Concentration lock-on microdevices for the investigation of neutrophil chemotaxis

描述（由申请人提供）：中性粒细胞是防止微生物在人体内侵入和传播的最有效屏障。中性粒细胞不能迅速到达感染或炎症部位可导致不可控制的感染，而过度的嗜中性粒细胞浸润可不必要地损害正常组织并损害器官功能，例如严重形式的哮喘和关节炎、急性肝炎或缺血-再灌注损伤。这是我们的长期目标，阐明细胞水平的机制，参与定向传感在中性粒细胞作为一个必要的先决条件，合理的治疗方法，能够微调中性粒细胞反应适当的疾病状态的发展。我们小组以前的观察表明，两种不同的传感机制可能同时负责中性粒细胞的定向迁移，一种是空间的，用于感测细胞周围微环境空间的不对称性，另一种是时间的，用于感测刺激物浓度的时间变化。这项研究的驱动假设是，中性粒细胞协同联合收割机的两个传感机制，空间和时间，优化迁移反应在复杂的环境。检验这一假设的特殊挑战是如何解耦空间和时间分量，知道中性粒细胞在空间化学梯度中的运动导致细胞水平浓度的时间变化，并且扩散空间中趋化因子浓度的任何时间变化也会影响空间梯度。为了应对这一挑战，我们将开发一种计算机控制的微流体装置，该装置将连续改变中性粒细胞移动的条件，以实现消除移动中性粒细胞将经历的时间刺激。这项新技术是独一无二的，因为它是第一个主动趋化装置，能够锁定移动的中性粒细胞目标的化学梯度。该技术也完全不同于传统的被动趋化装置，后者具有相同的梯度，而不管细胞的运动性。通过外部反馈回路将细胞外扰动和细胞反应联系起来，我们将解耦空间和时间刺激的影响，并能够首次识别和测量中性粒细胞感知机制的空间和时间分量的个体贡献。这一新的知识将拓宽我们对动态炎症过程背景下中性粒细胞反应的理解，并在中性粒细胞分子生物学现有知识的背景下，将帮助我们开发预防和治疗感染性和炎症性疾病的新方法。 公共卫生相关性：中性粒细胞如何能够对各种各样的刺激作出反应尚不完全清楚。尽管大多数参与细胞内信号传导的分子已经被鉴定出来，但我们仍然不知道当中性粒细胞响应炎症信号的梯度而移动时，所有这些分子是如何一起工作的。为了更好地在系统水平上研究这些机制，我们将开发一种计算机控制的微型系统，用于在细胞移动时精确刺激细胞。辨别中性粒细胞中的梯度感应机制可以为新药治疗正在产生耐药性的细菌感染开辟可能性，或者避免中性粒细胞在无菌炎症（如哮喘，关节炎或缺血再灌注损伤）期间未经检查可能造成的破坏。

DESCRIPTION (provided by applicant): Neutrophils are the most effective barrier in preventing the invasion and spreading of microorganisms within the human body. Failure of neutrophils to promptly arrive at sites of infection or inflammation can result in uncontrollable infections, while overzealous neutrophilic infiltration can unnecessarily damage normal tissues and impair organ function e.g. in severe forms of asthma and arthritis, acute hepatitis, or ischemia-reperfusion injury. It is our long term goal to elucidate the cellular-level mechanisms involved in directional sensing in neutrophils as a necessary prerequisite to the development of rational therapeutic approaches capable of fine tuning the neutrophil responses appropriate to the disease state. Previous observations from our group have suggested that two distinct sensing mechanisms could be simultaneously responsible for neutrophil directional migration, one spatial, for sensing the asymmetry in space of the micro-environment surrounding the cells, and one temporal, for sensing the changes in time of concentration of the stimuli. The driving hypothesis for this study is that neutrophils synergistically combine the two sensing mechanisms, spatial and temporal, for optimized migratory responses in complex environments. The particular challenge for testing this hypothesis is about how to decouple the spatial and temporal components, knowing that the movement of the neutrophil in spatial chemical gradient results in a temporal change in the concentration at the cell level, and any temporal change of chemokine concentration in a diffusive space also affects the spatial gradients. To address this challenge, we will develop a computer controlled microfluidic device that will continuously altering the conditions in which the neutrophil move to achieve a cancellation of the temporal stimulus that moving neutrophil would otherwise experience. This new technology is unique because it is the first active chemotaxis device, able to lock a chemical gradient on a moving neutrophil target. The technology is also radically different from the traditional, passive chemotaxis devices that have the same gradient, regardless of cell motility. By linking extracellular perturbations and cellular responses through external feed-back loops we will decouple the effects of spatial and temporal stimuli, and be able for the first time to identify and measure the individual contribution of the spatial and temporal components of the neutrophil sensing mechanism. This new knowledge will broaden our understanding of the neutrophil responses in the context of dynamic inflammatory processes and in the context of current knowledge of the molecular biology of the neutrophil will help us develop novel approaches to the prevention and treatment of infectious and inflammatory diseases. PUBLIC HEALTH RELEVANCE: How neutrophils are able to respond to a wide variety of stimuli is not entirely known. Despite the fact that most of the molecules involved in intracellular signaling have been identified, we still do not know how all these molecules work together when neutrophils move in response to gradients of inflammatory signals. To better study these mechanisms at systems level, we will develop a computer controlled microscale system for precise stimulation of the cell while they are moving. Discerning the mechanisms of gradient sensing in neutrophil could open the possibility for new drugs to treat infections with bacteria that are becoming drug resistant, or avoid the destruction neutrophils could cause if unchecked during sterile inflammation like asthma, arthritis, or ischemia-reperfusion injury.