Group IIA secreted phospholipase A2 controls skin carcinogenesis and psoriasis by shaping the gut microbiota.

Besides promoting inflammation by mobilizing lipid mediators, group IIA secreted phospholipase A2 (sPLA2-IIA) prevents bacterial infection by degrading bacterial membranes. Here, we show that, despite the restricted intestinal expression of sPLA2-IIA in BALB/c mice, its genetic deletion leads to amelioration of cancer and exacerbation of psoriasis in distal skin. Intestinal expression of sPLA2-IIA is reduced after treatment with antibiotics or under germ-free conditions, suggesting its upregulation by gut microbiota. Metagenome, transcriptome, and metabolome analyses have revealed that sPLA2-IIA deficiency alters the gut microbiota, accompanied by notable changes in the intestinal expression of genes related to immunity and metabolism, as well as in the levels of various blood metabolites and fecal bacterial lipids, suggesting that sPLA2-IIA contributes to shaping of the gut microbiota. The skin phenotypes in Pla2g2a-/- mice are lost (a) when they are cohoused with littermate WT mice, resulting in the mixing of the microbiota between the genotypes, or (b) when they are housed in a more stringent pathogen-free facility, where Pla2g2a expression in WT mice is low and the gut microbial compositions in both genotypes are nearly identical. Thus, our results highlight a potentially new aspect of sPLA2-IIA as a modulator of gut microbiota, perturbation of which affects distal skin responses.

Crosslinking Efficacy and Cytotoxicity of Genipin and Its Activated Form Prepared by Warming It in a Phosphate Buffer: A Comparative Study.

The aim of the present study was to compare the acute and cumulative cytotoxicity of intact (n-GE) and warmed genipin (w-GE), while investigating the differences in crosslinking capabilities of these two genipins by rheological and mechanical tests. The n-GE solution was prepared by dissolving genipin powder in a sodium phosphate buffer solution. The w-GE solution was prepared by warming the n-GE solution at 37 °C for 24 h. The mechanical tests for chitosan (CH)/genipin gels showed the crosslinking rate of w-GE was much greater than that of n-GE up until 6 h after preparation, whereas the degree of crosslinking of CH/n-GE gels became higher at 12 h. The ISO 10993-5 standard method, which is established specifically for evaluating cumulative cytotoxicity, determined equivalent IC50 for w-GE (0.173 mM) and n-GE (0.166 mM). On the other hand, custom-made cytotoxicity tests using a WST-8 assay after 1 h of cultivation showed that the acute cytotoxicity of w-GE was significantly higher than that of n-GE at concentrations between 0.1-5 mM. The acute cytotoxicity of w-GE should be taken into consideration in its practical uses, despite the fact that the much faster crosslinking of w-GE is useful as an effective cross linker for in-situ forming gels.

Utility of a rotation/revolution-type agitator for chondrocyte isolation during preparation of engineered cartilage.

During the manufacture of cell- and tissue-based products, such as engineered cartilage for autologous chondrocyte implantation, maximizing the number of cells isolated from donor tissue substantially improves the productivity of these products. The method used for agitating tissues with digestive fluid and enzymes can considerably affect both the quality and quantity of isolated cells. This study aimed to investigate the effectiveness of a rotation/revolution-type agitator for chondrocyte isolation following the enzymatic digestion of rat costal cartilage. Cartilage tissue cut into 1 mm3-thick sections was equally divided between two groups and placed in 50-mL conical tubes; sections in both groups were digested using 0.1 mg/mL liberase TH (collagenase/thermolysin) at 37 °C for 4 h with either rotation/revolution or conventional orbital agitation method. Compared with using conventional orbital agitator, using the rotation/revolution-type agitator resulted in a significant (>two-fold) increase in the number of isolated cells. In subsequent primary cultures, chondrocytes obtained by rotation/revolution agitation showed superior initial attachment to tissue culture dish on day 1 and 2 compared with those obtained by conventional agitation; however, no differences in cell proliferation or cartilage-related molecule expression patterns were observed between cells derived from either method after 3 days of subculture. These findings suggested that there are no disadvantages to the proposed rotation/revolution agitation method. Rotation/revolution-type agitators are a promising apparatus for preparing chondrocytes for primary cultures and cartilage tissue engineering.

Characterization of poly(L-glutamic acid)-grafted hyaluronan as a novel candidate medicine and biomedical device for intra-articular injection.

