Atsushi Takamiyagi

Low-dose UVB contributes to host resistance against Leishmania amazonensis infection in mice through induction of gamma interferon and tumor necrosis factor alpha cytokines.

ABSTRACT UV radiation suppresses the immune response, a fact which raises the question of whether the phenomenon may find practical applications in the outcome of infectious diseases. In this study, BALB/c mice were exposed to low-dose UVB (250 J/m2) from Dermaray M-DMR-100 for 4 consecutive days. Twelve hours after the last UV exposure, groups of mice were injected with 2 × 106Leishmania amazonensis promastigotes. The development of skin lesions, as assessed by measurement of visible cutaneous lesions, was significantly suppressed in low-dose UVB-irradiated mice compared to nonirradiated controls. In order to characterize the cytokines involved in this phenomenon, BALB/c mice were irradiated with identical doses of UVB, and gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 4 cytokine levels in blood serum and skin were examined at different times by a sandwich enzyme-linked immunosorbent assay, immunohistochemical analysis, and reverse transcription (RT)-PCR. Upregulated expression of serum IFN-γ and TNF-α was observed from 6 to 24 h. Positive results for IFN-γ and TNF-α in UVB-irradiated mice were obtained by immunohistochemical analysis. By RT-PCR, the mRNA expression of both IFN-γ and TNF-α cytokines was detected in a time-dependent manner only in UVB-irradiated mice. Histopathological analysis and electron microscopy revealed that cellular infiltration, tissue parasitism, and parasitophorus vacuoles in irradiated mice were markedly less noticeable than those in nonirradiated controls. These results suggested that low-dose UVB irradiation played a pathogen-suppressing role in Leishmania-susceptible BALB/c mice via systemic and local upregulation of Th1 (IFN-γ and TNF-α) cytokines.

Association of human papillomavirus type 16 with malignant melanoma

We report a case of malignant melanoma associated with human papillomavirus (HPV) in a 37-year-old woman. The patient has had numerous brown papular and nodular tumors, 5 to 30 mm in diameter, on her left leg for > 15 years, some of them coalescing rapidly in the last 12 months to a multilobulated black nodule diagnosed as malignant melanoma by histology and immunohistochemistry. HPV type 16 DNA was detected in the melanoma specimen by reverse transcriptase polymerase chain reaction (rt-PCR) and in situ hybridization (ISH) of the tumor tissues. This is the first report of melanoma associated with HPV 16.

Pre-exposure with low-dose UVA suppresses lesion development and enhances Th1 response in BALB/c mice infected with Leishmania (Leishmania) amazonensis

This study was conducted to determine whether exposing mice to ultraviolet (UV) radiation would alter the pathogenesis of infection with Leishmania (Leishmania) amazonensis (L. amazonensis) which causes progressive cutaneous disease in susceptible mouse strains. BALB/c mice were irradiated with 10 and 30 J/cm(2) UVA on shaved skin of the back from Dermaray (M-DMR-100) for 4 consecutive days before infection with Leishmania promastigotes. The course of disease was recorded by measuring the size of lesions at various times after infection. Mice groups irradiated with UVA 10 and 30 J/cm(2) showed significantly suppressed lesion development compared with the non-irradiated mice. Light and electron microscopy revealed a few parasites at the site of inoculation in UVA-irradiated subjects. Sandwich enzyme-linked-immunosorbent-assay (ELISA) examination of sera showed dose dependently upregulated interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12, and downregulated interleukin (IL)-4 and interleukin (IL)-10 levels in UVA-irradiated as compared with the non-irradiated mice. Positive signals for IFN-gamma mRNA in irradiated mice were obtained by RT-PCR, while non-irradiated mice showed negative results. None of the examined samples showed signal for IL-4 mRNA. The present study disclosed that exposure of mice to different low-doses of UVA irradiation prior to infection may interfere with immunity to L. amazonensis in the murine model. This indicates that the cell-mediated response switch from Th2 to Th1 pattern suppressed the cutaneous lesions of L. amazonensis.

