Synergistic anti-tumor efficacy of mutant isocitrate dehydrogenase 1 inhibitor SYC-435 with standard therapy in patient-derived xenograft mouse models of glioma
Clinical outcomes in patients with WHO grade II/III astrocytoma, oligodendroglioma or secondary glioblastoma remain poor.
Isocitrate dehydrogenase 1 (IDH1) is mutated in > 70% of these tumors, making it an attractive therapeutic target.
To determine the efficacy of our newly developed mutant IDH1 inhibitor, SYC-435 (1-hydroxypyridin-2-one), we treated orthotopic glioma xenograft model (IC-BT142AOA) carrying R132H mutation and our newly established orthotopic patient-derived xenograft (PDX) model of recurrent anaplastic oligoastrocytoma (IC-V0914AOA) bearing R132C mutation.
In addition to suppressing IDH1 mutant cell proliferation in vitro, SYC-435 (15 mg/kg, daily x 28 days) synergistically prolonged animal survival times with standard therapies (Temozolomide + fractionated radiation) mediated by reduction of H3K4/H3K9 methylation and expression of mitochondrial DNA (mtDNA)-encoded molecules.
Furthermore, RNA-seq of the remnant tumors identified genes (MYO1F, CTC1 and BCL9) and pathways (base excision repair, TCA cycle II, sirtuin signaling, protein kinase A, eukaryotic initiation factor 2 and α-adrenergic signaling) as mediators of therapy resistance.
Our data demonstrated the efficacy SYC-435 in targeting IDH1 mutant gliomas when combined with standard therapy and identified a novel set of genes that should be prioritized for future studies to overcome SYC-435 resistance.
Flow cytometric analysis of CD4+ T cell reactivation following anti-PD1 immunotherapy in a transgenic mouse model
This protocol uses the Tg4 Nr4a3-Tocky mouse model to recalibrate T cell activation thresholds and reveals the role that immune checkpoints play in controlling T cell activation.
The example approach here uses flow cytometry to characterize quantitative and qualitative changes in splenic CD4+ T cells reactivated in the presence of anti-PD1 immunotherapy.
The protocol is optimized for studying anti-PD1 pathway blockade only.
The protocol is not compatible with cellular fixation, and T cells should be analyzed immediately after staining. For complete details on the use and execution of this protocol, please refer to Elliot et al. (2021).
Brain capillary obstruction during neurotoxicity in a mouse model of anti-CD19 chimeric antigen receptor T-cell therapy
Immunotherapy for haematologic malignancies with CD19-directed chimeric antigen receptor T cells has been highly successful at eradicating cancer but is associated with acute neurotoxicity in ∼40% of patients.
This neurotoxicity correlates with systemic cytokine release syndrome, endothelial activation and disruption of endothelial integrity, but it remains unclear how these mechanisms interact and how they lead to neurologic dysfunction.
We hypothesized that dysfunction of the neurovascular unit is a key step in the development of neurotoxicity.
To recapitulate the interaction of the intact immune system with the blood-brain barrier, we first developed an immunocompetent mouse model of chimeric antigen receptor T-cell treatment-associated neurotoxicity.
We treated wild-type mice with cyclophosphamide lymphodepletion followed by escalating doses of murine CD19-directed chimeric antigen receptor T cells.
Within 3-5 days after chimeric antigen receptor T-cell infusion, these mice developed systemic cytokine release and abnormal behaviour as measured by daily neurologic screening exams and open-field testing.
Histologic examination revealed widespread brain haemorrhages, diffuse extravascular immunoglobulin deposition, loss of capillary pericyte coverage and increased prevalence of string capillaries.
To measure any associated changes in cerebral microvascular blood flow, we performed in vivo two-photon imaging through thinned-skull cranial windows.
Unexpectedly, we found that 11.9% of cortical capillaries were plugged by Day 6 after chimeric antigen receptor T-cell treatment, compared to 1.1% in controls treated with mock transduced T cells. The capillary plugs comprised CD45+ leucocytes, a subset of which were CD3+ T cells. Plugging of this severity is expected to compromise cerebral perfusion.
