Micro-liquid enclosure array and its semi-automated assembling system for x-ray free-electron laser diffractive imaging of samples in solution.

We developed micro-liquid enclosure arrays (MLEAs) for holding solution samples in coherent diffractive imaging (CDI) using x-ray free-electron lasers (XFELs). Hundreds of fully isolated micro-liquid enclosures are arranged in a single MLEA chip for efficient measurement, where each enclosure is destroyed after exposure to a single XFEL pulse. A semi-automated MLEA assembling system was also developed to enclose solution samples into MLEAs efficiently at high precision. We performed XFEL-based CDI experiments using MLEAs and imaged in-solution structures of self-assembled gold nanoparticles. The sample hit rate can be optimized by adjusting solution concentration, and we achieved a single-particle hit rate of 31%, which is not far from the theoretical upper limit of 37% derived from the Poisson statistics. MELAs allow us to perform CDI measurement under controlled solution conditions and will help reveal the nanostructures and dynamics of particles in solution.

Design of a liquid cell toward three-dimensional imaging of unidirectionally-aligned particles in solution using X-ray free-electron lasers.

X-ray free-electron lasers (XFELs) opened up a possibility for molecular-scale single particle imaging (SPI) without the need for crystallization. In SPI experiments, the orientation of each particle has to be determined from the measured diffraction pattern. Preparing unidirectionally-aligned particles can facilitate the determination of the sample orientation. Here, we show the design principles of a liquid cell for three-dimensional imaging of unidirectionally-aligned particles in solution with XFELs. The liquid cell was designed so that neither incident X-rays nor diffracted X-rays are blocked by the substrate of the liquid cell even at high tilt angles. As a feasibility evaluation, we performed coherent diffraction measurements using the cells with a 1 µm focused XFEL beam. We successfully measured coherent diffraction patterns of a nano-fabricated metal pattern at 70° tilt angle and obtained the reconstructed image by applying iterative phase retrieval. The liquid cell will be usefully applied to molecular-scale SPI by using more tightly focused XFELs. In particular, imaging of membrane proteins embedded in lipid membranes is expected to have an enormous impact on life science and medicine.

Dual wavelength 5-aminolevulinic acid photodynamic therapy using a novel flexible light-emitting diode unit.

BACKGROUND: Photosensitizers used for photodynamic therapy (PDT) to treat dermatologic disease are metabolized into mainly protoporphyrin IX (PpIX), which has five absorption wavelength peaks: 410 nm, 510 nm, 545 nm, 580 nm, and 630 nm. Although only red light around 635 nm and blue light around 400 nm are used as light sources for PDT, the efficiency of PDT might be improved by using multiple wavelengths, including those that correspond to the other absorption peaks of PpIX. Furthermore, because the target disease often occurs on the face, a flexible-type light-source unit that can be fitted to the lesion without unnecessarily exposing the mucous membranes, e.g., the eyes, nostrils, and mouth, is preferred. OBJECTIVE: We investigated the efficacy of a flexible light-emitting diode (LED) unit that emits multiple wavelengths to improve PDT effects. METHODS: HaCaT cells were incubated with 5-ALA and subsequently irradiated with either a single wavelength or sequentially with two wavelengths. Cell viability and reactive oxygen species were analyzed. Nude mice were implanted with COLO679 cells by subcutaneous injection into the flank. 5-ALA was subcutaneously injected into the tumor. The tumor was irradiated with 50 J/cm2 (day 0) and assessed daily until day 21. RESULTS: The synergistic PDT effects of dual-wavelength irradiation and reactive oxygen species production were highest with the 405-nm and 505-nm wavelength combination. This dual wavelength combination was also the most effective in vivo. CONCLUSION: We could therefore conclude that dual-wavelength PDT is an efficient strategy for improving the therapeutic effects of PDT. Using a flexible LED unit is expected to achieve more uniform irradiation of uneven areas.

Ras homolog gene family H (RhoH) deficiency induces psoriasis-like chronic dermatitis by promoting TH17 cell polarization.

