Revealing mitf functions and visualizing allografted tumor metastasis in colorless and immunodeficient Xenopus tropicalis.

Transparent immunodeficient animal models not only enhance in vivo imaging investigations of visceral organ development but also facilitate in vivo tracking of transplanted tumor cells. However, at present, transparent and immunodeficient animal models are confined to zebrafish, presenting substantial challenges for real-time, in vivo imaging studies addressing specific biological inquiries. Here, we employed a mitf-/-/prkdc-/-/il2rg-/- triple-knockout strategy to establish a colorless and immunodeficient amphibian model of Xenopus tropicalis. By disrupting the mitf gene, we observed the loss of melanophores, xanthophores, and granular glands in Xenopus tropicalis. Through the endogenous mitf promoter to drive BRAFV600E expression, we confirmed mitf expression in melanophores, xanthophores and granular glands. Moreover, the reconstruction of the disrupted site effectively reinstated melanophores, xanthophores, and granular glands, further highlighting the crucial role of mitf as a regulator in their development. By crossing mitf-/- frogs with prkdc-/-/il2rg-/- frogs, we generated a mitf-/-/prkdc-/-/il2rg-/- Xenopus tropicalis line, providing a colorless and immunodeficient amphibian model. Utilizing this model, we successfully observed intravital metastases of allotransplanted xanthophoromas and migrations of allotransplanted melanomas. Overall, colorless and immunodeficient Xenopus tropicalis holds great promise as a valuable platform for tumorous and developmental biology research.

Development and validation of a disulfidptosis and disulfide metabolism-related risk index for predicting prognosis in lung adenocarcinoma.

BACKGROUND: Disulfidptosis is a recently proposed novel cell death mode in which cells with high SLC7A11 expression induce disulfide stress and cell death in response to glucose deficiency. The purpose of the research was to explore the function of disufidptosis and disulfide metabolism in the progression of lung adenocarcinoma (LUAD). METHODS: The RNA-seq data from TCGA were divided into high/low expression group on the base of the median expression of SLC7A11, and the characteristic of differentially expressed disulfide metabolism-related genes. Least absolute shrinkage and selection operator (LASSO) algorithm was conducted the disulfidptosis and disulfide metabolism risk index. The tumor mutation burden (TMB), mechanism, pathways, tumor microenvironment (TME), and immunotherapy response were assessed between different risk groups. The role of TXNRD1 in LUAD was investigated by cytological experiments. RESULTS: We established the risk index containing 5 genes. There are significant differences between different risk groups in terms of prognosis, TMB and tumor microenvironment. Additionally, the low-risk group demonstrated a higher rate of response immunotherapy in the prediction of immunotherapy response. Experimental validation suggested that the knockdown of TXNRD1 suppressed cell proliferation, migration, and invasion of LUAD. CONCLUSION: Our research highlights the enormous potential of disulfidptosis and disulfide metabolism risk index in predicting the prognosis of LUAD. And TXNRD1 has great clinical translational ability.

DDX24 Is Essential for Cell Cycle Regulation in Vascular Smooth Muscle Cells During Vascular Development via Binding to FANCA mRNA.

BACKGROUND: The DEAD-box family is essential for tumorigenesis and embryogenesis. Previously, we linked the malfunction of DDX (DEAD-box RNA helicase)-24 to a special type of vascular malformation. Here, we aim to investigate the function of DDX24 in vascular smooth muscle cells (VSMCs) and embryonic vascular development. METHODS: Cardiomyocyte (CMC) and VSMC-specific Ddx24 knockout mice were generated by crossing Tagln-Cre mice with Ddx24flox/flox transgenic mice. The development of blood vessels was explored by stereomicroscope photography and immunofluorescence staining. Flow cytometry and cell proliferation assays were used to verify the regulation of DDX24 on the function of VSMCs. RNA sequencing and RNA immunoprecipitation coupled with quantitative real-time polymerase chain reaction were combined to investigate DDX24 downstream regulatory molecules. RNA pull-down and RNA stability experiments were performed to explore the regulation mechanism of DDX24. RESULTS: CMC/VSMC-specific Ddx24 knockout mice died before embryonic day 13.5 with defects in vessel formation and abnormal vascular remodeling in extraembryonic tissues. Ddx24 knockdown suppressed VSMC proliferation via cell cycle arrest, likely due to increased DNA damage. DDX24 protein bound to and stabilized the mRNA of FANCA (FA complementation group A) that responded to DNA damage. Consistent with the function of DDX24, depletion of FANCA also impacted cell cycle and DNA repair of VSMCs. Overexpression of FANCA was able to rescue the alterations caused by DDX24 deficiency. CONCLUSIONS: Our study unveiled a critical role of DDX24 in VSMC-mediated vascular development, highlighting a potential therapeutic target for VSMC-related pathological conditions.

