRs)-CysLT1, CysLT2, and OXGR1 (also known as CysLT3 or GPR99). While CysLT-mediated functions in the effector phase of allergic inflammation and asthma have been established for some time, recent work has demonstrated novel roles for these mediators and their receptors in the induction and amplification of type 2 inflammation. Additionally, in vitro studies and murine models have uncovered diverse regulatory mechanisms that restrain or amplify CysLT receptor activation and CysLT receptor function. This review provides an overview of CysLT biosynthesis and its regulation, the molecular and functional pharmacology of CysLT receptors, and an overview of the established and emerging roles of CysLTs in asthma, aspirin-exacerbated respiratory disease, and type 2 inflammation.

Modified Hemocyanins from Rapana thomasiana and Helix aspersa Exhibit Strong Antitumor Activity in the B16F10 Mouse Melanoma Model.

Melanoma is one of the most common tumors worldwide, and new approaches and antitumor drugs for therapy are being investigated. Among the promising biomolecules of natural origin for antitumor research are gastropodan hemocyanins-highly immunogenic multimeric glycoproteins used as antitumor agents and components of therapeutic vaccines in human and mouse cancer models. A murine melanoma model established in C57BL/6 mice of the B16F10 cell line was used to study anticancer modified oxidized hemocyanins (Ox-Hcs) that were administered to experimental animals (100 μg/mouse) under different regimens: mild, intensive, and with sensitization. The solid tumor growth, antitumor response, cell infiltration in tumors, and survival were assessed using flow cytometry, ELISA, and cytotoxicity assays. Therapy with Ox-RtH or Ox-HaH resulted in the generation of enhanced specific immune response (increased levels of tumor-infiltrated mature NK cells (CD27+CD11b+) in sensitized groups and of macrophages in the intensively immunized animals) and tumor suppression. Beneficial effects such as delayed tumor incidence and growth as well as prolonged survival of tumor-bearing animals have been observed. High levels of melanoma-specific CTLs that mediate cytotoxic effects on tumor cells; tumor-infiltrating IgM antibodies expected to enhance antibody-dependent cellular cytotoxicity; type M1 macrophages, which stimulate the Th1 response and cytotoxic cells; and proinflammatory cytokines, were also observed after Ox-Hcs administration. The modified Hcs showed strong antitumor properties in different administration regimens in a murine model of melanoma with potential for future application in humans.

Optimized Fabrication of Dendritic Mesoporous Silica Nanoparticles as Efficient Delivery System for Cancer Immunotherapy.

In the past decade, cancer immunotherapy has revolutionized the field of oncology. Major immunotherapy approaches such as immune checkpoint inhibitors, cancer vaccines, adoptive cell therapy, cytokines, and immunomodulators have shown great promise in preclinical and clinical settings. Among them, immunomodulatory agents including cancer vaccines are particularly appealing; however, they face limitations, notably the absence of efficient and precise targeted delivery of immune-modulatory agents to specific immune cells and the potential for off-target toxicity. Nanomaterials can play a pivotal role in addressing targeting and other challenges in cancer immunotherapy. Dendritic mesoporous silica nanoparticles (DMSNs) can enhance the efficacy of cancer vaccines by enhancing the effective loading of immune modulatory agents owing to their tunable pore sizes. In this work, an emulsion-based method is optimized to customize the pore size of DMSNs and loaded DMSNs with ovalbumin (OVA) and cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (CpG-OVA-DMSNs). The immunotherapeutic effect of DMSNs is achieved through controlled chemical release of OVA and CpG in antigen-presenting cells (APCs). The results demonstrated that CpG-OVA-DMSNs efficiently activated the immune response in APCs and reduced tumor growth in the murine B16-OVA tumor model.

Modelling a Western Lifestyle in Mice: A Novel Approach to Eradicating Aerobic Spore-Forming Bacteria from the Colonic Microbiome and Assessing Long-Term Clinical Outcomes.

The environmentally acquired aerobic spore-forming (EAS-Fs) bacteria that are ubiquitous in nature (e.g., soil) are transient colonisers of the mammalian gastro-intestinal tract. Without regular exposure, their numbers quickly diminish. These species of bacteria have been suggested to be essential to the normal functioning of metabolic and immunogenic health. The modern Western lifestyle restricts exposure to these EAS-Fs, possibly explaining part of the pathogenesis of many Western diseases. To date, the only animal studies that address specific microbiome modelling are based around germ-free animals. We have designed a new animal model that specifically restricts exposure to environmental sources of bacteria. A new protocol, termed Super Clean, which involves housing mice in autoclaved individually ventilated cages (IVCs), with autoclaved food/water and strict ascetic handling practice was first experimentally validated. The quantification of EAS-Fs was assessed by heat-treating faecal samples and measuring colony-forming units (CFUs). This was then compared to mice in standard conditions. Mice were housed in their respective groups from birth until 18 months. Stool samples were taken throughout the experiment to assess for abundance in transiently acquired environmental bacteria. Clinical, biochemical, histological, and gene expression markers were analysed for diabetes, hypercholesterolaemia, obesity, inflammatory bowel disease, and non-alcoholic fatty liver disease (the "diseases of the West"). Our results show that stringent adherence to the Super Clean protocol produces a significantly decreased abundance of aerobic spore-forming Bacillota after 21 days. This microbiomic shift was correlated with significantly increased levels of obesity and impaired glucose metabolism. There was no evidence of colitis, liver disease or hypercholesterolaemia. This new murine model successfully isolates EAS-Fs and has potential utility for future research, allowing for an investigation into the clinical impact of living in relative hygienic conditions.

