[Nationwide Survey of Medical Radiation Exposure on Cardiovascular Examinations in Japan].

PURPOSE: It is very important to manage the radiation dose of cardiovascular interventional (CVI) procedures. Overseas, the diagnostic reference levels for cardiac interventional procedures were established with the air kerma at the patient entrance reference point (Ka,r) and the air kerma-area product (PKA). Although the Japan DRLs 2015 was established by the Japan Network for Research and Information on Medical Exposure (J-RIME), the Japan DRL for CVIs were established by fluoroscopic dose rates of 20 mGy/min at the patient entrance reference point with 20 cm thickness polymethyl methacrylate (PMMA) phantom. In the present our study, we performed a questionnaire survey of indicated values of angiographic parameters in CVI procedures. METHODS: A nationwide questionnaire was sent by post to 765 facilities. Question focused on angiographic technology, exposure parameters and radiation doses as the displayed dosimetric parameters on the angiographic machine. RESULTS: The recovery rate was 22.8% at 175 out of 765 facilities. In total 1728 cases of the coronary angiography (CAG), 1703 cases of the percutaneous coronary intervention (PCI), 962 cases of the radiofrequency catheter ablation (RFCA) and 377 cases of pediatric CVI. The 75th percentile value of Ka,r, PKA, fluoroscopy time (FT) and number of cine images (CI) for CAG, PCI, RFCA and pediatric CVI were 702, 2042, 644, and 159 mGy, respectively, 59.3, 152, 81.3, and 14.9 Gy・cm2, respectively, 10.2, 35.6, 61.1, and 35.6 min, respectively and 1503, 2672, 722, and 2378 images, respectively. Our investigation showed that the angiographic parameters were different in several CVI procedures. CONCLUSIONS: The displayed dosimetric parameters on the angiographic machine in CVI procedures showed different values. We should classify the dosimetric parameters for each procedure.

Epidemiological and pathological study of feline morbillivirus infection in domestic cats in Japan.

BACKGROUND: Feline morbillivirus (FmoPV) is a novel paramyxovirus found to infect domestic cats. FmoPV has been isolated in several countries in Asia and Europe and is considered to have genetic diversity. Also, it is suspected to be associated with feline renal diseases including tubulointerstitial nephritis (TIN), which affects domestic cats with a high incidence rate. RESULTS: To clarify the state of FmoPV infection among domestic cats in Japan, an epidemiological survey was conducted. Twenty-one out of 100 cats were found to have serum antibodies (Ab) against FmoPV-N protein by indirect immunofluorescence assay (IF) using FmoPV-N protein-expressing HeLa cells. Twenty-two of the cats were positive for FmoPV RNA in the urine and/or renal tissues. In total, 29 cats were positive for Ab and/or viral RNA. These FmoPV-infected cats were classified into three different phases of infection: RNA+/Ab + (14 cats), RNA+/Ab- (8 cats) and RNA-/Ab + (7 cats). In immunohistochemistry (IHC), 19 out of 29 cats were positive for FmoPV-N protein in kidney tissues; however, the FmoPV-N protein was located in the inflammatory lesions with severe grade in only four out of the 19 cats. Since 15 out of 29 infected cats were positive for viral RNA and Ab, approximately half of the infected cats were persistently infected with FmoPV. CONCLUSIONS: A statistically significant difference was observed between infection of FmoPV and the presence of inflammatory changes in renal lesions, indicating a relationship between FmoPV infection and feline renal diseases. However, we could not obtain histopathological evidence of a relationship between FmoPV infection and TIN.

Age-related decrease in CD271(+) cells in human skin.

According to recent studies, stem cells are found in various tissues in our bodies. It has been reported that stem cells can reside in the skin tissues, including the epidermis, dermis, hair follicles and subcutaneous tissues. Homeostasis of the skin is maintained because these stem cells collaborate with each other to form new cells. We previously identified the CD271(p75NTR)(+) cell as a stem cell that was present in the epidermis, dermis and subcutaneous tissue, and further investigated the role of stem cells in wound healing and their association with skin disease. In this study, we investigated the localization of CD271(+) cells in human skin (epidermis and dermis) and its age-related changes in stem cells using CD271(+) cells. The study revealed that the number of CD271(+) cells in the epidermis and dermis decreased with aging. It is possible that such an age-related decrease in stem cells causes impaired regenerative ability and is associated with various skin diseases. If the relationship between stem cells and skin aging and diseases can be elucidated by investigations such as this study, it may lead to the development of novel anti-aging technologies and medical treatments for skin diseases in the future.