BACKGROUND: Ras homolog gene family H (RhoH) is a membrane-bound adaptor protein involved in proximal T-cell receptor signaling. Therefore RhoH plays critical roles in the differentiation of T cells; however, the function of RhoH in the effecter phase of the T-cell response has not been fully characterized. OBJECTIVE: We sought to explore the role of RhoH in inflammatory immune responses and investigated the involvement of RhoH in the pathogenesis of psoriasis. METHODS: We analyzed effector T-cell and systemic inflammation in wild-type and RhoH-null mice. RhoH expression in T cells in human PBMCs was quantified by using RT-PCR. RESULTS: RhoH deficiency in mice induced TH17 polarization during effector T-cell differentiation, thereby inducing psoriasis-like chronic dermatitis. Ubiquitin protein ligase E3 component N-recognin 5 (Ubr5) and nuclear receptor subfamily 2 group F member 6 (Nr2f6) expression levels decreased in RhoH-deficient T cells, resulting in increased protein levels and DNA binding activity of retinoic acid-related orphan receptor γt. The consequential increase in IL-17 and IL-22 production induced T cells to differentiate into TH17 cells. Furthermore, IL-22 binding protein/Fc chimeric protein reduced psoriatic inflammation in RhoH-deficient mice. Expression of RhoH in T cells was lower in patients with psoriasis with very severe symptoms. CONCLUSION: Our results indicate that RhoH inhibits TH17 differentiation and thereby plays a role in the pathogenesis of psoriasis. Additionally, IL-22 binding protein has therapeutic potential for the treatment of psoriasis.

Ultraviolet B-Induced Maturation of CD11b-Type Langerin- Dendritic Cells Controls the Expansion of Foxp3+ Regulatory T Cells in the Skin.

Skin dendritic cells (DCs) are divided into several subsets with distinctive functions. This study shows a previously unappreciated role of dermal CD11b-type Langerin- DCs in maintaining immunological self-tolerance after UVB exposure. After UVB exposure, dermal CD11b-type Langerin- DCs upregulated surface CD86 expression, induced proliferation of Foxp3+ regulatory T (Treg) cells without exogenous Ags, and upregulated a set of genes associated with immunological tolerance. This Treg-expansion activity was significantly hampered by CD80/CD86 blockade in vivo. These results indicate that CD11b-type Langerin- DCs from the UVB-exposed skin are specialized to expand Treg cells in the skin, which suppress autoimmunity.

Bath-PUVA therapy improves impaired resting regulatory T cells and increases activated regulatory T cells in psoriasis.

BACKGROUND: Bath-psoralen plus ultraviolet light A (PUVA) therapy is an effective, safe, and inexpensive treatment for psoriasis. Psoriasis might be due to an unbalanced ratio of Th17 cells and regulatory T cells (Treg). The Treg functional ratio is significantly lower in patients with psoriasis compared with controls and is inversely correlated with the Psoriasis Area and Severity Index score. We previously reported that bath-PUVA therapy significantly increases the number of Treg and restores Treg function to almost normal in most patients with psoriasis. OBJECTIVES: We examined the effects of bath-PUVA therapy on three distinct Foxp3+ subsets: activated Treg (aTreg), resting Treg (rTreg), and cytokine-secreting non-suppressive T cells. METHODS: We enrolled 15 patients with psoriasis and 11 healthy controls. We examined aTreg, rTreg, and cytokine-secreting non-suppressive T cells in peripheral blood obtained from the psoriasis patients before and after every fifth bath-PUVA therapy session. RESULTS: Levels of aTreg, which are considered to have the strongest suppressive activity in patients with psoriasis, were significantly increased in the early bath-PUVA therapy sessions, and then diminished. Levels of rTreg were lower in psoriasis patients than in healthy controls, and increased during bath-PUVA therapy. CONCLUSIONS: Bath-PUVA therapy induced aTreg and rTreg concomitantly with an improvement in the psoriatic lesions, suggesting a mechanism for the effectiveness of bath-PUVA therapy for psoriasis patients.

Homeostasis of thymus-derived Foxp3+ regulatory T cells is controlled by ultraviolet B exposure in the skin.

Accumulating evidence shows that immunological tolerance induced by Ag administration together with UVB irradiation is dependent on Foxp3(+) CD4(+) regulatory T (Treg) cells. However, the mechanisms by which UVB controls Treg cells in the skin are currently unclear. In this study, we have shown that exposure to UVB induced expansion of Treg cells up to 50-60% of the CD4(+) T cells in the irradiated skin. The Treg cell expansion in the skin lasted for 2 wk after exposure, which contributed to homeostasis of Treg cells in the periphery later. UVB-expanded Treg cells formed clusters with dendritic cells and proliferated in situ. Furthermore, the expanded Treg cells appeared to derive from neuropilin 1(+) thymus-derived Treg (tTreg) cells in the periphery because UVB-expanded Treg cells possessed Treg cell-specific CpG hypomethylation pattern, as seen in tTreg cells. These results collectively indicate that homeostasis of tTreg cells is controlled by UVB exposure in the skin. UVB therapy may be useful for not only inflammatory skin disorders, but also autoimmunity, transplantation, and allergy.