Preparation of a shared chip for cell capturing and imaging with nanoparticle scaffold, nanogap and circulating tumor cell detection.

Aim: To achieve accurate detection of circulating tumor cells (CTCs) in a large-volume sample. Materials & methods: Silica nanoparticles were crosslinked layer-by-layer on glass slides as the substrate of a chip using polyacrylic acid. Polyacrylic acid was immobilized as a spacer and capture ligands were immobilized on the spacer. Results: The chip can be integrally applied to capture, post-treatment and imaging detection for CTCs. The detected cell numbers were 33 and 40 for 9 cell/ml samples and clinical blood samples (7.5 ml), respectively. The detection ratio of positive samples was 100%. Conclusion: The significantly increased detected number for CTCs indicates that this methodology may avoid or greatly reduce the false-negative ratio of positive clinical samples.

Construction and validation of a T cell proliferation regulator-related signature for predicting prognosis and immunotherapy response in lung adenocarcinoma.

Background: As the main executor of immunotherapy, T cells significantly affect the efficacy of immunotherapy. However, the contribution of the T cell proliferation regulator to the prognosis of lung adenocarcinoma (LUAD) and immunotherapy is still unclear. Methods: Based on T cell proliferation regulators, LUAD samples from The Cancer Genome Atlas (TCGA) were divided into two different clusters by consensus clustering. Subsequently, the T cell proliferation regulator (TPR) signature was constructed according to the prognostic T cell proliferation regulators. Combined with clinical information, a nomogram for clinical practice was constructed. The predictive ability of the signature was verified by the additional Gene Expression Omnibus (GEO) dataset. We also analyzed the differences of tumor microenvironment (TME) in different subgroups and predicted the response to immunotherapy according to the TIDE algorithm. Finally, we further explored the role of ADA (Adenosine deaminase) in the lung adenocarcinoma cell lines through the knockdown of ADA. Results: According to the consensus clustering, there were differences in survival and tumor microenvironment between two different molecular subtypes. T cell proliferation regulator-related signature could accurately predict the prognosis of LUAD. The low-risk group had a higher level of immune infiltration and more abundant immune-related pathways, and its response to immunotherapy was significantly better than the high-risk group (Chi-square test, p<0.0001). The knockdown of ADA inhibited proliferation, migration, and invasion in lung adenocarcinoma cell lines. Conclusion: T cell proliferation regulators were closely related to the prognosis and tumor microenvironment of LUAD patients. And the signature could well predict the prognosis of LUAD patients and their response to immunotherapy. ADA may become a new target for the treatment of LUAD.

Novel Monomethoxy Poly(Ethylene Glycol) Modified Hydroxylated Tung Oil for Drug Delivery.