Senescent dermal fibroblasts enhance stem cell migration through CCL2/CCR2 axis.

During aging, increases in the number of senescent cells are seen in various tissues. On the other hand, stem cells play crucial roles in tissue repair and homeostasis. Therefore, it is likely that stem cells give rise to new cells that replace senescent cells. However, how stem cells contribute to homeostasis in the dermis has not been elucidated. Here, we investigated the effects of factors secreted from senescent fibroblasts on stem cells. We found that senescent human dermal fibroblast (HDF) conditioned medium (CM) significantly enhanced stem cell migration compared with young HDF CM. The senescent HDF CM strongly secreted chemokine ligand 2 (CCL2). Furthermore, CCL2 was found to enhance stem cell migration, and the inhibition of CCR2, a receptor for CCL2, reduced stem cell migration. These results suggest that senescent fibroblasts recruit stem cells by secreting various factors and that the CCL2/CCR2 axis is one of the mechanisms underlying this phenomenon.

Identification of a natural recombination in the F and H genes of feline morbillivirus.

Feline morbillivirus (FmoPV) has recently been identified in Hong Kong and Japan. FmoPV is considered to belong to the genus Morbillivirus, in the family Paramyxoviridae. In this study, the complete nucleotide sequences of three strains of FmoPV detected in cats in Japan were determined. Among the six genes in FmoPV; N, P/V/C, M, F, H and L, the P gene showed the highest polymorphism in the nucleotide and putative amino acid sequences among the FmoPV strains. There was no geographical association in terms of the FmoPV phylogeny; however, from extensive phylogenetic and recombination analyses, we found that one Japanese FmoPV strain, MiJP003, was a probable recombinant between two virus strains in the independent lineages found in Japan and Hong Kong, respectively. The recombination was considered to have occurred within the F and H genes. Such recombination is thought to be involved in the evolution of FmoPV.

Accelerated differentiation of melanocyte stem cells contributes to the formation of hyperpigmented maculae.

It has been reported that the abnormal regulation of melanocyte stem cells (McSCs) causes hair greying; however, little is known about the role of McSCs in skin hyperpigmentation such as solar lentigines (SLs). To investigate the involvement of McSCs in SLs, the canonical Wnt signalling pathway that triggers the differentiation of McSCs was analysed in UVB-induced delayed hyperpigmented maculae in mice and human SL lesions. After inducing hyperpigmented maculae on dorsal skin of F1 mice of HR-1× HR/De, which was formed long after repeated UVB irradiation, the epidermal Wnt1 expression and the number of nuclear ß-catenin-positive McSCs were increased as compared to non-irradiated control mice. Furthermore, the expression of dopachrome tautomerase (Dct), a downstream target of ß-catenin, was significantly upregulated in McSCs of UVB-irradiated mice. The Wnt1 expression and the number of nuclear ß-catenin-positive McSCs were also higher in human SL lesions than in normal skin. Recombinant Wnt1 protein induced melanocyte-related genes including Dct in early-passage normal human melanocytes (NHEMs), an in vitro McSC model. These results demonstrate that the canonical Wnt signalling pathway is activated in SL lesions and strongly suggest that the accelerated differentiation of McSCs is involved in SL pathogenesis.

ZIP2 protein, a zinc transporter, is associated with keratinocyte differentiation.

Zinc is essential for the proper functioning of various enzymes and transcription factors, and its homeostasis is rigorously controlled by zinc transporters (SLC39/ZIP, importers; SLC30/ZnT, exporters). Skin disease is commonly caused by a zinc deficiency. Dietary and inherited zinc deficiencies are known to cause alopecia and the development of vesicular or pustular dermatitis. A previous study demonstrated that zinc played crucial roles in the survival of keratinocytes and their unique functions. High levels of zinc have been detected in the epidermis. Epidermal layers are considered to use a mechanism that preferentially takes in zinc, which is involved with the unique functions of keratinocytes. However, few studies have investigated the ZIP (Zrt- and Irt-like protein) proteins specifically expressed in keratinocytes and their functions. We explored the ZIP proteins specifically expressed in the epidermis and analyzed their functions. Gene expression analysis showed that the expression of ZIP2 was consistently higher in the epidermis than in the dermis. Immunohistochemistry analysis confirmed the expression of ZIP2 in differentiating keratinocytes. The expression of ZIP2 was found to be up-regulated by the differentiation induction of cultured keratinocytes. Intracellular zinc levels were decreased in keratinocytes when ZIP2 was knocked down by siRNA, and this subsequently inhibited the differentiation of keratinocytes. Moreover, we demonstrated that ZIP2 knockdown inhibited the normal formation of a three-dimensional cultured epidermis. Taken together, the results of this study suggest that ZIP2, a zinc transporter expressed specifically in the epidermis, and zinc taken up by ZIP2 are necessary for the differentiation of keratinocytes.

