Feasibility study of direct CT lymphangiography in mice: comparison with interstitial CT/MR lymphangiography.

OBJECTIVES: To establish a CT lymphangiography method in mice via direct lymph node puncture. METHODS: We injected healthy mice (n = 8) with 50 µl of water-soluble iodine contrast agent (iomeprol; iodine concentration, 350 mg/mL) subcutaneously into the left-rear foot pad (interstitial injection) and 20 µl of the same contrast agent directly into the popliteal lymph node (direct puncture) 2 days later. Additionally, we performed interstitial MR lymphangiography on eight mice as a control group. We calculated the contrast ratio for each lymph node and visually assessed the depiction of lymph nodes and lymphatic vessels on a three-point scale. RESULTS: The contrast ratios of 2-min post-injection images of sacral and lumbar-aortic lymph nodes were 20.7 ± 16.6 (average ± standard deviation) and 17.1 ± 12.0 in the direct puncture group, which were significantly higher than those detected in the CT or MR interstitial lymphangiography groups (average, 1.8-3.6; p = 0.008-0.019). The visual assessment scores for sacral lymph nodes, lumbar-aortic lymph nodes, and cisterna chyli were significantly better in the direct puncture group than in the CT interstitial injection group (p = 0.036, 0.009 and 0.001, respectively). The lymphatic vessels between these structures were significantly better scored in direct puncture group than in the CT or MR interstitial lymphangiography groups at 2 min after injection (all p ≤ 0.05). CONCLUSIONS: In CT lymphangiography in mice, the direct lymph node puncture provides a better delineation of the lymphatic pathways than the CT/MR interstitial injection method. KEY POINTS: • The contrast ratios of 2-min post-injection images in the direct CT lymphangiography group were significantly higher than those of CT/MR interstitial lymphangiography groups. • The visibility of lymphatic vessels in subjective analysis in the direct CT lymphangiography group was significantly better in the direct puncture group than in the CT/MR interstitial lymphangiography groups. • CT lymphangiography with direct lymph node puncture can provide excellent lymphatic delineation with contrast being maximum at 2 min after injection.

Invariant NKT cells promote skin wound healing by preventing a prolonged neutrophilic inflammatory response.

The wound-healing process consists of the inflammation, proliferation, and remodeling phases. In chronic wounds, the inflammation phase is prolonged with persistent neutrophil infiltration. The inflammatory response is critically regulated by cytokines and chemokines that are secreted from various immune cells. Recently, we showed that skin wound healing was delayed and the healing process was impaired under conditions lacking invariant natural killer T (iNKT) cells, an innate immune lymphocyte with potent immuno-regulatory activity. In the present study, we investigated the effect of iNKT cell deficiency on the neutrophilic inflammatory response during the wound healing process. Neutrophil infiltration was prolonged in wound tissue in mice genetically lacking iNKT cells (Jα18KO mice) than in wild-type (WT) control mice on days 1 and 3 after wounding. MIP-2, KC, and IL-17A were produced at a significantly higher level in Jα18KO mice than in WT mice. In addition, neutrophil apoptosis was significantly reduced in the wound tissue in Jα18KO mice than in WT mice. Treatment with anti-IL-17A mAb, anti-Gr-1 mAb, or neutrophil elastase inhibitor reversed the impaired wound healing in Jα18KO mice. These results suggest that iNKT cells may promote the wound healing process through preventing the prolonged inflammatory response mediated by neutrophils.

Contribution of CARD9-mediated signalling to wound healing in skin.

The inflammatory response after skin injury involves the secretion of a variety of cytokines and growth factors that are necessary for tissue repair. Caspase recruitment domain-containing protein 9 (CARD9) is an essential signalling adaptor molecule for NF-κB activation upon triggering through C-type lectin receptors (CLRs), which are expressed in macrophages and dendritic cells. However, the role of CARD9 in inflammatory responses at the wound site has not been elucidated. In this study, we analysed the role of CARD9 in the healing process of skin wounds. Wounds were created on the backs of wild-type (WT) C57BL/6 mice and CARD9 gene-disrupted (knockout [KO]) mice. We analysed per cent wound closure, and the wound tissues were harvested for analysis of leucocyte accumulation and cytokine and chemokine expressions. CARD9KO mice exhibited significant attenuation of wound closure compared with WT mice on days 5, 7 and 10 postwounding, which was associated with decreased macrophage accumulation and reduced TNF-α, IL-1ß, CCL3 and CCL4 expressions. These results suggest that CARD9 may be involved in the wound-healing process through the regulation of macrophage-mediated inflammatory responses.

Promotion of acute-phase skin wound healing by Pseudomonas aeruginosa C4 -HSL.