Novel monomethoxy poly(ethylene glycol) (mPEG) modified hydroxylated tung oil (HTO), denoted as mPEG-HTO-mPEG, was designed and synthesized for drug delivery. mPEG-HTO-mPEG consists of a hydroxylated tung oil center joined by two mPEG blocks via a urethane linkage. The properties of mPEG-HTO-mPEG were affected by the length of the mPEG chain. Three mPEG with different molecular weights were used to prepare mPEG-HTO-mPEG. The obtained three mPEG-HTO-mPEG polymers were characterized by nuclear magnetic resonance (NMR), Fourier transformation infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and gel permeation chromatography (GPC), respectively. Furthermore, the particle sizes of mPEG-HTO-mPEG micelles were evaluated by dynamic light scattering (DLS) and transmission electron microscope (TEM). A critical aggregation concentration (CAC) ranged from 7.28 to 11.73 mg/L depending on the chain length of mPEG. The drug loading and release behaviors of mPEG-HTO-mPEG were investigated using prednisone acetate as a model drug, and results indicated that hydrophobic prednisone acetate could be effectively loaded into mPEG-HTO-mPEG micelles and exhibited a long-term sustained release. Moreover, compared with HTO, mPEG-HTO-mPEG had no obvious cytotoxicity to HeLa and L929 cells. Therefore, monomethoxy poly(ethylene glycol) modified hydroxylated tung oil mPEG-HTO-mPEG may be a promising drug carrier.

MiR-125b-5p modulates the function of regulatory T cells in tumor microenvironment by targeting TNFR2.

BACKGROUND: Tumor necrosis factor receptor type 2 (TNFR2) is primarily expressed by CD4+FoxP3+ regulatory T cells (Tregs), especially those present in tumor microenvironment. There is compelling evidence that TNFR2 plays a crucial role in the activation, expansion, and phenotypic stability of Tregs and promotes tumor immune evasion. Understanding of epigenetic regulation of TNFR2 expression in Tregs may help device a novel strategy in cancer immunotherapy. METHODS: MiR-125b-5p-overexpressing or knockdown murine CD4 T cells and Tregs were constructed, and the effect of miR-125b-5p on Tregs proliferation, suppressive function and TNFR2 expression were examined. In vivo antitumor efficacy of Ago-125b-5p (miR-125b-5p agomir) was evaluated in MC38 tumor bearing mice, and tumor-infiltrating Tregs and CD8+ cytotoxic T lymphocytes (CTLs) were analyzed. RNA-seq analysis was applied to reveal the genes and signaling pathways regulated by miR-125b-5p in Tregs. RESULTS: In this study, we found that TNFR2 was a direct target of miR-125b-5p. Overexpression of miR-125b-5p decreased the proportion of Tregs and their expression of TNFR2 and consequently inhibited its proliferation and suppressive function by regulating the metabolism-related signaling pathways. Moreover, in colon cancer bearing mice, the administration of Ago-125b-5p markedly inhibited the tumor growth, which was associated with reduction of Tregs and increase of IFNγ+CD8+ T cells in tumor environment. Furthermore, in human colon adenocarcinoma patients, we verified that miR-125b-5p expression was downregulated, and low levels of miR-125b-5p were associated with poor prognosis. Interestingly, the expression of miR-125b-5p and TNFR2 were negatively correlated. CONCLUSIONS: Our study for the first time found that the expression of TNFR2 by Tregs was regulated by miR-125b-5p. Our results showed that miR-125b-5p had the capacity to inhibit the expression of TNFR2 and immunosuppressive activity of Tregs and consequently enhanced the antitumor efficacy. This property of miR-125b-5p may be therapeutically harnessed in the treatment of human cancers.

The Impact of Different Rose Bengal Formulations on Corneal Thickness and the Efficacy of Rose Bengal/Green Light Corneal Cross-linking in the Rabbit Eye.

PURPOSE: To examine central corneal thickness (CCT) changes during in vivo rose bengal-green light corneal cross-linking (RG-CXL) and compare the CXL efficacy of different rose bengal formulations. METHODS: After epithelium removal, the right eyes of rabbits were immersed in rose bengal solution prepared by different solvents (water, phosphate buffered saline, dextran, and hydroxypropyl methylcellulos [HPMC]) for 2 or 20 minutes, then the rose bengal distribution in the corneal stroma was analyzed by confocal fluorescence detection. During the RG-CXL process, the CCT was measured at seven time points. The left eyes served as the untreated control group. Corneal enzymatic resistance and corneal biomechanics were tested to compare the RG-CXL efficacy. RESULTS: The rose bengal infiltration depths were 120 and 200 µm for the 2- and 20-minute groups, respectively. CCT increased significantly after infiltration, then decreased significantly in the first 200 seconds of irradiation and decreased slowly for the next 400 seconds. The CCT of the 20-minute groups was significantly thicker than that of the 2-minute groups (P < .0001). All RG-CXL treatments improved the corneal enzymatic resistance and corneal biomechanics, with the effects being greater in the 20-minute groups. The inclusion of 1.1% HPMC in the rose bengal formulation helped to maintain CCT during irradiation while not affecting either the infiltration of rose bengal or the efficacy of RG-CXL. CONCLUSIONS: Within the range studied, RG-CXL efficacy increased with infiltration time. The incorporation of a 20-minute infiltration of 0.1% rose bengal-1.1% HPMC into the RG-CXL procedure may further improve the safety of the treatment and its prospects for clinical use. [J Refract Surg. 2022;38(7):450-458.].

