설사 환아에서의 로타바이러스와 아데노바이러스의 감염에 관한 연구

영유아 설사 중 바이러스에 의한 가장 흔한 원인으로 알려진 로타바이러스와 아데노바이러스는 외국의 경우 이에 대한 많은 연구가 이루어지고 있으나 우리나라에서의 두 바이러스의 감염에 대한 연구는 거의 없는 실정이다. 따라서 1997년 8월부터 1998년 7월까지 이화여자대학교 의과대학 부속 목동병원 소아과에 심한 설사로 입원한 영유아의 대변검체에서 로타바이러스와 아데노바이러스의 검출을 시도하여 각각의 검출법에 의한 설사환아에서의 로타바이러스와 아데노바이러스의 감염의 빈도를 알아보고자 하였다. 대변검체에서 로타바이러스의 검출은 간접 효소면역측정법 (Enzyme linked immunosorbent assay, 이하 ELISA라 함)으로 항원을 측정하였고, polyacrylamide gel electrophoresis (이하 PAGE라 함)로 로타바이러스의 RNA segment를 확인하고 (electropherotyping), 로타바이러스의 VP7 유전자에 대한 역전사 중합효소 연쇄반응(reverse transcription polymerase chain reaction; 이하 RT-PCR이라 함)을 시행하여 감염의 유무를 검사하였다. 또한 RT-PCR에 의해 VP7 유전자가 검출되면, G1-G4 각각의 특이적인 유전자를 증폭할 수 있는 primer를 사용하는 nested-PCR법으로 로타바이러스의 G 단백의 혈청형을 결정하였다. 대변검체에서 아데노바이러스 항원의 검출은 ELISA를 시행하고 HEp-2 세포 또는 293 세포에 대변 부유액을 배양하여, 세포병변이 확인되면 아데노바이러스의 hexon 유전자에 대한 PCR을 시행하여 아데노바이러스의 감염을 확인하였다. 또한 PCR로 장관계 아데노바이러스인 type 40, 41의 long-fiber 유전자를 증폭시켜 아데노바이러스 40, 41에 의한 감염의 유무를 다시 확인하였다. 그 결과 설사로 입원한 영유아 103개의 설사 대변 검체 중 ELISA법에 의한 로타바이러스의 검출은 5.8% (6/103)이었다. 로타바이러스의 경우 PAGE에서 특징적인 RNA 분절을 보이는 것은 15.5% (16/103)이었고, 이 중 long type이 10.7% (11/103), short type이 4.9% (5/103)이었다. 또한 RT-PCR에 의한 로타바이러스 VP7 유전자가 검출된 것은 8.7% (9/103) 이었다. VP7 gene의 nested-PCR에 의한 G serotype은 type 1 (G1)이 1% (1/103)였으며, type 3 (G3)이 1% (1/103), type 4 (G4)가 6.8% (7/103)이었고 1%의 경우는 G1과 G3의 혼합된 양상을 나타내었다. 아데노바이러스의 경우는 ELISA로는 2.9% (3/103)가 아데노바이러스의 감염을 나타내었고, 아데노바이러스의 hexon 유전자에 대한 PCR로는 2.9% (3/103)에서 증폭된 유전자를 확인하였다. 또한 103개의 검체 중 로타바이러스와 아데노바이러스가 동시에 검출된 것은 없었다. ; Acute diarrhea is quite common in infancy and early childhood. Rotavirus is the most common enteric viruses that cause severe diarrhea in infants and young children worldwide. Adenovirus is now recognized as the second most common identified agents in stools and young children with gasteroenteritis. A total of 103 stool specimens were collected from Ewha Womans University Mok-Dong Hospital in Seoul, from Aug. 1997 to Jul. 1998. To detect rotavirus, stool samples were analyzed by enzyme-linked immunoabsorbent assay (ELISA), polyacrylamide gel electrophoresis of viral RNA and RT-PCR to amplify gene 8, encoding VP7 glycoprotein of the rotavirus. To detect adenovirus, ELISA and PCR were performed. Hexon genes of the adenovirus and long-fiber genes of adenovirus 40 and 41 were amplified by PCR. Six of the 103 specimens were found positive by ELISA for rotavirus. The RT-PCR products of the VP7 genes of the rotavirus were detected in 9 samples of the 103 samples, followed by serotype specific region targeted PCR to identify the G serotype of human rotavirus, especially G1, G2, G3, and G4. Three of the 103 specimens were found positive in adenovirus ELISA test. When common adenovirus hexon primers were used, 3 of the 103 specimens were positive. In case of long-fiber genes of the adenovirus subgroup F (type 40, 41) specific primers were used, none of the 103 stool specimens were found to be positive by PCR. Rotaviruses and adenoviruses were responsible for 18.4% and 4.9% of cases of acute diarrhea in hospitalized children in the present study, but coinfection of the two viruses were not detected. In case of rotavirus infection, the highest number of cases occurred from March to June. PAGE of rotavirus dsRNA yielded high detection rate, but RT-PCR could detect serotypes of rotavirus. Serotyping is useful tool to study the epidemiologic characterisrics of rotaviruses. Annual and seasonal variations in the occurance and distribution of serotypes may provide useful information on modes of transmission of the virus, and could lead to incorporation of multiple serotypes in candidate vaccines. In adenovirus infection, PCR amplification support the potential of this technique as an additional method for detection of adenovirus infections, including type 40, 41 adenovirus infection, which often could be fatal.감사의 말 = vi 논문개요 = vii I. 서론 = 1 II. 연구재료 및 방법 = 5 A. 연구재료 = 5 1. 대변검체 = 5 B. 연구방법 = 5 1. 간접 효소면역 측정법에 의한 로타바이러스와 아데노바이러스 항원 검출 = 5 2. 로타바이러스 VP7 유전자의 역전사중합효소연쇄반응과 혈청형 분석 = 6 가. 대변 검체에서의 로타바이러스 RNA 추출 = 6 나. 로타바이러스 RNA의 정제 = 6 다. 전기영동 및 은염색 = 7 라. 역전사 중합효소연쇄반응과 혈청형 분석 = 7 3. 아데노바이러스의 중합효소연쇄반응 = 8 가. HEp-2 세포주에서의 대변 검체 배양 = 9 나. 아데노바이러스의 DNA 추출 = 9 다. 아데노바이러스의 중합효소연쇄반응 = 10 III. 결과 = 15 A. 로타바이러스의 검출 빈도 = 15 B. 아데노바이러스의 검출 빈도 = 16 IV. 고찰 = 25 V. 결론 = 28 참고문헌 = 30 영문 초록 = 3

