Nao Nitta

Role of CC chemokine receptor 2 in bone marrow cells in the recruitment of macrophages into obese adipose tissue.

The MCP-1 (monocyte chemoattractant protein-1)/CCR2 (CC motif chemokine receptor-2) pathway may play a role in macrophage infiltration into obese adipose tissue. Here we investigated the role of CCR2 in the recruitment of bone marrow-derived macrophages into obese adipose tissue in vitro and in vivo. Using the TAXIScan device, which can measure quantitatively the directionality and velocity of cell migration at time lapse intervals in vitro, we demonstrated that bone marrow cells (BMCs) from wild type mice migrate directly toward MCP-1 or culture medium conditioned by adipose tissue explants of genetically obese ob/ob mice, which are efficiently suppressed by pharmacological blockade of CCR2 signaling. The number of F4/80-positive macrophages was reduced in the adipose tissue from high fat diet-fed obese KKAy or ob/ob mice treated with a CCR2 antagonist propagermanium relative to vehicle-treated groups. We also found that the number of macrophages is reduced in the adipose tissue from ob/ob mice reconstituted with CCR2-/- BMCs (ob/ob + CCR2-/- BMCs) relative to those with CCR2+/+ BMCs (ob/ob + CCR2+/+ BMCs). Expression of mRNAs for CD11c and TLR4 (Toll-like receptor 4) markers of proinflammatory M1 macrophages was also decreased in the adipose tissue from ob/ob + CCR2-/- BMCs relative to ob/ob + CCR2+/+ BMCs, whereas mannose receptor and CD163, markers of anti-inflammatory M2 macrophages, were unchanged. This study provides in vivo and in vitro evidence that CCR2 in bone marrow cells plays an important role in the recruitment of macrophages into obese adipose tissue.

Differential Regulatory Function of Resting and Preactivated Allergen-Specific CD4+CD25+ Regulatory T Cells in Th2-Type Airway Inflammation

Although CD4+CD25+ regulatory T (Treg) cells are known to suppress Th1 cell-mediated immune responses, their effect on Th2-type immune responses remains unclear. In this study we examined the role of Treg cells in Th2-type airway inflammation in mice. Depletion and reconstitution experiments demonstrated that the Treg cells of naive mice effectively suppressed the initiation and development of Th2-driven airway inflammation. Despite effective suppression of Th2-type airway inflammation in naive mice, adoptively transferred, allergen-specific Treg cells were unable to suppress airway inflammation in allergen-presensitized mice. Preactivated allergen-specific Treg cells, however, could suppress airway inflammation even in allergen-presensitized mice by accumulating in the lung, where they reduced the accumulation and proliferation of Th2 cells. Upon activation, allergen-specific Treg cells up-regulated CCR4, exhibited enhanced chemotactic responses to CCR4 ligands, and suppressed the proliferation of and cytokine production by polarized Th2 cells. Collectively, these results demonstrated that Treg cells are capable of suppressing Th2-driven airway inflammation even in allergen-presensitized mice in a manner dependent on their efficient migration into the inflammatory site and their regulation of Th2 cell activation and proliferation.

Autonomous Biomolecular Computer Modeled after Retroviral Replication

We designed a retroviral computer, of which hardware is composed of DNA/RNA dependent DNA polymerase, transcriptase, RNaseH, and DNA and RNA strands. Sequences of DNA strands define functions and RNA single strands work as arguments and return values for each function. In this paper, we show that computational jobs, such as encoding of input data and AND/OR operation, can work on this computer. By combining multiple functions, more complex molecular programs for gene analysis can be constructed. Experimental study showed that some functions were actually executed in vitro autonomously. Since this computer has originally derived from the retrovirus mechanism, we expect an in vivo computer will be realized from this technology, which detects the cell state through gene expression patterns, and controls the cell conditions with output RNA. It may provide a powerful tool for both research and clinical application.

Image analysis of mast cell degranulation in a concentration gradient of stimuli formed in the channel between a glass plate and a silicon substrate.

Measurement of released granule components, popularly used to quantify mast cell exocytosis, does not deliver real-time information about degranulation at the single-cell level nor the ratio of responding/non-responding cells. Rather it provides, only end-point, bulk-population data. Here we studied degranulation of rat peritoneal mast cells dispersed in a narrow horizontal channel between a silicon substrate and a glass plate. Upon exposure to a concentration gradient of a soluble stimulus, degranulation started from those cells facing towards the highest concentration of stimulus. We captured images of exocytosing cells without the need for phase-contrast or differential interference-contrast microscopy. This was achieved using the reflection caused by the silicon substrate. The time-lapse images of cells in the channel were segmented into multiple concentration belts to identify the proportion of degranulated cells in each belt region. Maximum ratios of degranulated cells in the belt regions determined by time-course curve fitting calculations were then plotted against the distance from the stimulus injection site, resulting in a sigmoidal response curve. This method provides a powerful means for real-time analysis of concentration- and stimulus-dependent degranulation of mast cells and allows comparison of cell responses under different conditions. To show its effectiveness, we evaluated the effect of a protein kinase C (PKC) inhibitor, Gö6976, on degranulation induced by various stimuli. In contrast to stimulation with concanavalin A+lysophosphatidylserine (lysoPS) or nerve growth factor+lysoPS (completely inhibited by Gö6976 over the whole range of stimulus concentrations used) or compound 48/80 and mastoparan (no inhibition by Gö6976), stimulation with ionomycin, a known Ca(2+) ionophore, showed a concentration-dependent inhibition by Gö6976, with a major inhibition at low stimulus concentrations and a diminished one at higher ionomycin concentrations. The results indicate that ionomycin-induced degranulation is mainly induced via a PKC-independent signal cascade at high stimulus concentrations, whereas below a certain concentration, degranulation is completely dependent on PKC.

Retrovirus-based computer

A practical intra-cellular biomolecular computer should work inside living cells without damaging them. The retrovirus-based biomolecular computer has been designed, by mimicking the natural processes of living cells, to enable the creation of a practical intra-cellular computer. Here, I review the idea of retrovirus-based computing, and examine its feasibility. This idea might lead to the development of new technologies not only for biological research, but also for medical purposes. In future, through the development of the in vivo retrovirus-based computer, gene diagnosis and gene therapy is expected to cooperatively work inside living cells, enabling intelligent gene therapy technology that uses biomolecular computing.

Single-Cell Analysis of Morphological and Metabolic Heterogeneity in Euglena gracilis by Fluorescence-Imaging Flow Cytometry

Microalgal biofuels and biomass have ecofriendly advantages as feedstocks. Improved understanding and utilization of microalgae require large-scale analysis of the morphological and metabolic heterogeneity within populations. Here, with Euglena gracilis as a model microalgal species, we evaluate how fluorescence- and brightfield-derived-image-based descriptors vary during environmental stress at the single-cell level. This is achieved with a new multiparameter fluorescence-imaging cytometric technique that allows the assaying of thousands of cells per experiment. We track morphological changes, including the intensity and distribution of intracellular lipid droplets, and pigment autofluorescence. The combined fluorescence-morphological analysis identifies new metrics not accessible with traditional flow cytometry, including the lipid-to-cell-area ratio (LCAR), which shows promise as an indicator of oil productivity per biomass. Single-cell metrics of lipid productivity were highly correlated ( R2 > 0.90, p < 0.005) with bulk oil extraction. Such chemomorphological atlases of algal species can help optimize growth conditions and selection approaches for large-scale biomass production.

Intelligent Image-Activated Cell Sorting

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.