Deregulation of salivary glandular epithelial cells by oral bacteria

학위논문 (석사)-- 서울대학교 대학원 : 치의과학과, 2017. 2. 최영님.Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease characterized by dryness of mouth. Lymphocytic infiltrates develop around the epithelial cells in salivary and lacrimal glands, leading to dysfunction of exocrine glands. The pathogenesis of SS involves many components, including environmental and hormonal factors together with genetic background. Especially, the dysregulated salivary glandular epithelial cells (SGECs) as a non-professional antigen presenting cells (APCs) play a key role in the activation and recruitment of lymphocytes as well as in the perpetuation of the inflammatory process. However, the potential roles of oral bacteria in the induction of deregulation of SGECs were not fully understood. Here, we hypothesized that specific oral bacteria may be responsible for induction of APC marker expression and production of SS-related molecules in SGECs. Previously, the communities of oral bacteria collected by mouth rinse from SS patients (SS, n = 25), healthy controls (HC, n = 15), and patients with dry mouth due to medication as sicca controls (SC, n = 10) were analyzed by pyrosequencing. Based on the results of microbiota analysis, Streptococcus salivarius, Streptococcus oralis, Rothia mucilaginosa, Fusobacterium nucleatum, Prevotella melaninogenica, and P. histicola were selected for further study. In particular, an increase in P. melaninogenica was associated with SS risk by logistic regression analysis (odd ratio 5.4, p = 0.003). Human salivary gland (HSG) cells were infected with the selected bacteria in the absence or presence of IFNfor 3 days. To prevent the outgrowth of bacteria, gentamicin was added to the culture 6 h after infection with the bacteria. Expression of major histocompatibility complex class I (MHC I), MHC class II (MHC II), CD80, and CD86 was then examined by flow cytometry. The amounts of IL-6, IP-10, and IFNλ secreted into the culture supernatant were also measured by ELISA. To investigate mechanisms for immune modulation by oral bacteria, invasion ability and location of bacteria within HSG cells were analyzed by flow cytometry and confocal microscopy. The expression of Toll-like receptors (TLRs) in HSG cells and induction of immunoactive molecules in stimulated HSG cells with various ligands to pattern recognition receptors (PRRs) were also examined by flow cytometry and ELISA. To assess the influence of inflammatory cytokines and oral bacteria on physical epithelial barrier function, transepithelial electrical resistance (TER) value of stimulated HSG cells was measured for 3 days after a confluent monolayer of HSG cells was formed. The expression of APC markers such as MHC class I, class II, and costimulatory molecules on HSG cells was induced by IFN, a major cytokine present in the salivary gland of SS. HSG cells expressed very low levels of CD80 and CD86. These molecules were slightly upregulated by IFN. F. nucleatum, a highly immune stimulatory species used as a positive control, and P. melaninogenica upregulated MHC class I and CD86 in the absence of IFN and further upregulated the IFN–induced expression of MHC class I and CD80. Interestingly, P. melaninogenica and F. nucleatum upregulated the IFN–induced expression of MHC class II. They also induced the production of IL-6 and IP-10 both in the absence or presence of IFN. In contrast, S. salivarius, the most abundant commensal species, and two species S. oralis and R. mucilaginosa that were increased in sicca condition compared to HC showed suppressive effects on the expression of APC markers and IP-10. Surprisingly, both P. melaninogenica and F. nucleatum significantly increased the secretion levels of IFNλ compared to control. However, R. mucilaginosa and P. histicola downregulated the expression of this molecule in the absence of IFN. Three bacteria P. melaninogenica, F. nucleatum, and R. mucilaginosa, which induced deregulation of HSG cells, highly invaded into HSG cells within few hours, and they were detected in the endosomal maturation process. This finding suggested oral bacteria inside the cells could differentially modulate the HSG cells by signaling pathway through TLRs and/or NOD-like receptors (NLRs). The basal expression levels of TLR2, -4, and -9 were confirmed, and those were further upregulated by IFN. Deregulation of HSG cells was induced by stimulation with various PRR ligands. Bacterial components may potentially play a role in the deregulation of SGECs. Treatment of pro-inflammatory cytokines and the SS-associated species decreased the TER value of HSG cells, suggesting that inflammatory meiliu made by active immune response in target tissue and specific oral bacteria could disrupt the physical epithelial barrier. Changes in the oral microbiota associated with SS patients may potentially induce deregulation of SGECs and contribute to the pathogenesis of SS.1. Introduction 1 2. Materials and Methods 9 2.1. Cell culture 9 2.2. Bacteria culture 9 2.3. Bacterial infection 10 2.4. Stimulation of HSG cell with IFN or ligands to pattern recognition receptors 10 2.5. Flow cytometry 11 2.6. Immunofluorescent microscopy 12 2.7. Cytokine and chemokine enzyme-linked immunosorbent assay (ELISA) 12 2.8. Flow cytometric invasion assay and detection of bacteria in late endosomes 13 2.9. Transepithelial electrical resistance (TER) measurement 14 2.10. Statistical analysis 14 3. Results 16 3.1. Expression of APC markers on HSG cells was upregulated by IFN 16 3.2. Expression of APC markers on HSG cells was modulated by oral bacteria in the absence or presence of IFN 19 3.3. Production of cytokine and chemokine was modulated by oral bacteria in the absence or presence of IFN 22 3.4. Production of type III interferon was modulated by oral bacteria in the absence or presence of IFN 25 3.5. Invasion ability of oral bacteria into HSG cells varies depending on species 27 3.6. Invaded bacteria were detected in the late endosomes of HSG cells 30 3.7. HSG cells express bacteria-sensing Toll-like receptors (TLRs) 32 3.8. Expression of APC markers on HSG cells was regulated by various ligands to PRRs 35 3.9. Production of cytokine and chemokine was regulated by various ligands to PRRs 37 3.10. Physical barrier of HSG cells was disrupted by inflammatory cytokines and specific bacteria 39 4. Discussion 42 5. Reference 48Maste