Corrigendum: DDX19A Promotes Metastasis of Cervical Squamous Cell Carcinoma by Inducing NOX1-Mediated ROS Production.

[This corrects the article DOI: 10.3389/fonc.2021.629974.].

RNA Helicase DDX24 Stabilizes LAMB1 to Promote Hepatocellular Carcinoma Progression.

Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies. Elucidating the underlying mechanisms of this disease could provide new therapeutic strategies for treating HCC. Here, we identified a novel role of DEAD-box helicase 24 (DDX24), a member of the DEAD-box protein family, in promoting HCC progression. DDX24 levels were significantly elevated in HCC tissues and were associated with poor prognosis of HCC. Overexpression of DDX24 promoted HCC migration and proliferation in vitro and in vivo, whereas suppression of DDX24 inhibited both functions. Mechanistically, DDX24 bound the mRNA618-624nt of laminin subunit beta 1 (LAMB1) and increased its stability in a manner dependent upon the interaction between nucleolin and the C-terminal region of DDX24. Moreover, regulatory factor X8 (RFX8) was identified as a DDX24 promoter-binding protein that transcriptionally upregulated DDX24 expression. Collectively, these findings demonstrate that the RFX8/DDX24/LAMB1 axis promotes HCC progression, providing potential therapeutic targets for HCC. SIGNIFICANCE: The identification of a tumor-promoting role of DDX24 and the elucidation of the underlying regulatory mechanism provide potential prognostic indicators and therapeutic approaches to help improve the outcome of patients with hepatocellular carcinoma.

Mechanisms of action of triptolide against colorectal cancer: insights from proteomic and phosphoproteomic analyses.

Triptolide is a potent anti-inflammatory agent that also possesses anticancer activity, including against colorectal cancer (CRC), one of the most frequent cancers around the world. In order to clarify how triptolide may be effective against CRC, we analyzed the proteome and phosphoproteome of CRC cell line HCT116 after incubation for 48 h with the drug (40 nM) or vehicle. Tandem mass tagging led to the identification of 403 proteins whose levels increased and 559 whose levels decreased in the presence of triptolide. We also identified 3,110 sites in proteins that were phosphorylated at higher levels and 3,161 sites phosphorylated at lower levels in the presence of the drug. Analysis of these differentially expressed and/or phosphorylated proteins showed that they were enriched in pathways involving ribosome biogenesis, PI3K-Akt signaling, MAPK signaling, nucleic acid binding as well as other pathways. Protein-protein interactions were explored using the STRING database, and we identified nine protein modules and 15 hub proteins. Finally, we identified 57 motifs using motif analysis of phosphosites and found 16 motifs were experimentally verified for known protein kinases, while 41 appear to be novel. These findings may help clarify how triptolide works against CRC and may guide the development of novel treatments.

SARS-CoV-2 spike spurs intestinal inflammation via VEGF production in enterocytes.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can cause gastrointestinal (GI) symptoms that often correlate with the severity of COVID-19. Here, we explored the pathogenesis underlying the intestinal inflammation in COVID-19. Plasma VEGF level was particularly elevated in patients with GI symptoms and significantly correlated with intestinal edema and disease progression. Through an animal model mimicking intestinal inflammation upon stimulation with SARS-CoV-2 spike protein, we further revealed that VEGF was over-produced in the duodenum prior to its ascent in the circulation. Mechanistically, SARS-CoV-2 spike promoted VEGF production through activating the Ras-Raf-MEK-ERK signaling in enterocytes, but not in endothelium, and inducing permeability and inflammation. Blockage of the ERK/VEGF axis was able to rescue vascular permeability and alleviate intestinal inflammation in vivo. These findings provide a mechanistic explanation and therapeutic targets for the GI symptoms of COVID-19.