Becker muscular dystrophy-like myopathy regarded as so-called "fatty muscular dystrophy" in a pig: a case report and its diagnostic method.

We describe a case of human Becker muscular dystrophy (BMD)-like myopathy that was characterized by the declined stainability of dystrophin at sarcolemma in a pig and the immunostaining for dystrophin on the formalin-fixed, paraffin-embedded (FFPE) tissue. The present case was found in a meat inspection center. The pig looked appeared healthy at the ante-mortem inspection. Muscular abnormalities were detected after carcass dressing as pale, discolored skeletal muscles with prominent fat infiltrations and considered so-called "fatty muscular dystrophy". Microscopic examination revealed following characteristics: diffused fat infiltration into the skeletal muscle and degeneration and regeneration of the remaining skeletal muscle fibers. Any lesions that were suspected of neurogenic atrophy, traumatic muscular degeneration, glycogen storage disease or other porcine muscular disorders were not observed. The immunostaining for dystrophin was conducted and confirmed to be applicable on FFPE porcine muscular tissues and revealed diminished stainability of dystrophin at the sarcolemma in the present case. Based on the histological observations and immunostaining results, the present case was diagnosed with BMD-like myopathy associated with dystrophin abnormality in a pig. Although the genetic properties were not clear, the present BMD-like myopathy implied the occurrence of dystrophinopathy in pigs. To the best of our knowledge, this is the first report of a natural case of myopathy associated with dystrophin abnormalities in a pig.

Comprehensive analysis of melanogenesis and proliferation potential of melanocyte lineage in solar lentigines.

BACKGROUND: Solar lentigines (SLs) are characterized by hyperpigmented macules, commonly seen on sun-exposed areas of the skin. Although it has been reported that an increase in the number of melanocytes and epidermal melanin content was observed in the lesions, the following questions remain to be answered: (1) Is acceleration of melanogenesis in the epidermis caused by an increased number of melanocytes or the high melanogenic potential of each melanocyte? (2) Why does the number of melanocytes increase? OBJECTIVE: To elucidate the pathogenic mechanism of SLs by investigating the number, melanogenic potential and proliferation status of the melanocyte lineage in healthy skin and SL lesions. METHODS: Immunostaining for melanocyte lineage markers (tyrosinase, MART-1, MITF, and Frizzled-4) and a proliferation marker, Ki67, was performed on skin sections, and the obtained images were analyzed by image analysis software. RESULTS: The expression level of tyrosinase to MART-1 of each melanocyte was significantly higher in SL lesions than healthy skin. The numbers of melanocytes in the epidermis, melanoblasts in the hair follicular infundibulum and melanocyte stem cells in the bulge region were increased in SL; however, no significant difference was observed in the Ki67-positive rate of these cells. CONCLUSION: The melanogenic potential of each melanocyte was elevated in SL lesions. It was suggested that the increased number of melanocytes in the SL epidermis might be attributed to the abnormal increase of melanocyte stem cells in the bulge.

Analysis of the effects of hydroquinone and arbutin on the differentiation of melanocytes.

Hydroquinone (HQ) is a chemical compound that inhibits the functions of melanocytes and has long been known for its skin-whitening effect. According to previous studies, the Tyrosinase (Tyr) activity inhibitory effect and melanocyte-specific cell toxicity are known depigmenting mechanisms; however, details of the underlying mechanisms are unknown. Arbutin (Arb) is also known for its Tyr activity inhibitory effect and is commonly used as a skin-whitening agent. However, the detailed depigmenting mechanism of Arb is also not yet fully understood. Few studies have attempted to elucidate the effects of HQ and Arb on undifferentiated melanocytes. In this study, we examined the effects of HQ and Arb throughout each stage of differentiation of melanocytes using a mouse embryonic stem cell (ESC) culture system to induce melanocytes. The results showed that HQ in particular downregulated the early stage of differentiation, in which neural crest cells were generated, and the late stage of differentiation, in which melanogenesis became active. On the other hand, Arb had no effect on the differentiation of melanocytes, and only suppressed melanogenesis by specifically suppressing elevations in Tyr expression in the late stage of differentiation.