Antitumor activity of an interleukin-2 monoclonal antibody in a murine osteosarcoma transplantation model.

BACKGROUND: Injection of monoclonal antibody to interleukin-2 (S4B6) into mice depletes regulatory T-cells (Tregs) and exhibits antitumor activities mediated through an autoimmune reaction. In this study, we demonstrate that administration of S4B6 suppresses the murine osteosarcoma cell line, LM8. MATERIALS AND METHODS: LM8 osteosarcoma cells were transplanted subcutaneously into C3H mice (n=58). C3H mice were injected intraperitoneally with S4B6 starting at 7 days before LM8 transplantation (pre-S4B6 group), 2 days after transplantation, or 5 days after transplantation. Control group mice were injected with normal rat IgG. Mice were sacrificed and examined 4 weeks later. RESULTS: The number of pulmonary metastatic colonies and the tumor size were significantly reduced in the pre-S4B6 group compared to the control group. In addition, pulmonary metastases were inhibited in mice injected with S4B6 2 days, but not 5 days, after tumor transplantation. CONCLUSION: S4B6 administration inhibited metastasis even when injected 2 days after LM8 transplantation. Our data suggest that treatment with S4B6 might be suitable in a postoperative clinical setting.

Tobacco smoke is related to Th17 generation with clinical implications for psoriasis patients.

Environmental factors contribute to the increased prevalence of autoimmune diseases via T helper type-17 cell (Th17) activation. Tobacco smoking increases the risk of psoriasis, but the mechanisms are not clear. We evaluated the percentage of circulating Th17 among CD3(+) cells in peripheral blood mononuclear cells (PBMC) obtained from 27 healthy volunteers (2.58±0.80%), 33 smoker (3.55±1.33%) and 21 non-smoker (3.10±1.14%) patients with psoriasis to elucidate the relation between smoking and psoriasis. More smokers (19/33) than non-smokers (6/21) had high Th17 levels (Th17/CD3>3.38%, mean+1 SD of healthy volunteers). Tobacco smoke extract (TSE, 7µl/ml) induced Th17 generation from central memory T cells in vitro. TSE increased interleukin 17 and 22 expression. These findings demonstrate the relation between tobacco smoke and IL-17 and IL-22, which exacerbate psoriasis.

Increased peripheral Th17 in patients with pustulosis palmaris et plantaris.

Pustulosis palmaris et plantaris (PPP) is a chronic recurrent dermatitis characterized by intraepidermal pustules with erythematous scaling on the palms and soles. PPP shares many characteristics with psoriasis, but has a different genetic background. T helper 17 cells (Th17) have an important role in the pathogenesis of psoriasis. In psoriasis, regulatory T cells (Treg) are dysfunctional and circulating Th17 are increased. Whether Th17 are involved in PPP, however, is unclear. Therefore, we examined the Th17 population in peripheral blood mononuclear cells (PBMC) of patients with PPP. Foxp3(+) Treg was also analyzed. We examined circulating Th17 and Treg in the peripheral blood of PPP patients. PBMC were obtained from healthy volunteer controls (n = 26, mean ± SD age 33.11 ± 9.80 years) and PPP patients (n = 24, age 55.00 ± 12.26 years). The proportion of Th17 among the PBMC was 2.52 ± 0.811% (mean ± SD) in healthy controls and 3.23 ± 1.45% in PPP patients. The proportion of Th17 in the PPP patients was significantly higher than that in the healthy controls (p < 0.05, Student's t test). PPP patients had significantly fewer Treg (5.69 ± 1.86%) than healthy controls (7.10 ± 1.78%). Th17 was inversely correlated with Treg.

Suppression of tumour metastasis in a murine osteosarcoma model with anti-CD25 monoclonal antibody treatment.

BACKGROUND: Mouse regulatory T cells (Treg) may be deleted by intraperitoneal injection of anti-CD25 monoclonal antibody (mAb) (PC61). When Treg populations are thus suppressed, the immune system attacks tumours in autoimmune reactions. MATERIALS AND METHODS: An osteosarcoma (LM8) was transplanted subcutaneously into C3H/He mice. Serial injections of PC61 were conducted from seven days before (pre-PC61 group) or from two days thereafter (post-PC61 group) and tumour growth and metastasis were examined four weeks later. A control group received PBS injections. RESULTS: Subcutaneous tumours were reduced in size and the numbers of lung and liver metastatic colonies were significantly decreased in both pre- and post-PC61 groups compared to the control group. CONCLUSION: Tumour suppression was effective even when injection of PC61 was performed two days after LM8 transplantation. These results indicate that such treatments might be suitable to be applied in the clinic after surgical operations.