Novel process of intrathymic tumor-immune tolerance through CCR2-mediated recruitment of Sirpα+ dendritic cells: a murine model

Immune surveillance system can detect more efficiently secretory tumor-specific antigens, which are superior as a target for cancer immunotherapy. On the contrary, immune tolerance can be induced in the thymus when a tumor antigen is massively secreted into circulation. Thus, the secretion of tumor-specific antigen may have contradictory roles in tumor immunity in a context-dependent manner. However, it remains elusive on the precise cellular mechanism of intrathymic immune tolerance against tumor antigens. We previously demonstrated that a minor thymic conventional dendritic cell (cDC) subset, CD8α(-)Sirpα(+) cDCs, but not the major subset, CD8α(+)Sirpα(-) cDCs can selectively capture blood-borne antigens and crucially contribute to the self-tolerance. In the present study, we further demonstrated that Sirpα(+) cDCs can capture a blood-borne antigen leaking inside the interlobular vascular-rich regions (IVRs). Blood-borne antigen selectively captured by Sirpα(+) cDCs can induce antigen-specific Treg generation or negative selection, depending on the immunogenicity of the presented antigen. Furthermore, CCR2 expression by thymic Sirpα(+) cDCs and abundant expression of its ligands, particularly, CCL2 by tumor-bearing mice prompted us to examine the function of thymic Sirpα(+) cDCs in tumor-bearing mice. Interestingly, tumor-bearing mice deposited CCL2 inside IVRs in the thymus. Moreover, tumor formation induced the accumulation of Sirpα(+) cDCs in IVRs under the control of CCR2-CCL2 axis and enhanced their capacity to take up antigens, resulting in the shift from Treg differentiation to negative selection. Finally, intrathymic negative selection similarly ensued in CCR2-competent mice once the tumor-specific antigen was secreted into bloodstream. Thus, we demonstrated that thymic Sirpα(+) cDCs crucially contribute to this novel process of intrathymic tumor immune tolerance.     

Single cell electroporation method for axon tracing in cultured slices

Axon tracing is an essential method to reveal neuronal connection patterns and characteristics of growing axons during development. Here we introduce an electroporation-based gene transfer technique with a fluorescent protein (FP), which enables us to observe a small number of axons. The result also demonstrates that dynamics of axon behavior can be followed for more than a week. Thus, such labeling with FP is a powerful tool for axon tracing.     

Role of Phe1010 in light-induced structural changes of the neo1-LOV2 domain of Adiantum

Phototropin (phot) is a blue-light sensor protein that elicits several photo responses in plants. Phototropin has two flavin mononucleotide (FMN)-binding domains, LOV1 and LOV2, in its N-terminal half. The C-terminal half is a blue-light-regulated Ser/Thr kinase. Various functional studies have reported that only LOV2 is responsible for the kinase activity, whereas the X-ray crystallographic structures of the LOV1 and LOV2 domains are almost identical. How does such a functional difference emerge? Our previous FTIR study of the LOV domains of Adiantum neochrome1 (neo1) showed that light-induced protein structural changes are small and temperature independent for neo1-LOV1, whereas the structural changes are large and highly temperature dependent for neo1-LOV2, which involve loops, alpha-helices, and beta-sheets. These observations successfully explained the different functions in terms of protein structural changes. They also suggested the presence of some crucial amino acids responsible for greater protein structural changes in the LOV2 domain. Here, we focused on phenylalanine-1010 (Phe1010) in neo1-LOV2, where FMN is sandwiched between Phe1010 and the reactive cysteine. Phenylalanine at this position is conserved for LOV2 domains, while the corresponding amino acid is leucine for LOV1 domains in almost all plant phototropins. We observed that unlike wild-type LOV2, the FTIR spectra of F1010L LOV2 exhibited no temperature dependence in the alpha-helical and beta-sheet regions and that spectral changes in amide-I of these regions were significantly reduced, which was similar to LOV1. Thus, the replacement of phenylalanine with leucine converts neo1-LOV2 into neo1-LOV1 in terms of protein structural changes that must be related to the different functions. We will discuss the roles of phenylalanine and leucine in the LOV2 and LOV1 domains, respectively.     

