A highly conserved multisubunit enzymic complex, SWI/SNF, participates in the regulation of eukaryote gene expression through its ability to remodel chromatin. While a single component of SWI/SNF, Swi2 or a related protein, can perform this function in vitro, the other components appear to modulate the activity and specificity of the complex in vivo. Here we describe the cloning of hELD/OSA1, a 189KDa human homologue of Drosophila Eld/Osa protein, a constituent of Drosophila SWI/SNF. By comparing conserved peptide sequences in Eld/Osa homologues we define three domains common to all family members. A putative DNA binding domain, or ARID (AT-rich DNA-interacting domain), may function in targetting SWI/SNF to chromatin. Two other domains unique to Eld/Osa proteins, EHD1 and EHD2, map to the C-teminus. We show that EHD2 mediates binding to Brahma-related gene 1 (BRG1), a human homologue of yeast Swi2. EHD1 and EHD2 also appear capable of interacting with each other. Using an antibody raised against EHD2 of hELD/OSA1, we detected Eld/Osa1 in endogenous SWI/SNF complexes derived from mouse brain.
Intracerebral haemorrhage (ICH) is a devastating condition with limited treatment options, and current understanding of pathophysiology is incomplete. Spontaneous cerebral bleeding is a characteristic of the human condition that has proven difficult to recapitulate in existing pre-clinical rodent models. Zebrafish larvae are frequently used as vertebrate disease models and are associated with several advantages, including high fecundity, optical translucency and non-protected status prior to 5 days post-fertilisation. Furthermore, other groups have shown that zebrafish larvae can exhibit spontaneous ICH. The aim of this study was to investigate whether such models can be utilised to study the pathological consequences of bleeding in the brain, in the context of pre-clinical ICH research. Here, we compared existing genetic (bubblehead) and chemically inducible (atorvastatin) zebrafish larval models of spontaneous ICH and studied the subsequent disease processes. Through live, non-invasive imaging of transgenic fluorescent reporter lines and behavioural assessment we quantified brain injury, locomotor function and neuroinflammation following ICH. We show that ICH in both zebrafish larval models is comparable in timing, frequency and location. ICH results in increased brain cell death and a persistent locomotor deficit. Additionally, in haemorrhaged larvae we observed a significant increase in macrophage recruitment to the site of injury. Live in vivo imaging allowed us to track active macrophage-based phagocytosis of dying brain cells 24 hours after haemorrhage. Morphological analyses and quantification indicated that an increase in overall macrophage activation occurs in the haemorrhaged brain. Our study shows that in zebrafish larvae, bleeding in the brain induces quantifiable phenotypic outcomes that mimic key features of human ICH. We hope that this methodology will enable the pre-clinical ICH community to adopt the zebrafish larval model as an alternative to rodents, supporting future high throughput drug screening and as a complementary approach to elucidating crucial mechanisms associated with ICH pathophysiology.
Application of tumor immunotherapy has achieved an unprecedented success in patients with hematopoietic malignancies. However, it remains challenging to treat the majority of solid tumors with immunotherapy due to low lymphocyte infiltration, treatment resistance, and immunosuppressive tumor microenvironment. Improving the clinical efficacy of immunotherapy in solid tumors requires a deeper understanding of immune evasion. Neuroblastoma is the most common extracranial tumor in children and is responsible for ~15% of childhood cancer-related deaths. MYCN is a member of the MYC oncogene family, and its aberrant expression in neuroblastoma predicts aggressive disease and poor prognosis. This largely results from the low infiltration rates of cytotoxic lymphocytes in MYCN-amplified neuroblastoma. Elucidation of the mechanism by which MYCN suppresses antitumor response in the neuroblastoma microenvironment will provide useful information to guide immunotherapy. Here we generated a compound transgenic zebrafish, Tg(dβh:MYCN;dβh:EGFP;lck:EGFP;CD4:mCherry), in which lymphocytes and tumor cells are differentially fluorescently labeled. We first monitored the infiltration of mCherry-labeled CD4+ T cells in EGFP-positive MYCN-overexpressing premalignant neural crests at 7, 14, and 21 days post fertilization (dpf). We found that at day 14 and 21, MYCN-overexpressing premalignant neural crests can attract a significantly higher number of lymphocytes, compared to control neural crests without MYCN overexpression. Interestingly, we also found the increased number of infiltrated T cells was MYCN dose dependent by using low MYCN-expressing transgenic fish, Tg(dβh:MYCN;dβh:mCherry;lck:EGFP). To determine if T-cell development is impacted, we imaged the thymus of MYCN-overexpressing or control fish at day 7, 14, and 21 dpf. We found that fluorescent intensity of thymus is significantly higher in MYCN-overexpressing fish compared to control fish. Taken together, our study identified MYCN-overexpressing premalignant neural crests can not only affect T-cell development but also attract T cells to disease site. We will perform further experiments to understand how and why MYCN-overexpressing premalignant neural crests impact T-cell development and which population of T cells is attracted to disease site. Citation Format: Xiaodan Qin, Andrew Lam, Xike Zhang, Adam Hurlstone, Hui Feng. Exploiting the zebrafish to study lymphocyte infiltration in MYCN-driven neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B12.
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