Bleomycin inhibits adipogenesis and accelerates fibrosis in the subcutaneous adipose layer through TGF-ß1.

Systemic sclerosis [scleroderma (SSc)]-associated skin fibrosis is characterized by increased fibrosis in the dermis and a reduction in the thickness of the subcutaneous adipose tissue layer. Although many studies have examined fibrosis in SSc, only a few studies have focused on the associated reduction in the thickness of the subcutaneous adipose tissue layer. In this study, we investigated the effects of SSc-induced fibrosis on adipose tissue. We found that bleomycin suppresses adipogenesis in adipose-derived stem cells (ASCs) and stimulates ASCs to express transforming growth factor ß1 (TGF-ß1), which suppresses adipogenesis and promotes fibrosis. Furthermore, we found that adipocyte-conditioned medium suppressed collagen synthesis by fibroblasts in fibrosis-like conditions. We concluded that in the skin affected by bleomycin-induced fibrosis, increased TGF-ß1 expression suppresses adipogenesis and promotes adipocyte fibrosis. It was also suggested that adipocytes have an inhibitory effect on the progression of fibrosis.

Epithelial-mesenchymal transition of the eccrine glands is involved in skin fibrosis in morphea.

Morphea is a type of localized scleroderma. It is a skin disease involving the development of fibrosis in the dermis and subcutaneous fat tissue beneath without a visceral lesion, and the cause is still unclear. An involvement of epithelial-mesenchymal transition (EMT) has been reported as a cause of tissue fibrosis, but this was mostly observed in pulmonary and hepatic fibrosis, and the involvement of EMT in a skin disease, morphea, has not been studied . Thus, we analyzed the involvement of EMT in skin fibrosis in morphea patients using pathological techniques. Skin lesions of six morphea patients were analyzed (five female and one male patient). As a control, non-light-exposed skin lesions of 11 healthy females were analyzed. Concretely, tissue samples were prepared from these subjects and subjected to immunostaining of transforming growth factor (TGF)-ß1, α-smooth muscle actin (α-SMA) and fibronectin, which have been reported to be associated with fibrosis, and Snail1 and E-cadherin, which are considered to be involved in EMT, and expressions of these were analyzed. In morphea patients, dermal expression of TGF-ß1, α-SMA and fibronectin, which are involved in fibrosis, was enhanced, and, at the same time, enhanced expression of Snail1 and reduced expression of E-cadherin, which are involved in EMT, were observed in the dermal eccrine glands. These findings suggested the progression of EMT in the dermal eccrine glands in morphea.

Lignin Induces ES Cells to Differentiate into Neuroectodermal Cells through Mediation of the Wnt Signaling Pathway.

Embryonic stem cells (ES cells) are characterized by their pluripotency and infinite proliferation potential. Ever since ES cells were first established in 1981, there have been a growing number of studies aimed at clinical applications of ES cells. In recent years, various types of differentiation inducement systems using ES cells have been established. Further studies have been conducted to utilize differentiation inducement systems in the field of regenerative medicine. For cellular treatments using stem cells including ES cells, differentiation induction should be performed in a sufficient manner to obtain the intended cell lineages. Lignin is a high-molecular amorphous material that forms plants together with cellulose and hemicelluloses, in which phenylpropane fundamental units are complexly condensed. Lignin derivatives have been shown to have several bioactive functions. In spite of these findings, few studies have focused on the effects of lignin on stem cells. Our study aimed to develop a novel technology using lignin to effectively induce ES cells to differentiate into neuroectodermal cells including ocular cells and neural cells. Since lignin can be produced at a relatively low cost in large volumes, its utilization is expected for more convenient differentiation induction technologies and in the field of regenerative medicine in the future.

Wnt/ß-catenin and kit signaling sequentially regulate melanocyte stem cell differentiation in UVB-induced epidermal pigmentation.

