Control and Manipulation of Pathogens with an Optical Trap for Live Cell Imaging of Intercellular Interactions

The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.National Institute of Biomedical Imaging and Bioengineering (U.S.) (grant T32EB006348)Massachusetts General Hospital (Department of Medicine Internal Funds)Center for Computational and Integrative Biology (Development fund)Center for Computational and Integrative Biology (AI062773)Center for Computational and Integrative Biology (grant AI062773)Center for Computational and Integrative Biology (grant DK83756)Center for Computational and Integrative Biology (grant DK 043351)National Institute of Allergy and Infectious Diseases (U.S.)National Institutes of Health (U.S.) (grant AI057999

Activated MCTC mast cells infiltrate diseased lung areas in cystic fibrosis and idiopathic pulmonary fibrosis

<p>Abstract</p> <p>Background</p> <p>Although mast cells are regarded as important regulators of inflammation and tissue remodelling, their role in cystic fibrosis (CF) and idiopathic pulmonary fibrosis (IPF) has remained less studied. This study investigates the densities and phenotypes of mast cell populations in multiple lung compartments from patients with CF, IPF and never smoking controls.</p> <p>Methods</p> <p>Small airways, pulmonary vessels, and lung parenchyma were subjected to detailed immunohistochemical analyses using lungs from patients with CF (20 lung regions; 5 patients), IPF (21 regions; 7 patients) and controls (16 regions; 8 subjects). In each compartment the densities and distribution of MC_Tand MC_TCmast cell populations were studied as well as the mast cell expression of IL-6 and TGF-β.</p> <p>Results</p> <p>In the alveolar parenchyma in lungs from patients with CF, MC_TCnumbers increased in areas showing cellular inflammation or fibrosis compared to controls. Apart from an altered balance between MC_TCand MC_Tcells, mast cell in CF lungs showed elevated expression of IL-6. In CF, a decrease in total mast cell numbers was observed in small airways and pulmonary vessels. In patients with IPF, a significantly elevated MC_TCdensity was present in fibrotic areas of the alveolar parenchyma with increased mast cell expression of TGF-β. The total mast cell density was unchanged in small airways and decreased in pulmonary vessels in IPF. Both the density, as well as the percentage, of MC_TCcorrelated positively with the degree of fibrosis. The increased density of MC_TC, as well as MC_TCexpression of TGF-β, correlated negatively with patient lung function.</p> <p>Conclusions</p> <p>The present study reveals that altered mast cell populations, with increased numbers of MC_TCin diseased alveolar parenchyma, represents a significant component of the histopathology in CF and IPF. The mast cell alterations correlated to the degree of tissue remodelling and to lung function parameters. Further investigations of mast cells in these diseases may open for new therapeutic strategies.</p

Mechanisms and mechanics of cell competition in epithelia

When fast-growing cells are confronted with slow-growing cells in a mosaic tissue, the slow-growing cells are often progressively eliminated by apoptosis through a process known as cell competition. The underlying signalling pathways remain unknown, but recent findings have shown that cell crowding within an epithelium leads to the eviction of cells from the epithelial sheet. This suggests that mechanical forces could contribute to cell elimination during cell competition

A systematic review of the role of vitamin insufficiencies and supplementation in COPD

<p>Abstract</p> <p>Background</p> <p>Pulmonary inflammation, oxidants-antioxidants imbalance, as well as innate and adaptive immunity have been proposed as playing a key role in the development of COPD. The role of vitamins, as assessed either by food frequency questionnaires or measured in serum levels, have been reported to improve pulmonary function, reduce exacerbations and improve symptoms. Vitamin supplements have therefore been proposed to be a potentially useful additive to COPD therapy.</p> <p>Methods</p> <p>A systematic literature review was performed on the association of vitamins and COPD. The role of vitamin supplements in COPD was then evaluated.</p> <p>Conclusions</p> <p>The results of this review showed that various vitamins (vitamin C, D, E, A, beta and alpha carotene) are associated with improvement in features of COPD such as symptoms, exacerbations and pulmonary function. High vitamin intake would probably reduce the annual decline of FEV1. There were no studies that showed benefit from vitamin supplementation in improved symptoms, decreased hospitalization or pulmonary function.</p

Paxillin and Hic-5 Interaction with Vinculin Is Differentially Regulated by Rac1 and RhoA

Cell migration is of paramount importance to organism development and maintenance as well as multiple pathological processes, including cancer metastasis. The RhoGTPases Rac1 and RhoA are indispensable for cell migration as they regulate cell protrusion, cell-extracellular matrix (ECM) interactions and force transduction. However, the consequences of their activity at a molecular level within the cell remain undetermined. Using a combination of FRET, FRAP and biochemical analyses we show that the interactions between the focal adhesion proteins vinculin and paxillin, as well as the closely related family member Hic-5 are spatially and reciprocally regulated by the activity of Rac1 and RhoA. Vinculin in its active conformation interacts with either paxillin or Hic-5 in adhesions in response to Rac1 and RhoA activation respectively, while inactive vinculin interacts with paxillin in the membrane following Rac1 inhibition. Additionally, Rac1 specifically regulates the dynamics of paxillin as well as its binding partner and F-actin interacting protein actopaxin (α-parvin) in adhesions. Furthermore, FRET analysis of protein:protein interactions within cell adhesions formed in 3D matrices revealed that, in contrast to 2D systems vinculin interacts preferentially with Hic-5. This study provides new insight into the complexity of cell-ECM adhesions in both 2D and 3D matrices by providing the first description of RhoGTPase-coordinated protein:protein interactions in a cellular microenvironment. These data identify discrete roles for paxillin and Hic-5 in Rac1 and RhoA-dependent cell adhesion formation and maturation; processes essential for productive cell migration

Role of TNFα in pulmonary pathophysiology

Tumor necrosis factor alpha (TNFα) is the most widely studied pleiotropic cytokine of the TNF superfamily. In pathophysiological conditions, generation of TNFα at high levels leads to the development of inflammatory responses that are hallmarks of many diseases. Of the various pulmonary diseases, TNFα is implicated in asthma, chronic bronchitis (CB), chronic obstructive pulmonary disease (COPD), acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). In addition to its underlying role in the inflammatory events, there is increasing evidence for involvement of TNFα in the cytotoxicity. Thus, pharmacological agents that can either suppress the production of TNFα or block its biological actions may have potential therapeutic value against a wide variety of diseases. Despite some immunological side effects, anti-TNFα therapeutic strategies represent an important breakthrough in the treatment of inflammatory diseases and may have a role in pulmonary diseases characterized by inflammation and cell death