Engineering Hibiscus-Like Riboflavin/ZIF-8 Microsphere Composites to Enhance Transepithelial Corneal Cross-Linking.

Riboflavin-5-phosphate (RF) is the most commonly used photosensitizer in corneal cross-linking (CXL), but its hydrophilicity and negative charge limit its penetration through the corneal epithelium into the stroma. To enhance the corneal permeability of RF and promote its efficacy in the treatment of keratoconus, novel hibiscus-like RF@ZIF-8 microsphere composites [6RF@ZIF-8 NF (nanoflake)] are prepared using ZIF-8 nanomaterials as carriers, which are characterized by their hydrophobicity, positive potential, biocompatibility, high loading capacities, and large surface areas. Both hematoxylin and eosin endothelial staining and TUNEL assays demonstrate excellent biocompatibility of 6RF@ZIF-8 NF. In in vivo studies, the 6RF@ZIF-8 NF displayed excellent corneal permeation, and outstanding transepithelial CXL (TE-CXL) efficacy, slightly better than the conventional CXL protocol. Furthermore, the special hibiscus-like structures of 6RF@ZIF-8 NF meant that it has better TE-CXL efficacy than that of 6RF@ZIF-8 NP (nanoparticles) due to the larger contact area with the epithelium and the shorter RF release passage. These results suggest that the 6RF@ZIF-8 NF are promising for transepithelial corneal cross-linking, avoiding the need for epithelial debridement.

B cell heterogeneity, plasticity, and functional diversity in cancer microenvironments.

B cells constitute a major component of tumor-infiltrating leukocytes. However, the influence of these cells on malignancy is currently under debate, reflecting the heterogeneity of B cell subsets in tumors. With recent advances, it becomes apparent that this debate includes not only the evaluation of B cells themselves, but also the underlying immune microenvironment network, which scripts the highly heterogeneous B cell populations in tumors and directs the roles of those sub-populations in disease progression and clinical treatment. In this review, we summarize recent findings on the heterogeneous subset composition of B cells in both human and mouse tumor models and their different impacts on disease progression. We further describe the multidimensional interplays between B cells and other immune cells in the tumor microenvironment, which account for the regulation of B cell differentiation and function in situ. We also assess the potential influences of distinct sub-tumor locations on B cell function in primary tumors during development and those under immunotherapy treatment. Illuminating the heterogeneous nature of B cell subset composition, generation, localization, and related immune network in tumor is of immense significance for comprehensively understanding B cell response in tumor and designing more efficacious cancer immunotherapies.

DDX19A Promotes Metastasis of Cervical Squamous Cell Carcinoma by Inducing NOX1-Mediated ROS Production.

The major obstacle to treat cervical squamous cell carcinoma (CSCC) is the high prevalence of metastasis, which severely affects 5-year survival rate and quality of life for cancer patients. The DEAD-box helicase family has been reported to be a critical mediator in the development and metastasis of various cancers. DEAD-box helicase 19A (DDX19A) is a member of the DEAD-box helicase family; however, its functional role in CSCC is unclear. In this study, bioinformatics analysis of clinical samples from public databases demonstrated that the expression of DDX19A was elevated in CSCC tissues and that high expression of DDX19A was positively correlated with metastasis and poor clinical outcome. Functionally, we found that DDX19A promoted CSCC cell migration and invasion in vitro and lung metastasis in vivo. Mechanistically, overexpression of DDX19A increased NADPH oxidase 1 (NOX1) expression, enhanced reactive oxygen species (ROS) production, and induced the migration and invasion of CSCC cells. Rescue experiments revealed that DDX19A-induced CSCC functional alterations were dependent on NOX1 and that DDX19A-promoted CSCC metastasis was abrogated upon the inhibition of ROS. Our results demonstrated that DDX19A could promote CSCC metastasis by inducing NOX1-mediated ROS production and that blockage of the NOX1/ROS axis might serve as a potential therapeutic target for patients with DDX19A-overexpressed CSCC.