Indeed, we found widely distributed patchy hypoxia by hypoxyprobe immunolabelling. Increased serum levels of soluble ICAM-1 and VCAM-1 support a putative mechanism of increased leucocyte-endothelial adhesion.
These data reveal that brain capillary obstruction may cause sufficient microvascular compromise to explain the clinical phenotype of chimeric antigen receptor T-cell neurotoxicity.
The translational impact of this finding is strengthened by the fact that our mouse model closely approximates the kinetics and histologic findings of the chimeric antigen receptor T-cell neurotoxicity syndrome seen in human patients.
This new link between systemic immune activation and neurovascular unit injury may be amenable to therapeutic intervention.
The Anti-Adiposity Mechanisms of Ampelopsin and Vine Tea Extract in High Fat Diet and Alcohol-Induced Fatty Liver Mouse Models
Ampelopsis grossedentata (AG) is an ancient medicinal plant that is mainly distributed and used in southwest China. It exerts therapeutic effects, such as antioxidant, anti-diabetic, and anti-inflammatory activities, reductions in blood pressure and cholesterol and hepatoprotective effects.
Researchers in China recently reported the anti-obesity effects of AG extract in diet-induced obese mice and rats.
To verify these findings, we herein investigated the effects of AG extract and its principal compound, ampelopsin, in high-fat diet (HFD)- and alcohol diet-fed mice, olive oil-loaded mice, and differentiated 3T3-L1 cells.
The results obtained showed that AG extract and ampelopsin significantly suppressed increases in the weights of body, livers and abdominal fat and also up-regulated the expression of carnitine palmitoyltransferase 1A in HFD-fed mice.
In olive oil-loaded mice, AG extract and ampelopsin significantly attenuated increases in serum triglyceride (TG) levels.
In differentiated 3T3-L1 cells, AG extract and ampelopsin promoted TG decomposition, which appeared to be attributed to the expression of hormone-sensitive lipase. In alcohol diet-fed mice, AG extract and ampelopsin reduced serum levels of ethanol, glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) and liver TG.
An examination of metabolic enzyme expression patterns revealed that AG extract and ampelopsin mainly enhanced the expression of aldehyde dehydrogenase and suppressed that of cytochrome P450, family 2, subfamily e1.
In conclusion, AG extract and ampelopsin suppressed diet-induced intestinal fat accumulation and reduced the risk of fatty liver associated with HFD and alcohol consumption.
Anti-inflammatory actions of acetate, propionate, and butyrate in fetal mouse jejunum cultures ex vivo and immature small intestinal cells
Necrotizing enterocolitis (NEC) is an intestinal disease that frequently occurs in premature infants. Presently, there is no effective therapy for NEC.
Therefore, the key to reduce the incidence rate of NEC is to take effective intervention measures as early as possible. Short-chain fatty acids (SCFAs) (acetate, propionate, and butyrate), the principal terminal products of enterobacteria fermentation, play anti-inflammatory actions in mature intestinal cells.
However, few studies focus on their roles in immature intestine.
Here, we evaluated the anti-inflammatory actions of SCFAs ex vivo with ICR fetal mouse jejunum cultures and explored the potential anti-inflammatory regulators through RNA-seq and then verified them in vitro with human fetal small intestinal epithelial FHs 74 Int cells.
In this study, we found that acetate, propionate, and butyrate decreased IL-1β-induced production of CXCL2 ex vivo and IL-8 and IL-6 in vitro significantly (p < .05).
Furthermore, the inhibitors of NF-κB p65, JNK1/2, and ERK1/2 pathways, which were selected from RNA-seq and depressed by SCFAs, also significantly decreased IL-8 and IL-6 productions induced by IL-1β (p < .05).
Therefore, our results showed that acetate, propionate, and butyrate ameliorated the fetal small intestine inflammatory response induced by IL-1β through inhibiting ERK1/2 pathway; NF-κB p65, JNK1/2, and ERK1/2 pathways; or NF-κB p65 and ERK1/2 pathways, respectively. These findings suggested that SCFAs may be a new therapy agent for NEC.
