Collagen of chronically inflamed skin is over-modified and upregulates secretion of matrix metalloproteinase 2 and matrix-degrading enzymes by endothelial cells and fibroblasts

In order to investigate the properties of collagen in chronically inflamed tissue, we isolated collagen from the ear skin of mice with chronic contact dermatitis and examined its biochemical characteristics and the functions that regulate the secretion of matrix metalloproteinase 2 and collagen-degrading enzymes from endothelial cells and fibroblasts. Collagen in skin with chronic contact dermatitis comprised 60% type I collagen and 40% type III collagen, which latter is higher than the content of type III collagen in control skin (35%). The denaturation temperature was higher (42 degrees C) than that of control skin (39 degrees C). The alpha2 chain of type I collagen was over-hydroxylated at both proline and lysine residues. Segment-long-spacing crystallites of type I collagen were unusually connected in tandem. Collagen of chronically inflamed skin was less susceptible to matrix metalloproteinase 2 after heat denaturation. Endothelial cells and fibroblasts secreted an increased amount of matrix metalloproteinase 2 when cultured on a gel formed from the collagen of chronically inflamed skin. Collagen-degrading activity secreted from fibroblasts was also upregulated when cells were in contact with collagen of chronically inflamed skin. These results suggest that the collagen in chronically inflamed tissue has altered biochemical characteristics and functions, which may affect the pathogenesis of the chronic skin disease.     

Distribution of the longevity gene product, SIRT1, in developing mouse organs

A longevity gene product, Sir2 (silent information regulator 2) is a NAD-dependent histone deacetylase involved in longevity in yeasts, worms and flies. The mammalian homolog of Sir2, SIRT1(sirtuin 1), has been shown to play important roles related to anti-aging effects (regulating apoptosis, stress tolerance, insulin resistance, and fat metabolism). Recently, SIRT1 expression has been demonstrated to occur at as early as embryonic day 10.5 in mice. SIRT1 during developing period may be involved in the mechanism of developmental origins of adult diseases, such as diabetes and cardiovascular disease. To investigate the contribution of SIRT1, it is important to reveal the distribution of this protein during development. In the present study, we demonstrated the distribution of immunoreactivity of SIRT1 in mouse organs during prenatal and neonatal development by staining a wide variety of serial sections. The SIRT1 immunoreactivity was strongly observed in the neuroepithelial layer, dorsal root ganglion, trigeminal ganglion, eyes, roots of whiskers, and internal organs, including the testis, liver, heart, kidney, and lung during the fetal period. Neurons which had finished migrating still showed relatively strong immunoreactivity. The immunoreactivity was completely absorbed by the blocking peptide in an absorption test. During the postnatal period, the immunoreactivities in most of these organs, except the heart and testis weakened, with the liver most dramatically affected. As SIRT1 expression was demonstrated in a wide variety of developing organs, further study to investigate prenatal factors which affect SIRT1 expression and its activity is important.     

Summary of 17 chemicals evaluated by OECD TG229 using Japanese Medaka,Oryzias latipes in EXTEND 2016

In June 2016, the Ministry of the Environment of Japan announced a program “EXTEND2016” on the implementation of testing and assessment for endocrine active chemicals, consisting of a two-tiered strategy. The aim of the Tier 1 screening and the Tier 2 testing is to identify the impacts on the endocrine system and to characterize the adverse effects to aquatic animals by endocrine disrupting chemicals detected in the aquatic environment in Japan. For the consistent assessment of the effects on reproduction associated with estrogenic, anti-estrogenic, androgenic, and/or anti-androgenic activities of chemicals throughout Tier 1 screening to Tier 2 testing, a unified test species, Japanese medaka (Oryzias latipes), has been used. For Tier 1 screening, the in vivo Fish Short-Term Reproduction Assay (OECD test guideline No. 229) was conducted for 17 chemicals that were nominated based on the results of environmental monitoring, existing knowledge obtained from a literature survey, and positive results in reporter gene assays using the estrogen receptor of Japanese medaka. In the 17 assays using Japanese medaka, adverse effects on reproduction (i.e., reduction in fecundity and/or fertility) were suggested for 10 chemicals, and a significant increase of hepatic vitellogenin in males, indicating estrogenic (estrogen receptor agonistic) potency, was found for eight chemicals at the concentrations in which no overt toxicity was observed. Based on these results, and the frequency and the concentrations detected in the Japanese environment, estrone, 4-nonylphenol (branched isomers), 4-tert-octylphenol, triphenyl phosphate, and bisphenol A were considered as high priority candidate substances for the Tier 2 testing.     