UV radiation is a well-known inducer of epidermal pigmentation that is utilized in therapy for vitiligo, one of the skin depigmentation disorders. Although it has been reported that melanocyte stem cells (McSCs) play essential roles in hair pigmentation, the relationship between McSCs and epidermal pigmentation remains unclear. Repetitive UVB irradiation on the dorsal skin of F1 mice of HR-1 × HR/De caused apparent epidermal pigmentation, and it was characterized by increase in the number of melanocytes. Interestingly, differentiation of McSCs into melanoblasts in hair follicles was followed by induction of epidermal melanocyte differentiation. Administration of a neutralizing antibody for Kit receptor that depletes resident melanoblasts could not suppress increased number of melanocytes. UVB irradiation also induced robust expression of Wnt7a as well as Kitl in epidermis, and ß-catenin translocation into nucleus in McSCs. Intradermal injection of IWR-1 (inhibitor of Wnt response 1), a chemical inhibitor of ß-catenin activation, and small interfering RNA (siRNA) against Wnt7a suppressed increase in the number of epidermal melanocytes. Taken altogether, it was demonstrated that Wnt7a triggered McSCs differentiation through ß-catenin activation, and Kitl might induce following migration of melanoblasts to epidermis. These findings will help in developing therapeutic technologies for vitiligo and other pigmentary disorders.

Major amino acids in collagen hydrolysate regulate the differentiation of mouse embryoid bodies.

To take advantage of the therapeutic potential of embryonic stem cells (ESCs), it is necessary to regulate their differentiation in response to defined factors. In this study, in order to explore novel molecules that regulate the differentiation of ESCs, we investigated whether collagen hydrolysate, collagen-characteristic amino acids, glycine (Gly), l-proline and trans-4-hydroxy-l-proline (l-Hyp); or dipeptides, proline-hydroxyproline and hydroxyproline-glycine regulate the differentiation of mouse embryoid bodies (EBs). We identified that treatment with collagen hydrolysate or Gly repressed the expression of the mesendodermal markers, Brachyury and Foxa2 in EBs and maintained the undifferentiated state of mESCs in a feeder-free monolayer culture. In contrast, l-Hyp promoted the expression of Brachyury, Mixl1, Gsc and Foxa2 in EBs. And the treatment with l-Hyp promoted cardiac differentiation within EBs, which was proven by the spontaneous contraction of cardiomyocytes and the expression of the cardiac markers, α-MHC, MLC-2v and Nkx2.5. Results suggest that l-Hyp is a promising new inducer for reproducible and efficient differentiation of mesendoderm lineages.

Increased hydrophobicity in Malassezia species correlates with increased proinflammatory cytokine expression in human keratinocytes.

Malassezia cells stimulate cytokine production by keratinocytes, although this ability differs among Malassezia species for unknown reasons. The aim of this study was to clarify the factors determining the ability to induce cytokine production by human keratinocytes in response to Malassezia species. M. furfur NBRC 0656, M. sympodialis CBS 7222, M. dermatis JCM 11348, M. globosa CBS 7966, M. restricta CBS 7877, and three strains each of M. globosa, M. restricta, M. dermatis, M. sympodialis, and M. furfur maintained under various culture conditions were used. Normal human epidermal keratinocytes (NHEKs) (1 × 10(5) cells) and the Malassezia species (1 × 10(6) cells) were co-cultured, and IL-1α, IL-6, and IL-8 mRNA levels were determined. Moreover, the hydrophobicity and ß-1,3-glucan expression at the surface of Malassezia cells were analyzed. The ability of Malassezia cells to trigger the mRNA expression of proinflammatory cytokines in NHEKs differed with the species and conditions and was dependent upon the hydrophobicity of Malassezia cells not ß-1,3-glucan expression.

[The comparison between dose rates at the interventional reference point of the angiography systems in many facilities].

The management of the radiation dose is very important in interventional radiology (IVR), especially in percutaneous coronary intervention (PCI). Therefore, we measured entrance surface doses at the interventional reference point of 27 cardiac intervention procedures in 22 cardiac catheterization laboratories around Hiroshima, and compared these doses. Recently, for cardiac interventional radiology, the X-ray machines using flat-panel detectors (FPD) instead of image intensifiers (I.I.) is increasing; 13 systems used FPD and 14 systems used I.I. For fluoroscopy rate, the difference between laboratories was 9 times. For cineangiography rate, the difference between laboratories was 7 times. In addition, between both devices, the I.I. group is bigger than the FPD group. When comparing by the same condition, for the dose at the interventional reference point, no significant difference was detected between the FPD group and the I.I. group. This study shows that FPD is not available for reducing the radiation dose simply. Therefore, it is necessary that we think of the balance with image quality and radiation dose. The optimization of the devices and cardiac intervention procedures becomes very important.