Cell-Surface Interactions in Pathogenesis

我们在这个项目中的主要研究目标是表征细胞与细胞外基质的相互作用以及外源添加的细胞外分子在发病机制中的作用。一个主要的焦点是肿瘤细胞的侵袭和转移，目标是确定新的分子调节因子和介体。癌症生物学中的一个基本问题是局部基质环境与癌症进展的关系。例如，晚期癌症的一个共同特征是在结缔组织发育过程中诱导肿瘤周围致密的胶原质基质。在完成了致密纤维胶原在诱导蛋白分解活性侵袭足细胞中的作用的研究后，我们转向了基质基质物理刚性的变化如何改变各种人类肿瘤细胞系细胞迁移的性质的问题。为了量化细胞迁移的各种参数，开发了一种基于计算机的无偏倚自动系统，该系统使用视频荧光显微镜跟踪各种肿瘤细胞系的Hoechst标记的核的算法，以提供快速量化的速度、持久性和趋化性迁移。这个名为“FastTrack”的程序是用MatLab软件开发的；然而，在最终发布后，它可以在没有专有软件的情况下下载到普通计算机上使用。 上皮-间充质转化(EMT)可能促进肿瘤细胞的侵袭性。在对正常组织形态发生的研究中，我们发现新的调控因子Btbd7是EMT转录因子Snail2的诱导者和E-cadherin介导的细胞-细胞黏附的干扰物。我们目前正在评估Btbd7或其上游细胞外基质调节因子纤维连接蛋白是否在某些肿瘤细胞的侵袭性中发挥作用。 细胞表面与可溶性或基质因子的分子相互作用可能在许多疾病中发挥重要作用。我们继续与FDA CBER的Subhash Dhawan博士长期合作，以确定与传染病的发病或抑制有关的细胞-表面和细胞外相互作用。一些分子被发现可以增强传染性，而另一些分子则可以抑制这一过程。例如，宿主细胞对细胞外提供的氯化血红素的反应，是血红素加氧酶-1(HO-1)的诱导剂，引起宿主对包括艾滋病毒和痘病毒在内的病毒感染的保护反应。这一合作项目最近被扩展到探索和记录诱导HO-1介导的宿主反应以抑制埃博拉和寨卡病毒感染的有效性。由于氯化血红素是FDA批准的药物，它对HO-1的诱导可能提供一种潜在的针对各种病原体的一般宿主防御治疗策略。 具体地说，我们证实寨卡病毒可以感染原代人类单核细胞来源的巨噬细胞。氯化血红素在体外显著降低了寨卡病毒的复制。氯化高铁血红素在人巨噬细胞和LLC-MK2猴肾细胞中诱导HO-1表达，并使寨卡病毒复制显著减少90%，毒性最小。RNAi沉默HO-1或其上游调控基因Nrf2的表达，减弱氯化血红素诱导的寨卡病毒感染抑制。氯化血红素是FDA批准的一种治疗无关疾病(门静脉症)的药物，基于其刺激针对寨卡病毒感染的固有细胞反应的能力，可能提供一种有用的治疗方法。 在一个从FDA延伸到弗雷德里克的美国陆军传染病医学研究所的分子和翻译科学部以及霍华德大学医学系的多站点合作中，我们测试了氯化血红素是否可以通过诱导HO-1来抑制埃博拉病毒的复制。不仅治疗原代单核细胞来源的巨噬细胞(MDM)，而且治疗Vero细胞、HeLa细胞和人包皮成纤维细胞，使埃博拉病毒感染减少了90%，对感染细胞的毒性最小。HO-1酶活性的抑制和HO-1表达的RNAi沉默阻止了这种由氯化血红素介导的对埃博拉病毒感染的抑制。因此，重新调整氯化血红素的用途以刺激固有的HO-1细胞反应可能提供一种帮助缓解埃博拉病毒感染的替代方法。 由于我们在体外和器官移植中的细胞行为实时成像方面拥有丰富的专业知识，我们已与Ashok Kulkarni博士的实验室展开合作，使用GCaMP6小鼠系统直接可视化钙信号，以表征小鼠三叉神经节中的疼痛信号。

Our major research goals in this project are to characterize the roles of cell interactions with the extracellular matrix and exogenously added extracellular molecules in pathogenesis. One major focus is on tumor cell invasion and metastasis, with a goal of identifying novel molecular regulators and mediators. A fundamental question in cancer biology is the relationship of the local matrix environment to cancer progression. For example, a common feature of advanced carcinomas is the induction of a dense collagenous matrix surrounding tumors during the process of desmoplasia. After completing studies establishing the role of dense fibrillar collagen in induction of proteolytically active invadopodia, we have turned to the question of how alterations in the physical rigidity of the matrix substrate can alter the nature of cell migration by a variety of human tumor cell lines. For quantifying a variety of parameters of cell migration, a non-biased, automated computer-based system has been developed that uses algorithms for video fluorescence microscopy tracking of Hoechst-labeled nuclei of a variety of tumor cell lines to provide rapid quantification of speed, persistence, and durotactic migration. This program termed "FastTracks" was developed using MATLAB software; after final publication, however, it can be downloaded for use on regular computers without proprietary software. Tumor cell invasiveness may be promoted by epithelial-mesenchymal transition (EMT). In studies of normal tissue morphogenesis, we identified the novel regulator Btbd7 as an inducer of the EMT