An assessment of the malignant potential of actinic keratoses and Bowen's disease : p53 and PCNA expression pattern correlate with the number of desmosomes

Actinic keratoses (AK) and Bowens disease (BD), both intraepidermal skin tumors, have a potential progression to squamous cell carcinoma (SCC). To evaluate the malignant potential of AK and BD, the expression pattern of p53 protein and proliferating cell nuclear antigen (PCNA) were examined in five types of AK and BD by immunohistochemistry. The ultrastructural difference of epidermal cells between AK and BD lesions was investigated. In the study of p53 and PCNA expression, the atrophic and acantholytic types of AK showed lower positive rates compared to others. These two types did not demonstrate all layers expression pattern. The number of desmosomes of the epidermal cells was significantly reduced in BD, and in the bowenoid and hypertrophic types of AK compared with other types of AK. The number of hemi‐desmosomes showed greatest reduction in BD and the bowenoid type of AK. On the basis of our findings, it is hypothesized that atrophic and acantholytic types of AK may have the lowest, and the bowenoid type of AK and BD may have the highest, malignant potential.

Inhibitory effect of Rumex Japonicus Houtt on the porphyrin photooxidative reaction.

Rumex Japonicus Houtt (RJH), a plant indigenous to Okinawa, Japan, has been used traditionally by the local people for treatment of acute and chronic cutaneous diseases; however, its pharmacological effect is not clearly understood. To investigate its active function, we examined the antioxidative effect of RJH on the hematoporphyrin‐induced photooxidative reaction. We measured intracellular reactive oxygen species (ROS) in cultured transformed human vascular endothelial cells (ECV‐304) by flow cytometry, lipid peroxide (LPO) in erythrocyte ghosts by spectrofluorometry, and hemolysis by spectrophotometry. Results showed the generation of intracellular ROS in ECV‐304, LPO production in erythrocyte ghosts, and hemolysis after visible light irradiation in the presence of hematoporphyrin. In the RJH root extract treated group, generation of intracellular ROS, LPO production, and hemolysis were inhibited significantly.

Alteration of mRNA levels of δ-aminolevulinic acid synthase, ferrochelatase and heme oxygenase-1 in griseofulvin induced protoporphyria mice

Human erythropoietic protoporphyria (EPP) is an inherited disorder of porphyrin metabolism and its experimental murine model can be produced by treatment with griseofulvin (GF). We investigated the alteration of mRNA expression in ferrochelatase (FeC), delta-aminolevulinic acid synthase (ALAS) and heme oxygenase-1 (HO-1) in liver, skin and peripheral blood cells of GF-treated mice. In liver, ALAS mRNA was enhanced dramatically by GF administration, in accord with thesis that the expression of ALAS is regulated by feedback mechanism. The expression of HO-1 mRNA increased most rapidly and drastically in liver, however its mechanism of regulation may be different from that of ALAS mRNA. The level of FeC mRNA in liver was less affected with GF treatment. Our results indicate that the inhibition of FeC by GF administration might occur primarily at post-transcriptional level. Similar effects were observed in the ALAS and HO-1 mRNA expression in peripheral blood cells, 2-fold increase in the ALAS mRNA and increase from undetectable level to detectable level in the HO-1 mRNA. In skin of GF-treated mice, average increases of 1.3-fold in the ALAS mRNA and 1.6-fold in the HO-1 mRNA were statistically insignificant. The FeC mRNA level was not altered in peripheral blood or in skin of GF-treated mice. The present study indicates that the molecular analysis is practicable in skin and peripheral blood. In further study, this model could contribute to investigate the pathogenesis of clinical manifestation including possibly cutaneous changes in EPP.

Quantitative analysis of ferrochelatase mRNA in blood cells of erythropoietic protoporphyria patients

Ferrochelatase (FC; heme synthetase, EC 4.99.1.1.) catalyses the synthesis of heme from protoporphyrin IX, the final step in the heme synthetic pathway. The hereditary deficiency of this enzyme gives rise to erythropoietic protoporphyria (EPP). We developed a rapid, non-radioactive means of measuring human FC mRNA levels in the EPP patients. It is based on the reverse transcriptase-polymerase chain reaction (RT-PCR) performed on the RNA obtained from peripheral blood. The amplified DNA was detected by agarose gel electrophoresis with ethidium bromide staining and the fluorescent intensity was measured by scanning densitometry applied directly to Polaroid 665 negative film. The relative expression level of FC mRNA, compared with that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, was estimated at several points in the exponential phase of PCR cycles or at a point in the exponential phase of PCR performed on serially diluted the cDNA samples. The estimate of the FC mRNA by this method correlated well with the level of the FC mRNA measured by Northern blotting in the EB virus-transformed lymphocytes of the same patients. The level of the FC mRNA appeared to vary among the patients in whom a decreased level of enzymatic activity was indicated.

