Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2: Higher Affinity than That with MD-2 or CD14

Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4. Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2. Here, we demonstrate cell surface LPS–TLR4-MD-2 complexes. CD14 greatly enhances the formation of LPS–TLR4-MD-2 complexes, but is not coprecipitated with LPS–TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. A tentative dissociation constant (Kd) for LPS–TLR4-MD-2 complexes was ∼3 nM, which is ∼10–20 times lower than the reported Kd for LPS–MD-2 or LPS–CD14. The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2. E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14. These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14

The Toll-like Receptor Protein Rp105 Regulates Lipopolysaccharide Signaling in B Cells

The susceptibility to infections induced by Gram-negative bacteria is largely determined by innate immune responses to bacteria cell wall lipopolysaccharide (LPS). The stimulation of B cells by LPS enhances their antigen-presenting capacity and is accompanied by B cell proliferation and secretion of large quantities of LPS-neutralizing antibodies. Similar to macrophages and neutrophils, the LPS-induced activation of B cells is dependent on Toll-like receptor (TLR)4. Here, we demonstrate that the responses of B cells to LPS are also regulated by another TLR protein, RP105, which is predominantly expressed on mature B cells in mice and humans. The analysis of mice homozygous for the null mutation in the RP105 gene revealed impaired proliferative and humoral immune responses of RP105-deficient B cells to LPS. Using originally LPS-unresponsive Ba/F3 cells expressing exogenous TLR4 and RP105, we demonstrate the functional cooperation between TLR4 and RP105 in LPS-induced nuclear factor κB activation. These data suggest the existence of the TLR4–RP105 signaling module in the LPS-induced B cell activation

Neutralizing Anti-Hemagglutinin Monoclonal Antibodies Induced by Gene-Based Transfer Have Prophylactic and Therapeutic Effects on Influenza Virus Infection

Hemagglutinin (HA) of influenza virus is a major target for vaccines. HA initiates the internalization of the virus into the host cell by binding to host sialic acid receptors; therefore, inhibition of HA can significantly prevent influenza virus infection. However, the high diversity of HA permits the influenza virus to escape from host immunity. Moreover, the vaccine efficacy is poor in some high-risk populations (e.g., elderly or immunocompromised patients). Passive immunization with anti-HA monoclonal antibodies (mAbs) is an attractive therapy; however, this method has high production costs and requires repeated inoculations. To address these issues, several methods for long-term expression of mAb against influenza virus have been developed. Here, we provide an overview of methods using plasmid and viral adeno-associated virus (AAV) vectors that have been modified for higher expression of neutralizing antibodies in the host. We also examine two methods of injection, electro-transfer and hydrodynamic injection. Our results show that antibody gene transfer is effective against influenza virus infection even in immunocompromised mice, and antibody expression was detected in the serum and upper respiratory tract. We also demonstrate this method to be effective following influenza virus infection. Finally, we discuss the perspective of passive immunization with antibody gene transfer for future clinical trials

Evaluation of hair follicle stem cells in radiation damage.

Radiation-induced hair loss is a clinically important, but under-researched topic. We reported that radiation-induced hair follicle dystrophy was characterized by morphological similarities to dystrophic catagen induced by chemotherapy. The extent of this hair follicle damage occurred in a radiation dose-dependent manner. However, the effect of irradiation on stem cells still remains unknown in radiation-induced hair follicle damage. In this study, a portion of the dorsal skin from 7-week-old male BALB/c mice, harboring uniform telogen phase hair follicles, was depilated to induce the anagen phase of the hair growth cycle. Then these mice received total body irradiation (TBI) with gamma-rays, at doses in the range of 4 to 16 Gy, 6 days after depilation. Under these conditions, hair could grow after irradiation at 4 Gy, but not at more than 6 Gy. In contrast, hair could be regenerated when mice were depilated 1 h after irradiation at 6 Gy. K15 positive stem cells drastically decreased in anagen hair follicles after irradiation. On the other hand, K15 positive stem cells remained the same in telogen hair follicles even after irradiation at all tested doses. However post-irradiation plucking - that is, anagen phase induction - dramatically diminished the number of K15 positive stem cells in those hair follicles. In contrast, post-irradiation plucking did trigger hair follicles differentiation. These findings suggest that hair follicle stem cells are relatively resistant to radiation. However, irradiation may inhibit the capability for self-renewal of stem cells, resulting in a decrease or depletion of stem cells in their differentiation. Therefore, anagen hair follicles are more sensitive to radiation than telogen hair follicles.日本研究皮膚科学会第35回年次学術大会・総

Stage-Specific Characterization of Physiological Response to Heat Stress in the Wheat Cultivar Norin 61

Bread wheat (Triticum aestivum) is less adaptable to high temperatures than other major cereals. Previous studies of the effects of high temperature on wheat focused on the reproductive stage. There are few reports on yield after high temperatures at other growth stages. Understanding growth-stage-specific responses to heat stress will contribute to the development of tolerant lines suited to high temperatures at various stages. We exposed wheat cultivar “Norin 61” to high temperature at three growth stages: seedling–tillering (GS1), tillering–flowering (GS2), and flowering–maturity (GS3). We compared each condition based on agronomical traits, seed maturity, and photosynthesis results. Heat at GS2 reduced plant height and number of grains, and heat at GS3 reduced the grain formation period and grain weight. However, heat at GS1 reduced senescence and prolonged grain formation, increasing grain weight without reducing yield. These data provide fundamental insights into the biochemical and molecular adaptations of bread wheat to high-temperature stresses and have implications for the development of wheat lines that can respond to high temperatures at various times of the year