A novel hyaluronan (HA) derivative, poly(L-glutamic acid)-grafted hyaluronan (PGA-g-HA), was synthesized to improve the durability of conventional HA products for intra-articular injection. The purpose of this study was to investigate the characteristics of the novel HA derivative in terms of viscoelasticity, degradation behavior, non-immunogenicity, and bioactivity using preliminary in vitro and in vivo experiments. The storage modulus (G') and loss modulus (G″) of PGA-g-HA were similar to those of HA80 (approximately 8.0 × 105 Da) rather than those of original HA200 (approximately 2.0 × 106 Da). PGA-g-HA showed strong resistance against hyaluronidase hydrolysis compared to unmodified HA200. The immunogenicity resulting from grafting PGA to HA200 was not detected in bone marrow derived dendritic cells. The anti-inflammatory activity of PGA-g-HA was confirmed in IL-1ß-stimulated chondrocytes. In addition, compared to unmodified HA200, the intra-articular injection of PGA-g-HA produced greater chondroprotective effects on a monoiodoacetic acid-induced model of rat knee osteoarthritis at two weeks after a single treatment. Therefore, PGA-g-HA is expected to be a promising medicine and biomedical device for intra-articular injection. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3006-3016, 2017.

PNPLA1 has a crucial role in skin barrier function by directing acylceramide biosynthesis.

Mutations in patatin-like phospholipase domain-containing 1 (PNPLA1) cause autosomal recessive congenital ichthyosis, but the mechanism involved remains unclear. Here we show that PNPLA1, an enzyme expressed in differentiated keratinocytes, plays a crucial role in the biosynthesis of ω-O-acylceramide, a lipid component essential for skin barrier. Global or keratinocyte-specific Pnpla1-deficient neonates die due to epidermal permeability barrier defects with severe transepidermal water loss, decreased intercellular lipid lamellae in the stratum corneum, and aberrant keratinocyte differentiation. In Pnpla1-/- epidermis, unique linoleate-containing lipids including acylceramides, acylglucosylceramides and (O-acyl)-ω-hydroxy fatty acids are almost absent with reciprocal increases in their putative precursors, indicating that PNPLA1 catalyses the ω-O-esterification with linoleic acid to form acylceramides. Moreover, acylceramide supplementation partially rescues the altered differentiation of Pnpla1-/- keratinocytes. Our findings provide valuable insight into the skin barrier formation and ichthyosis development, and may contribute to novel therapeutic strategies for treatment of epidermal barrier defects.

Dual Roles of Group IID Phospholipase A2 in Inflammation and Cancer.

Phospholipase A2 enzymes have long been implicated in the promotion of inflammation by mobilizing pro-inflammatory lipid mediators, yet recent evidence suggests that they also contribute to anti-inflammatory or pro-resolving programs. Group IID-secreted phospholipase A2 (sPLA2-IID) is abundantly expressed in dendritic cells in lymphoid tissues and resolves the Th1 immune response by controlling the steady-state levels of anti-inflammatory lipids such as docosahexaenoic acid and its metabolites. Here, we show that psoriasis and contact dermatitis were exacerbated in Pla2g2d-null mice, whereas they were ameliorated in Pla2g2d-overexpressing transgenic mice, relative to littermate wild-type mice. These phenotypes were associated with concomitant alterations in the tissue levels of ω3 polyunsaturated fatty acid (PUFA) metabolites, which had the capacity to reduce the expression of pro-inflammatory and Th1/Th17-type cytokines in dendritic cells or lymph node cells. In the context of cancer, however, Pla2g2d deficiency resulted in marked attenuation of skin carcinogenesis, likely because of the augmented anti-tumor immunity. Altogether, these results underscore a general role of sPLA2-IID as an immunosuppressive sPLA2 that allows the microenvironmental lipid balance toward an anti-inflammatory state, exerting beneficial or detrimental impact depending upon distinct pathophysiological contexts in inflammation and cancer.

The role of group IIF-secreted phospholipase A2 in epidermal homeostasis and hyperplasia.