A Pseudomonas aeruginosa quorum-sensing system, which produces N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12 -HSL) and N-butanoyl-l-homoserine lactone (C4 -HSL), regulates the virulence factors. In our previous study, 3-oxo-C12 -HSL, encoded by lasI gene, was shown to promote wound healing. However, the effect of C4 -HSL, encoded by rhlI gene, remains to be elucidated. We addressed the effect of C4 -HSL on wounds in P. aeruginosa infection. Wounds were created on the backs of Sprague-Dawley SD rats, and P. aeruginosa PAO1 (PAO1) or its rhlI deletion mutant (ΔrhlI) or lasI deletion mutant (ΔlasI) was inoculated onto the wound. Rats were injected intraperitoneally with anti-C4 -HSL antiserum or treated with C4 -HSL at the wound surface. PAO1 inoculation led to significant acceleration of wound healing, which was associated with neutrophil infiltration and TNF-α synthesis. These responses were reversed, except for TNF-α production, when ΔrhlI was inoculated instead of PAO1 or when rats were co-treated with PAO1 and anti-C4 -HSL antiserum. In contrast, the healing process and neutrophil infiltration, but not TNF-α synthesis, were accelerated when C4 -HSL was administered in the absence of PAO1. This acceleration was not affected by anti-TNF-α antibody. These results suggest that C4 -HSL may be involved in the acceleration of acute wound healing in P. aeruginosa infection by modifying the neutrophilic inflammation.

Contribution of Invariant Natural Killer T Cells to Skin Wound Healing.

In the present study, we determined the contribution of invariant natural killer T (iNKT) cells to the skin wound healing process. In iNKT cell-deficient (Jα18KO) mice lacking iNKT cells, wound closure was significantly delayed compared with wild-type mice. Collagen deposition, expression of α-smooth muscle actin and CD31, and wound breaking strength were significantly attenuated in Jα18KO mice. The adoptive transfer of liver mononuclear cells from wild-type but not from Jα18KO or interferon (IFN)-γ gene-disrupted (IFN-γKO) mice resulted in the reversal of this impaired wound healing in Jα18KO mice. IFN-γ expression was induced in the wounded tissues, which was significantly decreased at 6, 12, and 24 hours, but increased on day 3 after wounding in Jα18KO mice. The main source of the late-phase IFN-γ production in Jα18KO mice were neutrophils rather than NK cells and T cells. Administration of α-galactosylceramide, an activator of iNKT cells, resulted in the acceleration of wound healing on day 3 in wild-type mice. This effect was not observed in IFN-γKO mice. These results indicate that iNKT cells play important roles in wound healing. The iNKT cell-induced IFN-γ production may regulate the wound healing process in the early phase.

Multimodal biopanning of T7 phage-displayed peptides reveals angiomotin as a potential receptor of the anti-angiogenic macrolide Roxithromycin.

Roxithromycin (RXM) is a semi-synthetic fourteen-membered macrolide antibiotic that shows anti-angiogenic activity in solid tumors. In the present study, we conducted biopanning of T7 phage-displayed peptides either on a 96-well formatted microplate, a flow injection-type quartz-crystal microbalance (QCM) biosensor, or a cuvette-type QCM. RXM-selected peptides of different sequence, length and number were obtained from each mode of screening. Subsequent bioinformatics analysis of the RXM-selected peptides consistently gave positive scores for the extracellular domain (E458-T596) of angiomotin (Amot), indicating that this may comprise a binding region for RXM. Bead pull down assay and QCM analysis confirmed that RXM directly interacts with Amot via the screen-guided region, which also corresponds to the binding site for the endogenous anti-angiogenic inhibitor angiostatin (Anst). Thus, multimodal biopanning of T7PD revealed that RXM binds to the extracellular domain on Amot as a common binding site with Anst, leading to inhibition of angiogenesis-dependent tumor growth and metastasis. These data might explain the molecular basis underlying the mechanism of action for the anti-angiogenic activity of RXM.

A new phenolic glycoside syringate from the bark of Juglans mandshurica MAXIM. var. sieboldiana MAKINO.

A new phenolic glycoside syringate, 4'-hydroxy-2',6'-dimethoxyphenol 1-O-beta-D: -(6-O-syringoyl) glucopyranoside (1), together with two known ones, 2'-hydroxy-4'-methoxyphenol 1-O-beta-D: -(6-O-syringoyl) glucopyranoside (2) and 4'-hydroxy-2'-methoxyphenol 1-O-beta-D: -(6-O-syringoyl) glucopyranoside (3), were isolated from the bark of Juglans mandshurica MAXIM. var. sieboldiana MAKINO. Their structures were established on the basis of spectral and chemical data.