Stability of dimer and domain-domain interaction of Arabidopsis phototropin 1 LOV2

Transient grating signals after photoexcitation of Arabidopsis phototropin 1 light-oxygen-voltage 2 (phot1LOV2) domain without the linker were found to be very sensitive to temperature. In particular, the diffusion signal drastically increased with rising temperature. The signal was consistently explained by the superposition of the photo-induced dissociation and association reactions. This observation indicated the presence of an equilibrium between the monomer and dimer forms of the phot1LOV2 domain in the dark. The equilibrium was confirmed by a gel chromatographic technique. The equilibrium constants at various temperatures were calculated from the fraction of the dimer, and the stabilization enthalpy and entropy were determined. Interestingly, the transient grating signal of phot1LOV2 with the linker (phot1LOV2-linker), which exists as the monomer form, was also temperature dependent; the diffusion signal intensity decreased with increasing temperature. Because the diffusion signal reflects a conformation change of the linker upon photoexcitation, this temperature dependence indicated that there were two forms of the phot1LOV2-linker. One form exhibited a conformational change upon photoexcitation whereas the other form showed no change. These two forms are not distinguishable spectroscopically. The fraction of these species depended on the temperature. Considering the monomer-dimer equilibrium of the phot1LOV2 domain, we suggest that the nonreactive form possesses the linker region that is dissociated from the LOV2 domain. Because the dissociation of the linker region from the LOV2 domain is a key step for the conformation change of the phot1LOV2-linker to induce biological activity, we proposed that the phototropins could have a role as a temperature sensor.     

Maintenance forced by a restriction-modification system can be modulated by a region in its modification enzyme not essential for methyltransferase activity

Several type II restriction-modification gene complexes can force their maintenance on their host bacteria by killing cells that have lost them in a process called postsegregational killing or genetic addiction. It is likely to proceed by dilution of the modification enzyme molecule during rounds of cell division following the gene loss, which exposes unmethylated recognition sites on the newly replicated chromosomes to lethal attack by the remaining restriction enzyme molecules. This process is in apparent contrast to the process of the classical types of postsegregational killing systems, in which built-in metabolic instability of the antitoxin allows release of the toxin for lethal action after the gene loss. In the present study, we characterize a mutant form of the EcoRII gene complex that shows stronger capacity in such maintenance. This phenotype is conferred by an L80P amino acid substitution (T239C nucleotide substitution) mutation in the modification enzyme. This mutant enzyme showed decreased DNA methyltransferase activity at a higher temperature in vivo and in vitro than the nonmutated enzyme, although a deletion mutant lacking the N-terminal 83 amino acids did not lose activity at either of the temperatures tested. Under a condition of inhibited protein synthesis, the activity of the L80P mutant was completely lost at a high temperature. In parallel, the L80P mutant protein disappeared more rapidly than the wild-type protein. These results demonstrate that the capability of a restriction-modification system in forcing maintenance on its host can be modulated by a region of its antitoxin, the modification enzyme, as in the classical postsegregational killing systems.     

Immunotherapy with dendritic cells pulsed with tumor-derived gp96 against murine lung cancer is effective through immune response of CD8+ cytotoxic T lymphocytes and natural killer cells

Background and purpose Immunization with heat shock proteins, gp96, elicits specific protective immunity against parent tumors. However, it is marginally effective as a therapeutic tool against established tumors. In the present study, we evaluated the efficacy and mechanism of immunotherapy with bone marrow-derived dendritic cells (DCs) pulsed with tumor-derived gp96 against murine lung cancer. Methods Mice were transplanted subcutaneously with ovalbumin (OVA)-transfected Lewis Lung Cancer (LLC-OVA) cells and immunized with gp96 derived from LLC-OVA, DCs, or DCs pulsed with gp96 derived from LLC-OVA. Results The antitumor effect was significantly enhanced in the mice immunized with DCs pulsed with gp96 derived from LLC-OVA, compared to mice immunized with gp96 or DCs (P < 0.05). The antitumor effect was significantly dependent on natural killer (NK) cells and CD8⁺ cells and partially dependent on CD4⁺ cells. Analysis by laser confocal microscopy demonstrated that gp96 was shown on the cell surface at 15 min, and after 30 min internalized in the endosomes and not in the endoplasmic reticulum or lysosomes. OVA-specific⁺ CD8⁺ cells were more readily recruited into the draining lymph nodes and higher CD8⁺ cytotoxic T cell activity against LLC-OVA was observed in splenocytes from mice immunized with DCs pulsed with gp96 derived from LLC-OVA. Re-challenge of the surviving mice with LLC-OVA tumors after the initial tumor inoculation showed dramatic retardation in tumor growth. Conclusion In conclusion, immunotherapy of DCs pulsed with tumor-derived gp96 against murine lung cancer is effective through immune response of CD8⁺ cytotoxic T lymphocytes and NK cells.     