Dexamethasone Promotes Keratinocyte Proliferation by Triggering Keratinocyte Growth Factor in Mast Cells

Background: The skin is a dynamic body organ that can be activated by both central and local hypothalamic-pituitaryadrenal axis systems. This phenomenon might be the crucial explanation why stress can cause relapse of chronic inflammatory skin diseases, such as psoriasis. Here, we determined the effects of mast cells on keratinocyte proliferation under stress hormone stimulation. Methods: We subcutaneously injected dexamethasone on the shaved back of mice and evaluated histological changes and keratinocyte growth factor (KGF) expression on dermal mast cells. Further, human mast cell line (HMC-1) and keratinocyte cell line (HaCaT) cells were treated with dexamethasone in vitro to observe the extent of proliferation and the expression of KGF. Finally, the supernatants of HMC-1 cells treated with dexamethasone were used for the culture of HaCaT cells to investigate the effect on proliferation. Results: We observed epidermal thickening in dexamethasone-injected mice, accompanied by an increase in the number of KGF-expressing dermal mast cells. Similar to mouse dermal mast cells, KGF was highly expressed in the human mast cell line HMC-1 following stimulation with dexamethasone. Further, dexamethasone-treated mast cells promoted keratinocyte proliferation in vitro. However, the effects of mast cells on keratinocytes were significantly diminished in the presence of anti-KGF-blocking antibodies. Conclusion: Taken together, our results show that a stressful environment may disturb skin barrier homeostasis through mast cell-derived KGF expression. (C) 2019 S. Karger AG, Base

Comparison of the expression profile of junb, c-Jun, and S100A8 (calgranulin A) in psoriasis vulgaris and guttate psoriasis

Background: Psoriasis is a chronic, inflammatory, immune-mediated skin disease. Recently, several psoriasis-linked genetic loci have been reported; PSORS4 contains S100A8 (calgranulin A), and PSOR6 (19p13) locus harbors JunB (19p13.2). S100A8 is considered to be a marker of inflammation in a variety of diseases. The expression of JunB and c-Jun have been reported to be reduced in psoriatic lesions. Objective: We attempted to assess the role and correlation of S100A8, JunB, and c-Jun in the pathogenesis of guttate psoriasis and psoriasis vulgaris by studying whether any difference of immunohistochemical expression existed. Methods: Skin biopsy specimens from patients with psoriasis vulgaris (n = 37) and guttate psoriasis (n = 17), and a normal skin controls (n = 9) were utilized in the study. Formalin-fixed and paraffin-embedded tissue sections were prepared and JunB, c-Jun, and calgranulin A were immunohistochemically stained in order to compare the expression of those three proteins in each group. Results: Reduced JunB expression was observed in patients with psoriasis vulgaris and guttate psoriasis, as compared to patients in the control group; however, c-Jun expression was reduced only in the psoriasis vulgaris group. The expression of S100A8 increased in the psoriasis groups as compared to the control group. In addition, the expression of S100A8 was different between the psoriasis vulgaris and guttate psoriasis groups; S100A8 was expressed more profoundly in the guttate psoriasis group (p<0.05). Conclusion: Our results indicate that S100A8 contributes to the pathogenesis of guttate psoriasis, and it may be a good target for therapy for guttate psoriasis provoked by microorganisms. (Ann Dermatol (Seoul) 21(1)35∼38,2009)

Therapeutic effects and immunomodulation of suanbo mineral water therapy in a murine model of atopic dermatitis

Atopic dermatitis; Balneotherapy; Mineral waters; NC/NGa mic

Immunomodulatory effects of Deokgu thermomineral water balneotherapy on oxazolone-induced atopic dermatitis murine model