Epidermal lipids are important for skin homeostasis. However, the entire picture of the roles of lipids, particularly nonceramide lipid species, in epidermal biology still remains obscure. Here, we report that PLA2G2F, a functionally orphan-secreted phospholipase A2 expressed in the suprabasal epidermis, regulates skin homeostasis and hyperplasic disorders. Pla2g2f(-/-) mice had a fragile stratum corneum and were strikingly protected from psoriasis, contact dermatitis, and skin cancer. Conversely, Pla2g2f-overexpressing transgenic mice displayed psoriasis-like epidermal hyperplasia. Primary keratinocytes from Pla2g2f(-) (/-) mice showed defective differentiation and activation. PLA2G2F was induced by calcium or IL-22 in keratinocytes and preferentially hydrolyzed ethanolamine plasmalogen-bearing docosahexaenoic acid secreted from keratinocytes to give rise to unique bioactive lipids (i.e., protectin D1 and 9S-hydroxyoctadecadienoic acid) that were distinct from canonical arachidonate metabolites (prostaglandins and leukotrienes). Ethanolamine lysoplasmalogen, a PLA2G2F-derived marker product, rescued defective activation of Pla2g2f(-/-) keratinocytes both in vitro and in vivo. Our results highlight PLA2G2F as a previously unrecognized regulator of skin pathophysiology and point to this enzyme as a novel drug target for epidermal-hyperplasic diseases.

Subacute systemic toxicity assessment of beta-tricalcium phosphate/carboxymethyl-chitin composite implanted in rat femur.

The efficacy of a composite of beta-tricalcium phosphate particles and carboxymethyl-chitin (beta-TCP/CM-chitin) for bone repair has already been established in animal experiments. In the present study, subacute systemic toxicity was evaluated to further assess the biological safety of the implanted composite. beta-TCP/CM-chitin (approximately 4 mg/kg and 7 mg/kg in male and female rats, respectively) was implanted for 28 days into penetrating defects (2 mm diameter) made artificially in the shaft of the right femur of rats. Sham operation groups with the defect only were prepared as controls. Haematology, blood chemistry, urinalysis, and the histopathology of 44 organs and tissues were investigated. Body weight measurements and clinical observations were performed daily throughout the study. No subacute systemic toxicity possibly caused by the implantation of beta-TCP/CM-chitin was detected. These findings indicate that beta-TCP/CM-chitin composite is a highly biocompatible bone substitute, at least with an implantation dosage of < 4-7 mg/kg.

In vitro cytocompatibility assessment of beta-tricalcium phosphate/carboxymethyl-chitin composite.

A novel bioabsorbable bone substitute composed of a beta-tricalcium phosphate (beta-TCP) and a carboxymethyl-chitin (CM-chitin) sodium has been developed. Rabbit tibia defects (4 mm in diameter) were repaired after 4 weeks more effectively by the composite compared with a sham-operation group. To further investigate the biological safety of the components, genotoxicity and carcinogenicity of an extract prepared from the composite were determined using four different in vitro assays. The main extract component was identified as CM-chitin sodium [average molecular weight (Mw) approximately 230 kDa] as determined by Fourier transform infrared spectroscopy and gel permeation chromatography analysis. The concentrations of P and Ca possibly derived from beta-TCP were 17.7 and 37.1 microg/g, respectively, as determined by inductively coupled plasma mass spectroscopy. Both the metabolic activation and nonactivation (-S9) systems of the rat microsome S9 fraction were used to perform a genotoxicity evaluation using the Ames test and chromosome aberration assay on Chinese hamster lung fibroblast cells treated with the extract. In these assays, no genotoxicity was detected with doses < or =5 mg/mL (maximum concentration). The cell transformation assay using BALB/c 3T3 cells and the metabolic cooperation assay with V79 cells both showed negative results for any tumor-promoting activity caused by the extract (approximately 5 mg/mL). These results indicate that the bioabsorbable beta-TCP/CM-chitin composite is a highly biocompatible bone substitute.

Thromboresistance of alkali- and heat-treated titanium metal formed with apatite.

Fibrin deposition and platelet adhesion onto alkali- and heat-treated titanium metal (AH-Ti), alkali- and water-treated titanium metal (Wa-Ti), and alkali- and heat-treated titanium metal formed with apatite (Ap-Ti) in simulated body fluid (SBF) were evaluated by exposure to anticoagulated blood or washed platelet suspension (WPS) under static conditions and subsequent observation with scanning electron microscopy (SEM). The results were compared with those for commercially pure titanium metal (cp-Ti). Thrombus formation on AH-Ti and Wa-Ti, which were exposed to heparinized whole blood for 1 h, was significantly less than that on cp-Ti, on which pronounced depositions of fibrin-erythrocytes and lymphocytes were observed. No thrombus was observed on Ap-Ti, possibly because of a high adsorption of heparin. Morphological change of platelets attached to surfaces via adsorbed plasma proteins was found to a significant extent on AH-Ti and Wa-Ti exposed to WPS. However, there was almost no difference between cp-Ti and Ap-Ti in round morphology of adherent platelets. These findings suggested that Ap-Ti exhibits stronger antithrombogenic characteristics than cp-Ti and other materials examined in heparinized blood.