Effect of thyroid hormone-disrupting chemicals on swim bladder inflation and thyroid hormone-related gene expression in Japanese medaka and zebrafish

We compared the influence of thyroid hormone-disrupting chemicals (heptafluorobutanoic acid, PFBA and tris[1,3-dichloro-2-propyl] phosphate, TDCPP) and thyroid hormone (3,3′,5-triiodo-L-thyronine, T3) on swim bladder inflation and thyroid hormone-related gene expression in Japanese medaka and zebrafish. The swim bladder of most larvae had inflated at 4 h post hatching (hph) in Japanese medaka and at 48 hph in zebrafish in controls. In both fish species, the swim bladder inflation was inhibited in larvae exposed to PFBA (lowest observed effect concentration [LOEC] in medaka: 40 mg/L; in zebrafish: 80 mg/L), TDCPP (LOEC in medaka: 1 mg/L; in zebrafish: 0.5 mg/L), and T3 (no inhibition in Japanese medaka; LOEC in zebrafish: 7.5 μg/L). We also examined the influence of PFBA, TDCPP, and T3 on the expression of thyroid stimulating hormone subunit beta (tshβ) or thyroid hormone receptor alpha (trα) and beta (trβ). No changes were observed in the expression of genes after PFBA and TDCPP exposure; however, T3 exposure upregulated trα and trβ expression in both fish species. When the results were compared between Japanese medaka and zebrafish, swim bladder inflation in both species was found to be inhibited by exposure to thyroid hormone-disrupting chemicals. Our results show that inhibition of the swim bladder inflation at 4 hph in Japanese medaka and 48 hph in zebrafish is a potential indicator of thyroid hormone-disturbing activity of chemicals.     

Utility of real-time diagnosis using contact endoscopy for oral and lingual diseases

Objective

In this study, we prospectively investigated the diagnostic accuracy of CE findings in oral and lingual diseases.

Methods

Between January 2004 and December 2009, CE was used to examine 66 patients with oral and lingual diseases at Hyogo College of Medicine Hospital. Blood vessel networks and superficial cell layers in the mucosal epithelium of normal and lesion sites were observed after staining with 1% methylene blue. Endoscopic diagnoses (CE diagnosis) were compared with subsequent definitive diagnoses based on pathological findings. The sensitivity and specificity for CE diagnosis were calculated.

Results

On CE findings, SCC showed the characteristics of absent and tortuous blood vessels, heterogeneous distribution, and increased nucleus/cytoplasm (N/C) ratio. Leukoplakia showed no atypical cells, abundant cornified layers, or cytoplasm without nuclei. Lesions were pathologically classified into three groups: 46 squamous cell carcinomas (SCC), 10 leukoplakias, and 10 other benign lesions (n = 66). In 4 patients with SCC, malignancy was underestimated by CE findings. The overall diagnostic rate of the CE was 93.9% (62/66 patients). The sensitivity and specificity of SCC were 0.913 (42/46 patients) and 1.0 (20/20 patients), respectively.

Conclusion

The usefulness of contact endoscopy (CE) as an in vivo real-time diagnostic instrument that can deliver results prior to pathological confirmation was suggested.     

Apoptotic cell death in erythrocytes of p53-deficient medaka (Oryzias latipes) after γ-irradiation

Purpose: Previous studies have examined the effects of γ-irradiation (γ-IR) on wild-type and p53 mutant Medaka (Oryzias latipes) 24?hours after irradiation and in the present work, apoptosis and alterations in erythrocytes of 4, 8 and 24?h and 14 days after gamma-ray irradiation were reported as genotoxic biomarkers of γ-irradiation.