transcription factor Snail2 and a disruptor of E-cadherin mediated cell-cell adhesion. We are currently evaluating whether Btbd7 or fibronectin, its upstream extracellular matrix regulator, can play a role in the invasiveness of certain tumor cells. Molecular interactions at the cell surface with soluble or matrix factors are likely to play important roles in many diseases. We have continued a long-term collaboration with Dr. Subhash Dhawan in CBER, FDA to characterize cell-surface and extracellular interactions involved in the pathogenesis or suppression of infectious diseases. Some molecules are found to enhance infectivity, while others can suppress this process. For example, the host cellular response to extracellularly provided hemin, an inducer of heme oxygenase-1 (HO-1), elicits host protective responses against viral infections including HIV and poxvirus. This collaborative project has recently been extended to explore and to document the effectiveness of inducing HO-1 mediated host responses to suppress Ebola and Zika virus infections. Since hemin is an FDA-approved drug, its induction of HO-1 might provide a potential general host-defense therapeutic strategy against a variety of pathogens. Specifically, we established that Zika virus can infect primary human monocyte-derived macrophages. Hemin dramatically reduced Zika virus replication in vitro. Hemin induced HO-1 expression and produced major reductions of >90% in Zika virus replication in human macrophages and LLC-MK2 monkey kidney cells with minimal toxicity. RNAi silencing of the expression of HO-1, or of its upstream regulatory gene Nrf2, attenuated hemin-induced suppression of Zika virus infection. Hemin, an FDA-approved drug for an unrelated disease (porphyria) may provide a useful therapeutic approach based on its ability to stimulate an innate cellular response against Zika virus infection. In a multi-site collaboration that extended beyond the FDA to include the Molecular and Translational Sciences Division of the United States Army Medical Research Institute of Infectious Diseases at Frederick and the Department of Medicine at Howard University, we tested whether hemin could inhibit Ebola virus replication through induction of HO-1. Treatment of not only primary monocyte-derived macrophages (MDM), but also Vero cells, HeLa cells, and human foreskin fibroblasts, reduced Ebola virus infection by >90% with minimal toxicity to infected cells. Inhibition of HO-1 enzymatic activity and RNAi silencing of HO-1 expression prevented this hemin-mediated suppression of Ebola virus infection. Consequently, re-purposing hemin to stimulate the innate HO-1 cellular response may provide an alternative approach to help alleviate Ebola virus infection. Because we have extensive expertise in real-time imaging of cell behavior in vitro and in organ explants, we have initiated a collaboration with Dr. Ashok Kulkarni's laboratory to use the GCaMP6 mouse system for direct visualization of calcium signaling for characterizing pain signaling in the mouse trigeminal ganglion.