Lactobacillus rhamnosus L34 attenuates chronic kidney disease progression in 5/6 nephrectomy mouse model through the excretion of anti-inflammatory molecules
Background: Although pathogenic gut microbiota causes gut leakage, increases translocation of uremic toxins into circulation, and accelerates CKD progression, the local strain of Lactobacillus rhamnosus L34 (L34) might attenuate gut leakage. We explored the effects of L34 on kidney fibrosis and levels of gut-derived uremic toxins (GDUTs) in 5/6-nephrectomy (5/6Nx) mice.
Methods: At 6 weeks post-5/6Nx in mice, either L34 (1 × 106 CFU) or phosphate buffer solution (as 5/6Nx control) were daily fed for 14 weeks. In vitro, the effects of L34-conditioned media with or without indoxyl sulfate (a representative GDUT) on inflammation and cell integrity (transepithelial electrical resistance; TEER) were assessed in Caco-2 (enterocytes). In parallel, the effects as such on pro-inflammatory cytokines and collagen expression were assessed in HK2 proximal tubular cells.
Results: At 20-weeks post-5/6Nx, L34-treated mice showed significantly lesser renal injuries, as evaluated by i) kidney fibrosis area (P < 0.01) with lower serum creatinine and proteinuria, ii) GDUT including trimethylamine-N-oxide (TMAO) (P = 0.02) and indoxyl sulfate (P < 0.01), and iii) endotoxin (P = 0.03) and serum TNF-α (P = 0.01), than 5/6Nx-controls. Fecal-microbiome analysis revealed an increased proportion of Bacteroidetes in 5/6Nx-controls.
Rabbit Polyclonal antibody Anti-CRBN |
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| Anti-CRBN | ImmunoStep | 50 µg | 418.8 EUR |
Polyclonal Goat anti-GST α-form |
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| GST-ANTI-1 | Detroit R&D | 50 uL | 336 EUR |
Polyclonal Goat anti-GST μ-form |
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| GST-ANTI-2 | Detroit R&D | 50 uL | 336 EUR |
Polyclonal Goat anti-GST p-form |
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| GST-ANTI-3 | Detroit R&D | 50 uL | 336 EUR |
Mouse anti IgA1 |
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| 10-I06A | Fitzgerald | 1 mg | 369.6 EUR |
SensiTek Anti-Mouse |
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| ABC008 | ScyTek Laboratories | 8 ml | 85.2 EUR |
SensiTek Anti-Mouse |
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| ABC015 | ScyTek Laboratories | 15 ml | 96 EUR |
SensiTek Anti-Mouse |
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| ABC125 | ScyTek Laboratories | 125 ml | 211.2 EUR |
SensiTek Anti-Mouse |
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| ABC500 | ScyTek Laboratories | 500 ml | 504 EUR |
SensiTek Anti-Mouse |
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| ABC999 | ScyTek Laboratories | 1000 ml | 853.2 EUR |
UltraTek Anti-Mouse |
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| ABJ008 | ScyTek Laboratories | 8 ml | 96 EUR |
UltraTek Anti-Mouse |
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| ABJ015 | ScyTek Laboratories | 15 ml | 117.6 EUR |
UltraTek Anti-Mouse |
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| ABJ125 | ScyTek Laboratories | 125 ml | 273.6 EUR |
UltraTek Anti-Mouse |
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| ABJ500 | ScyTek Laboratories | 500 ml | 664.8 EUR |
UltraTek Anti-Mouse |
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| ABJ999 | ScyTek Laboratories | 1000 ml | 1120.8 EUR |
After incubation with indoxyl sulfate, Caco-2 enterocytes had higher IL-8, NFκB expression, and lower TEER value, and HK2 cells demonstrated higher gene expression of TNF-α, IL-6, and collagen (type III and type IV).
These indoxyl sulfate-activated parameters were attenuated with L34-conditioned media indicating the protective role of L34 on enterocyte integrity and renal fibrogenesis.
Conclusion: Lactobacillus rhamnosus L34 attenuated uremia-induced systemic inflammation by reducing GDUTs and gut-leakage that provided reno-protective effects in CKD.
Keywords: Lactobacillus rhamnosus; 5/6 nephrectomy mouse model; chronic kidney disease; gut leakage; gut-derived uremic toxins; probiotics.