Inhibitory Effects of Alpinia speciosa K. SCHUM on the Porphyrin Photooxidative Reaction

It is thought that the β‐carotene defense mechanism against photosensitivity involves the inhibition of singlet oxygen formation, a kind of active oxygen. When we screened chemical substances obtained from plants indigenous to Okinawa, known to have residents with the longest life span in Japan, we found that Alpinia speciosa K. SCHUM (Japanese name: gettou), which is used as a food preservative, has an activity similar to that of β‐carotene. We measured the amount of lipid peroxide (LPO) formed from a hematoporphyrin‐containing rat liver microsomal suspension irradiated with visible light. The inhibitory effect of Alpinia speciosa on LPO formation was confirmed when the addition of increasing concentrations of Alpinia speciosa extract led to a decrease in the amount of LPO formed. Moreover, the reaction mechanism that affects the amount of singlet oxygen formed was measured, and the effect of the extract was determined by the ESR trapping technique. It was found that the extract effectively inhibited the formation of singlet oxygen. The extract of Alpinia speciosa contains dihydro‐5,6‐dehydrokawain. It was confirmed that dihydro‐5,6‐dehydrokawain, which is a water‐soluble compound, has singlet oxygen quenching activity. We synthesized five derivatives of kawain and found that dimethyl [6‐(2‐phenylethyl)‐2‐oxo‐2H‐pyran‐4‐yl] phosphorothionate has the strongest singlet oxygen quenching activity. The use of the compound from Alpinia speciosa that exhibits singlet oxygen quenching activity as an inhibitory agent of the phototoxic reaction in porphyria is expected.

Cutaneous Malignant Hemangioendothelioma: Clinical and Histopathological Observations of Nine Patients and a Review of the Literature

Nine patients with cutaneous malignant hemangioendothelioma (CMHE) were reported in Okinawa. All the patients were elderly, between 75 and 93 years of age. Four patients were males and five were female. The onset of the disease ranged from 1 to 9 months before the first visit. Eight patients had lesions on the scalp, and one, on the face and cheek. The lesions were in the form of exudative erythematous purpura, erythematous purpuric ulcers, and tumors. One patient developed a systematic metastasis involving the lungs, heart and intestine, and two patients had local metastasis to the cervical lymph nodes. Histopathologically, the tumor vessels were proliferated irregularly and showed anastomosis. The lumens were lined by large and atypical endothelial cells. Most of the specimens were infiltrated with large numbers of red blood cells. By electron microscope, Weibel‐palade bodies were found inside the tumor cells located at the peripheral part of the lesion. The patients were treated by irradiation, IL‐2 injection, and/or surgery. They were treated for 3 months to 2 years. Eight patients died between 4 to 24 months after the onset of disease and one has survived. The prognosis was poor.

A Case of Trichorrhexis Nodosa Developed in Winter

A young Japanese man developed localized trichorrhexis nodosa (LTN) of the scalp hair in the winter season. To investigate the roles of shampoo, severe sunlight exposure and/or mechanical injuries, we performed the following studies. Hair was collected from the patient and from a control. The study was performed in two steps. In the first step, hair was put into shampoo, rinsed with saline water, and then exposed to ultraviolet B (UVB) radiation once a day for one week. In the second step, the hair was similarly treated, but each shaft was bent gently with forceps before UVB exposure. Scanning electron microscopic studies revealed cuticular changes when the hair was treated only with shampoo and UVB. When it was treated with shampoo, UVB, and mechanical bending, the patients hair developed longitudinal and transverse fractures of the hair shafts, while the control hair showed only partial damage to the hair shaft. On the basis on the above findings, we conclude that mechanical bending may damage the hair shaft.