Hydrostatic Compression Effects on Fifth-Group Element Superconductors V, Nb, and Ta Subjected to High-Pressure Torsion

In fifth-group element superconductors V, Nb, and Ta, the increase in superconducting transition temperature (Tc) was attempted by using both high-pressure torsion (HPT) and additional hydrostatic pressure (HP) compression. The former brings about the grain refinement and strain accumulation in the unit-cell level. The additional compression for severely strained superconductors triggers strengthening intergrain-contact and/or structural deformation in the unit-cell level. The manner of the appearance of the above two effects depends on the kind of elements: First, in V, there is no prominent effect of HPT, comparing to the hydrostatic compression effects on its non-strained material. Next, in Ta, the effect of strengthening intergrain-contact appears at small hydrostatic compression, resulting in temporal increase in Tc. Finally, Nb exhibits prominent increase in Tc by both effects and, in particular, the structural deformation in the unit-cell level promotes the increase in Tc. Thus, the accumulation of residual strain in the level of starting material can be a promising work to manipulate Tc under HP compression

Murine Splenic Natural Killer Cells Do Not Develop Immunological Memory after Re-Encounter with Mycobacterium bovis BCG.

Several lines of evidence have recently suggested that natural killer (NK) cells develop immunological memory against viral infections. However, there is no apparent evidence that NK cells acquire specific memory against Mycobacterium bovis bacillus Calmette-Guérin (BCG), the only currently licensed vaccine for preventing tuberculosis. In the present study, we investigated whether murine splenic NK cells can be activated by BCG in a dendritic cell (DC)-independent or -dependent manner, and furthermore examined whether these NK cells acquire specific memory following BCG vaccination. NK cells isolated from spleens of BCG-immunized mice produced interferon (IFN)γ through direct BCG stimulation in the absence of antigen-presenting cells; however, NK cells from control animals similarly directly responded to BCG, and the response level was not statistically significant between the immunized and the naïve NK cells. When purified NK cells that had been exposed to BCG were cocultured with RAW murine macrophages infected with BCG, the antibacterial activity of the macrophages was strongly enhanced; however, its level was similar to that by naïve NK cells, which had not been exposed to BCG. When splenocytes harvested from BCG-immunized mice were stimulated with purified protein derivative (PPD) derived from Mycobacterium tuberculosis, a specific IFNγ response was clearly observed, mainly attributed to NK cells and memory CD4+ T cells. To investigate whether these NK cells as well as the T cells are activated by cell-cell interaction with DCs presenting mycobacterial antigens, NK cells isolated from BCG-immunized mice were cocultured with splenocytes harvested from naïve mice in the presence of PPD stimulation. However, no IFNγ response was found in the NK cells. These results suggest that murine splenic NK cells do not develop BCG-specific immunological memory in either a DC-independent or -dependent manner

BCG vaccination does not enhance specific IFNγ production of NK cells in response to a second BCG stimulation.

<p>Mice were immunized with BCG or phosphate-buffered saline (PBS), and 6 weeks later, NK cells were isolated from the spleens of these animals (<i>n</i> = 5 per group). The purified NK cells were cultured (1.5 × 10⁶ /mL) in the presence or absence of BCG (MOI = 1) at 37°C for 24 h, and then the culture supernatants were harvested. The production level of IFNγ was measured by ELISA. The data are presented as mean ± standard deviation, and <i>p</i> values < 0.05 were considered statistically significant. Similar results were obtained in three independent experiments. NS, not significant.</p

Spleen-resident natural killer (NK) cells are directly activated by <i>Mycobacterium bovis</i> BCG and purified protein derivative (PPD) antigen.

<p>NK cells isolated from spleens of naïve mice were cultured (1.5 × 10⁶ /mL) in the presence or absence of BCG (multiplicity of infections (MOI) = 1) (A) or PPD (50 μg/mL) (B) at 37°C for 24 h, and then the culture supernatants were harvested (<i>n</i> = 5 per group). The production level of IFNγ was measured by an enzyme-linked immunosorbent assay (ELISA). The data are presented as mean ± standard deviation, and <i>p</i> values < 0.05 were considered statistically significant. Similar results were obtained in three independent experiments. ***<i>p</i> < 0.0001.</p

NK cells in spleens of BCG-immunized mice are not activated in a DC-dependent manner.

<p>At 6 weeks after the single vaccination of mice with BCG or PBS, NK cells (3 × 10⁵) were isolated from spleens of these animals, and then were cocultured with splenocytes (3 × 10⁷) harvested from naïve mice in the presence or absence of PPD stimulation (50 μg/mL) at 37°C for 24 h (<i>n</i> = 5 per group). The cells were stained with anti-mouse NK1.1 followed by anti-mouse IFNγ mAbs, and then analyzed with flow cytometry. The data are presented as mean ± standard deviation, and <i>p</i> values <0.05 were considered statistically significant. Similar results were obtained in two independent experiments. NS, not significant.</p

NK cells markedly enhance the ability of macrophages to eradicate BCG.

<p>RAW 264.7 murine macrophage cells were infected with BCG (MOI = 3) at 37°C for 2 h, washed with PBS three times, and then plated at 1 × 10⁶ cells/mL in a 12 well plate. Purified NK cells (3 × 10⁵), which had been stimulated with BCG (MOI = 1) at 37°C for 4 h, were added to the BCG-infected RAW cell culture. As controls, unstimulated naïve NK cells were added to the BCG-infected RAW cell culture, and the BCG-infected RAW cells alone were additionally prepared. Forty eight hours later, these cells were harvested and lysed with 1 mL of 0.067% SDS solution. Serial dilutions were plated on Middlebrook 7H10 agar plates, and 3 weeks later, the number of bacterial colonies grown on the agar plates were counted (A). As in (A), IFNγ (B) and TNF-α (C) in the culture supernatants were measured by ELISA. Purified naïve NK cells were cultured in medium supplemented with either the culture supernatant of the BCG-infected RAW cells or uninfected control RAW cells at a ratio of 1:1 at 37°C for 24 h, and IFNγ in the culture supernatants was measured using ELISA (D). The data are presented as mean ± standard deviation, and <i>p</i> values < 0.05 were considered statistically significant. Similar results were obtained in three independent experiments. Horizontal bar in (A), mean value; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.0001; NS, not significant.</p

T cells and NK cells in spleens of BCG-immunized mice provoke specific IFNγ responses upon stimulation with PPD antigen.

<p>At 6 weeks after the single vaccination of mice with BCG or PBS, splenocytes were harvested from the immunized and the control mice (<i>n</i> = 5 per group). The cells were cultured (2 × 10⁷ cells/mL) in the presence or absence of PPD (50 μg/mL) at 37°C for 24 h. A portion of the culture supernatants was harvested for ELISA, and then brefeldin A (10 μg/mL) was added to the remaining cell cultures. The production level of IFNγ in the culture supernatants was measured by ELISA (A). The splenocytes harvested were stained with anti-mouse CD4 (B) or anti-mouse NK1.1 (C) followed by anti-mouse IFNγ mAbs, and then analyzed with flow cytometry. The data are presented as mean ± standard deviation, and <i>p</i> values <0.05 were considered statistically significant. Similar results were obtained in three independent experiments. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.0001.</p