Background: Although the therapeutic mechanism of balneotherapy for atopic dermatitis has not been clarified, many atopic patients who visit thermomineral springs have shown clinical improvements. Objective: This study was aimed to evaluate the immunomodulatory effect of thermomineral water balneotherapy on the atopic dermatitis murine model. Methods: The oxazolone-induced atopic dermatitis murine model was used to evaluate the therapeutic effect of balneotherapy with Deokgu thermomineral water compared with distilled water. Histologic evaluation and confocal microscopic imaging were performed to analyze the lesional expression of cluster-of-differentiation (CD)4 and forkhead box p3 (Foxp3). Lesional mRNA expression of interleukin (IL) 33, thymic stromal lymphopoietin (TSLP), and Foxp3 was evaluated by real-time reverse transcription polymerase chain reaction. Results: Compared with the distilled water bath group, confocal microscopic evaluation of CD4 and Foxp3 merged images showed increased expression of regulatory T cells in the thermomineral balneotherapy group. The lesional mRNA level of IL-33 showed a reduced trend in the thermomineral balneotherapy group, whereas the level of mRNA of Foxp3 was increased. TSLP showed a decreased trend in both distilled water and thermomineral water bath groups. There was a trend of reduced expression in lesional IL-33 mRNA but increased cell count of CD4+ Foxp3+ regulatory T cells in thermomineral balneotherapy compared with distilled water bath. Conclusion: Therefore, thermomineral balneotherapy can be an effective and safe adjuvant therapeutic option for atopic dermatitis. Copyright © The Korean Dermatological Association and The Korean Society for Investigative Dermatology

Angiostatin works as immune modulatory molecules via inhibition of neutrophil activation and migration

And apoptosis; Angiostatin; Lipid raft; Neutrophi

Heat shock protein 90 is involved in IL-17-mediated skin inflammation following thermal stimulation

The pathogenesis of inflammatory skin diseases involves interactions between immune cells and keratinocytes, including the T helper 17 (Th17)-mediated immune response. Several chemokines [chemokine (C-X-C motif) ligand (CXCL)1, CXCL5 and CXCL8] and antimicrobial peptides [-defensin 1 (BD1), LL-37, S100A8 and S100A9] were transcriptionally upregulated in the keratinocyte cell line HaCaT upon stimulation with interleukin (IL)-17. Balneotherapy, the treatment of disease by bathing, is an alternative therapy that has frequently been used for the treatment of inflammatory skin diseases. Immersion in pools of thermal mineral water is often considered to have chemical, thermal, mechanical and immunomodulatory benefits. We examined the effect of thermal treatment on IL-17-mediated inflammation in a model of skin disease. As Act1 is required for IL-17 signaling and is a client protein of heat shock protein 90 (HSP90), we evaluated the effect of HSP90 inhibition on IL-17-mediated cytokine and antimicrobial peptide expression in keratinocytes following heat treatment. We found that after thermal stimulation, Act1 binding to HSP90 was significantly increased in the presence of IL-17 (100 ng/ml) and 17-N-allylamino-17-demethoxygeldanamycin (17-AAG, 1 mu M). Antimicrobial peptide and chemokine expression generally increased after heat treatment; Act1 knockdown and 17-AAG reversed this effect. These observations demonstrate the possible immunomodulatory effect of heat on keratinocytes during the progression of IL-17-mediated inflammatory skin diseases

Detection of Streptococcus pneumoniae pneumolysin gene by PCR in sera and cerebrospinal fluids from hospitalized patients

Streptococcus pneumoniae is the worldwide leading causative agent of community acquired pneumonia and meningitis with high morbidity and mortality. Pneumonia and meningitis are the two most frequent manifestations of S. pneumoniae. However, only a small proportion of the cases can be detected by conventional methods such as blood culture. The recently developed pneumolysin PCR method can detect low numbers of organisms or even DNA fragments even without live cells of the pathogen. Penumolysin is a species specific 52 kDa protein found in all strains of S. pneumoniae and is an ideal target for antigen detection. We detected S. pneumoniae in sera (n=29) from hospitalized children with respiratory infection, and cerebrospinal fluids (n=57) from hospitalized adults and children with viral meningitis. ELISA using anti-S. pneumoniae monoclonal antibody and PCR detection of the pneumolysin gene were applied to the clinical specimens from October 1998 to July 2001. In the children with respiratory infection, PCR detection of the peripheral blood monocytes showed 37.9% (11/29) of positivity, while ELISA using sera gave only 18.5% (5/27) of positivity. In the adult patients with viral meningitis, PCR detection of cerebrospinal fluids showed 41.4% (12/29) of positivity, and ELISA gave 35.3% (6/17) of positivity. With cerebrospinal fluids of children with viral menigitis, PCR showed 42.3% (11/26) of postivity, while ELISA gave 26.9% (7/26) of positivity. PCR detection of S. pneumoniae pneumolysin gene applied to peripheral blood monocytes and heated cerebrospinal fluid samples appeared to be a more specific diagnostic test than the conventional cultural methods in S. pneumoniae infections