Materials and methods: Sexually mature wild-type, WT (Hd-rR) and p53(?/?) adult female medaka (O. latipes) were exposed to 4?Gy dose of γ-IR and sampling were collected after 4, 8 and 24?h and 14 days.

Results: Apoptosis and morphological alterations were observed from 4?h after irradiation and remarkably increased 8?h after irradiation in the wild-type. Apoptotic cell death has been observed 8?h after irradiation most prominently but subtle in p53 mutant medaka. All these phenotypes were recovered 14 days after irradiation in both strains. Although no micronuclei were seen in any group, nuclear abnormalities were observed in red blood cells. Both apoptosis and morphological alterations in erythrocytes were decreased after 24 and 14 days after γ-irradiation.

Conclusions: We conclude that apoptosis and malformations caused by 4?Gy γ-irradiation in the erythrocytes of medaka fish occurs from 4–24?h and the initial response until 8?h was p53-dependent.     

A computer simulation method for low-dose CT images by use of real high-dose images: a phantom study

Practical simulations of low-dose CT images have a possibility of being helpful means for optimization of the CT exposure dose. Because current methods reported by several researchers are limited to specific vendor platforms and generally rely on raw sinogram data that are difficult to access, we have developed a new computerized scheme for producing simulated low-dose CT images from real high-dose images without use of raw sinogram data or of a particular phantom. Our computerized scheme for low-dose CT simulation was based on the addition of a simulated noise image to a real high-dose CT image reconstructed by the filtered back-projection algorithm. First, a sinogram was generated from the forward projection of a high-dose CT image. Then, an additional noise sinogram resulting from use of a reduced exposure dose was estimated from a predetermined noise model. Finally, a noise CT image was reconstructed with a predetermined filter and was added to the real high-dose CT image to create a simulated low-dose CT image. The noise power spectrum and modulation transfer function of the simulated low-dose images were very close to those of the real low-dose images. In order to confirm the feasibility of our method, we applied this method to clinical cases which were examined with the high dose initially and then followed with a low-dose CT. In conclusion, our proposed method could simulate the low-dose CT images from their real high-dose images with sufficient accuracy and could be used for determining the optimal dose setting for various clinical CT examinations.     

Development of novel in vitro photosafety assays focused on the Keap1–Nrf2–ARE pathway

Although photoallergens require UV energy for antigen formation, the subsequent immune response is considered to be the same as in ordinary skin sensitization. Therefore, in vitro tests for skin sensitization should also be applicable for photoallergy testing. In this study, we examined whether activation of the Keap1 (Kelch‐like ECH‐associated protein 1)–Nrf2 (nuclear factor‐erythroid 2‐related factor 2)–ARE (antioxidant response element) pathway could be used to assess the photoallergenic potential of chemicals, using the reporter cell line AREc32 or KeratinoSens^TM. First, we identified an appropriate UVA irradiation dose [5 J cm^–2 irradiation in phosphate‐buffered saline (PBS)] by investigating the effect of UV irradiation on ARE‐dependent gene induction using untreated or 6‐methylcoumarin (6‐MC)‐treated cells. Irradiation of well‐known photoallergens under this condition increased ARE‐dependent gene expression by more than 50% compared with both vehicle and non‐irradiated controls. When the cut‐off value for detecting photoallergens was set at 50% induction, the accuracy of predicting photoallergenic/phototoxic chemicals was 70% in AREc32 cells and 67% in KeratinoSens^TM cells, and the specificity was 100% in each case. We designate these assays as a photo‐ARE assay and photo‐KeratinoSens^TM, respectively. Our results suggest that activation of the Keap1‐Nrf2‐ARE pathway is an effective biomarker for evaluating both photoallergenic and phototoxic potentials. Either of the above tests might be a useful component of a battery of in vitro tests/in silico methods for predicting the photoallergenicity and phototoxicity of chemicals. Copyright © 2015 John Wiley & Sons, Ltd.