Chronic hyperglycemia alters retinal astrocyte microstructure and uptake of cholera toxin B in a murine model of diabetes.

Astrocytes are the principle glial cells of the central nervous system and play an active role in maintaining proper metabolism in surrounding neurons. Because of their involvement in metabolic control, it is likely that their physiology changes in response to metabolic diseases such as diabetes and associated diabetic retinopathy. Here, we investigated whether microstructural changes in astrocyte morphology occur during the early stages of chronic hyperglycemia that may be indicative of early pathogenic programs. We used MORF3 mice in conjunction with streptozotocin-induced hyperglycemia to investigate the morphology of single retinal astrocytes at an early timepoint in diabetic disease. We report that astrocytes initiate a morphological remodeling program, which depends on both the glycemic background and the presence of intravitreal injury, to alter the amount of the neuronal-associated pad and bristle microstructural motifs. Additionally, hyperglycemia increases astrocyte uptake of cholera toxin B, possibly reflecting changes in glycolipid and glycoprotein biosynthesis. Chronic hyperglycemia coupled with intravitreal injection of cholera toxin B also causes extensive leukocyte infiltration into the retina. Our results have important clinical relevance as current therapies for diabetic retinopathy involve intravitreal injection of pharmaceuticals in individuals with often poorly controlled blood glucose levels.

The Thyroid Hormone Analog GC-1 Mitigates Acute Lung Injury by Inhibiting M1 Macrophage Polarization.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a life-threatening condition with a high mortality rate of ≈40%. Thyroid hormones (THs) play crucial roles in maintaining homeostasis of the cellular microenvironment under stress. The previous studies confirmed that the clinical-stage TH analog GC-1 significantly alleviates pulmonary fibrosis by improving the function of mitochondria in epithelial cells. However, the effects of GC-1 on macrophages in lung injury and the related mechanisms remain unclear. This study evaluated the therapeutic effects of GC-1 in two murine models of lipopolysaccharide (LPS)- or hydrochloric acid (HCl)-induced ALI. Additionally, mouse alveolar macrophages (AMs) and human THP-1-derived macrophages are utilized to investigate the impact of GC-1 on macrophage polarization. GC-1 effectively reduces the inflammatory response and lung injury in ALI mice, as evidenced by neutrophil infiltration, cytokine levels, alveolar fluid clearance, and pulmonary pathology. Notably, GC-1 selectively inhibits M1 macrophage polarization, which may be achieved by impeding NF-κB signaling activation through the DNMT3b-PPARγ-NF-κB pathway in a TH receptor β1 (TRβ1)-dependent manner, consequently suppressing the polarization of macrophages toward the M1 phenotype and overproduction of inflammatory cytokines. Overall, these findings highlight the immunomodulatory property of GC-1 as an anti-inflammatory strategy for ALI/ARDS and inflammation-related diseases.

Dual MAPK Inhibition Triggers Pro-inflammatory Signals and Sensitizes BRAF V600E Glioma to T Cell-Mediated Checkpoint Therapy.

BRAF V600E pediatric low-grade gliomas frequently transform into high-grade gliomas (HGG) and poorly respond to chemotherapy, resulting in high mortality. Although combined BRAF and MEK inhibition (BRAFi+MEKi) outperforms chemotherapy, ∼70% of BRAF V600E HGG patients are therapy resistant and undergo unbridled tumor progression. BRAF V600E glioma have an immune-rich microenvironment suggesting that they could be responsive to immunotherapy but effects of BRAFi+MEKi on anti-tumor immunity are unclear. Using patient tumor tissue before and after BRAFi+MEKi, two novel syngeneic murine models of BRAF V600E HGG, and patient-derived cell lines, we examined the effects of clinically relevant BRAFi+MEKi with dabrafenib and trametinib on tumor growth, cell states, and tumor-infiltrating T cells. We find that BRAFi+MEKi treatment: i) upregulated programmed cell death protein-1 (PD-1) signaling genes and PD-1 ligand (PD-L1) protein expression in murine BRAF V600E HGG by stimulating IFNγ and IL-27, ii) attenuated T cell activity by IL-23, IL-27 and IL-32 production, which can promote the expansion of regulatory T cells, and iii) induced glial differentiation linked to a therapy-resistant PD-L1+ compartment through Galectin-3 secretion by tumor cells. Murine BRAF V600E HGG shrinkage by BRAFi+MEKi is associated with the upregulation of interferon-gamma response genes, MHC class I/II expression, and antigen presentation and processing programs, indicative of increased anti-tumor immunity. Combined BRAFi+MEKi with therapeutic antibodies inhibiting the PD-1 and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) immune checkpoints re-activate T cells and provide a survival benefit over single therapy in a T cell-dependent manner. The quadruple treatment overcame BRAFi+MEKi resistance by invigorating T cell-mediated anti-tumor immunity in murine BRAF V600E HGG. PD-L1 expression was elevated in human BRAF-mutant versus BRAF-wildtype glioblastoma clinical specimen, complementing experimental findings and suggesting translational relevance for patient care.