Neutralization of acidified water by use of Rhizopus delemar immobilized with a cellulose membrane

To solve serious environmental problems caused by the acidification of pond and lake water by acid rain, remediation methods must be used to keep water pH values neutral. In this study, a microbial method to neutralize acidified water was developed. The neutralization activities of 30 strains of bacteria, yeasts and fungi were measured with a medium adjusted to pH 3.0. Because fungi showed high neutralization properties, the Rhizopus delemar fungus was used to study the characteristics of acidified water neutralization. When R. delemar cells were cultured in a media acidified with nitric, sulfuric and hydrochloric acids, the cells neutralized acids by secreting basic compounds including ammonia. The cells also assimilated nitric acid. R. delemar was used to neutralize pond water adjusted to pH 4.0 with nitric acid. R. delemar cells increased the pH value of pond water from 4.0 to around 7.0 within 2 days, although indigenous microorganisms had not been able to neutralize the same pond water. In this study, R. delemar immobilized in a cellulose tube neutralized acidified water repeatedly by the draw-fill method.     

Lung perfusion impairments in pulmonary embolic and airway obstruction with noncontrast MR imaging.

A noncontrast electrocardiography (ECG)-gated, fast-spin-echo magnetic resonance imaging was applied to noninvasively define perfusion impairments in pulmonary embolic and airway obstruction dog models. Two-phase ECG-gated lung images of the minimal lung signal intensity during systole and maximal signal intensity during diastole were acquired by using optimized R-wave triggering delay times in seven dogs anesthetized with pentobarbital sodium before, soon after, and 2 mo after embolization with enbucrilate and in another eight dogs before and after bronchial occlusion with balloon catheters, in combination with a gadolinium diethylenetriaminepentaacetic acid-enhanced dynamic study. An ECG-gated subtraction image between the two-phase lung images provided a uniform but gravity-dependent perfusion map in normal lungs. Furthermore, it defined all 13 variable-size perfusion deficits associated with pulmonary embolism and the dynamically decreased perfusion with time after bronchial occlusion in all the airway obstruction models. These results were consistent with contrast-enhanced pulmonary arterial perfusion phase images. This noncontrast imaging could be equivalent to a contrast-enhanced dynamic study in the definition of regionally impaired pulmonary arterial perfusion in pulmonary embolism and airway obstruction.     

Actin directly interacts with different membrane channel proteins and influences channel activities: AQP2 as a model

The interplay between actin and 10 membrane channel proteins that have been shown to directly bind to actin are reviewed. The 10 membrane channel proteins covered in this review are aquaporin 2 (AQP2), cystic fibrosis transmembrane conductance regulator (CFTR), ClC2, short form of ClC3 (sClC3), chloride intracellular channel 1 (CLIC1), chloride intracellular channel 5 (CLIC5), epithelial sodium channel (ENaC), large-conductance calcium-activated potassium channel (Maxi-K), transient receptor potential vanilloid 4 (TRPV4), and voltage-dependent anion channel (VDAC), with particular attention to AQP2. In regard to AQP2, most reciprocal interactions between actin and AQP2 occur during intracellular trafficking, which are largely mediated through indirect binding. Actin and the actin cytoskeleton work as cables, barriers, stabilizers, and force generators for motility. However, as with ENaC, the effects of actin cytoskeleton on channel gating should be investigated further. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.