Macrophage 3D migration: A potential therapeutic target for inflammation and deleterious progression in diseases.

Macrophages are heterogeneous cells that have different physiological functions, such as chemotaxis, phagocytosis, endocytosis, and secretion of various factors. All physiological functions of macrophages are integral to homeostasis, immune defense and tissue repair. However, in several diseases, macrophages are recruited from the blood towards inflammatory sites. This process is called macrophage migration, which promotes deleterious disease progression. Macrophage migration is a key player in many inflammatory diseases, autoimmune diseases and cancers because it contributes to the accumulation of proinflammatory factors, the destruction of tissues and the development of tumors. Therefore, macrophage migration is proposed to be a potential therapeutic target. Macrophages migrate between two-dimensional (2D) and three-dimensional (3D) environments, implying that distinct migratory features and mechanisms are involved. Compared with the 2D migration of macrophages, 3D migration involves more complex variations in cellular morphology and dynamics. The structure of the extracellular matrix, a key factor, is modified in diseases that influence macrophage 3D migration. Macrophage 3D migration relates to disease pathology. Research that focuses on macrophage 3D migration is an emerging field and was reviewed in this article to indicate the molecular and cellular mechanisms of macrophage migration in 3D environments and to provide potential targets for controlling disease progression associated with this migration.

Macrophage-mediated vascular permeability via VLA4/VCAM1 pathway dictates ascites development in ovarian cancer.

The development of ascites correlates with advanced stage disease and poor prognosis in ovarian cancer. Vascular permeability is the key pathophysiological change involved in ascites development. Previously, we provided evidence that perivascular M2-like macrophages protect the vascular barrier through direct contact with endothelial cells (ECs). Here, we investigated the molecular mechanism and its clinical significance in the ovarian cancer setting. We found that upon direct coculture with the endothelium, M2 macrophages tuned down their VLA4 and reduced the levels of VCAM1 in ECs. On the other hand, ectopically overexpressing VLA4 in macrophages or VCAM1 in ECs induced hyperpermeability. Mechanistically, downregulation of VLA4 or VCAM1 led to reduced levels of RAC1 and ROS, which resulted in decreased phosphorylation of PYK2 (p-PYK2) and VE-cadherin (p-VE-cad), hence enhancing cell adhesion. Furthermore, targeting the VLA4/VCAM1 axis augmented vascular integrity and abrogated ascites formation in vivo. Finally, VLA4 expression on the macrophages isolated from ascites dictated permeability ex vivo. Importantly, VLA4 antibody acted synergistically with bevacizumab to further enhance the vascular barrier. Taking these data together, we reveal here that M2 macrophages regulate the vascular barrier though the VCAM1/RAC1/ROS/p-PYK2/p-VE-cad cascade, which provides specific therapeutic targets for the treatment of malignant ascites.

DDX24 Mutations Associated With Malformations of Major Vessels to the Viscera.

Vascular malformations present diagnostic and treatment challenges. In particular, malformations of vessels to the viscera are often diagnosed late or incorrectly due to the insidious onset and deep location of the disease. Therefore, a better knowledge of the genetic mutations underlying such diseases is needed. Here, we evaluated a four-generation family carrying vascular malformations of major vessels that affect multiple organs, which we named "multiorgan venous and lymphatic defect" (MOVLD) syndrome. Genetic analyses identified an association between a mutation in DEAD-box helicase 24 (DDX24), a gene for which the function is largely unknown, and MOVLD. Next, we screened 161 patients with sporadic vascular malformations of similar phenotype to our MOVLD family and found the same mutation or one of the two additional DDX24 mutations in 26 cases. Structural modeling revealed that two of the mutations are located within the adenosine triphosphate-binding domain of DDX24. Knockdown of DDX24 expression in endothelial cells resulted in elevated migration and tube formation. Transcriptomic analysis linked DDX24 to vascular system-related functions. Conclusion: Our results provide a link between DDX24 and vascular malformation and indicate a crucial role for DDX24 in endothelial cell functions; these findings create an opportunity for genetic diagnosis and therapeutic targeting of malformations of vessels to the viscera.

Madecassoside protects BV2 microglial cells from oxygen-glucose deprivation/reperfusion-induced injury via inhibition of the toll-like receptor 4 signaling pathway.

In a previous study, the authors reported that madecassoside (MA) exerted a potent neuroprotective effect against cerebral ischemia-reperfusion (I/R) injury in rats, mediated by anti-oxidative, anti-inflammatory, and anti-apoptotic mechanisms. However, the cellular and molecular bases for its neuroprotective effects have not been fully elucidated. In this study, an in vitro ischemic model of oxygen-glucose deprivation followed by reperfusion (OGD/R) was used to investigate the role of the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB) pathway in the neuroprotective and anti-inflammatory effects of MA. BV2 microglia viability after OGD/R, treated with or without MA, was measured using the MTT assay. Messenger RNA and protein expression of pro-inflammatory cytokines (tumor necrosis factor α [TNF-α], interleukin-1ß [IL-1ß], interleukin-6 [IL-6]) were measured using real-time polymerase chain reaction (RT-PCR) and ELISA after OGD/R or lipopolysaccharide treatment. Expression of TLR4/MyD88 and NF-κB p65 were measured using RT-PCR, Western blotting, and immunofluorescence analysis. MA significantly rescued OGD/R-induced cytotoxicity in BV2 microglia. Meanwhile, MA suppressed the secretion of pro-inflammatory mediators, including TNF-α, IL-1ß, and IL-6, induced by OGD/R or lipopolysaccharide in BV2 microglia. The mechanism of its neuroprotection and anti-inflammation from OGD/R may involve the inhibition of activation of TLR4 and MyD88 in BV2 microglia, and the blockage of NF-κB p65 nuclear translocation. MA exhibited a significant neuroprotective effect against I/R injury in both in vivo and in vitro experiments by attenuating microglia-mediated neuroinflammation via inhibition of the TLR4/MyD88/NF-κB signaling pathway.

Imaging of Tumor-Associated Macrophages in a Transgenic Mouse Model of Orthotopic Ovarian Cancer.

PURPOSE: Tumor-associated macrophages (TAMs) are often associated with a poor prognosis in cancer. To gain a better understanding of cellular recruitment and dynamics of TAM biology during cancer progression, we established a novel transgenic mouse model for in vivo imaging of luciferase-expressing macrophages. PROCEDURES: B6.129P2-Lyz2tm1(cre)Ifo/J mice, which express Cre recombinase under the control of the lysozyme M promoter (LysM) were crossed to Cre-lox Luc reporter mice (RLG), to produce LysM-LG mice whose macrophages express luciferase. Cell-type-specific luciferase expression in these mice was verified by flow cytometry, and via in vivo bioluminescence imaging under conditions where macrophages were either stimulated with lipopolysaccharide or depleted with clodronate liposomes. The distribution of activated macrophages was longitudinally imaged in two immunocompetent LysM-LG mouse models with either B16 melanoma or ID8 ovarian cancer cells. RESULTS: In vivo imaging of LysM-LG mice showed luciferase activity was generated by macrophages. Clodronate liposome-mediated depletion of macrophages lowered overall bioluminescence while lipopolysaccharide injection increased macrophage bioluminescence in both the B16 and ID8 models. Tracking macrophages weekly in tumor-bearing animals after intraperitoneal (i.p.) or intraovarian (i.o.) injection resulted in distinct, dynamic patterns of macrophage activity. Animals with metastatic ovarian cancer after i.p. injection exhibited significantly higher peritoneal macrophage activity compared to animals after i.o. injection. CONCLUSION: The LysM-LG model allows tracking of macrophage recruitment and activation during disease initiation and progression in a noninvasive manner. This model provides a tool to visualize and monitor the benefit of pharmacological interventions targeting macrophages in preclinical models.