Studies on the development of IgE-secreting plasma cells in the thymus and its rStudies on the development of IgE-secreting plasma cells in the thymus and its role in the anaphylactic responses ole in the anaphylactic responses

Doctor정상 상태의 혈청 내 E 타입 항체는 기생충 감염으로부터 우리 몸을 보호하거나 알르레기 질환을 유발하는데 중요한 역할을 한다. E 타입 항체는 모든 타입의 항체 가운데 정상 상태의 혈중 내 가장 낮은 농도로 존재하지만 면역 반응 조절 장애를 동반하는 여러 질환에서는 그 농도가 급격하게 증가된다고 알려져 있다. 따라서, E 타입 항체 생산에 관여하는 B 세포의 조절 기전을 이해하는 것은 중요하지만, 현재까지 E 타입 항체를 분비하는 형질 세포가 정상상태에서 어떻게 생성되고 발달 되는지에 대한 구체적 메커니즘은 규명되지 않았다. 선행 연구 결과를 통해 C57BL/6 생쥐 보다 BALB/c 생쥐가 혈청 내 100배 많은 E 타입 항체를 가지고 있다는 것을 확인하였다. BALB/c 생쥐의 흉선에서 E 타입 항체 생성에 필수적인 인터류킨-4 (Interleukin-4)를 다량 분비하는 자연 살해 T 세포가 많이 존재하고, 해당 세포가 없을 때 혈중 내 E 타입 항체의 농도가 감소한다는 사실을 바탕으로 본 연구는 E 타입 항체를 분비하는 B 세포가 흉선에서 발달 될 것이라는 가설을 세우고 해당 세포의 조절 메커니즘, 발달 과정, 특징, 그리고 생리학적 역할을 규명하고자 하였다. 우선 자기장을 이용한 세포 분리 및 세포 내 면역글로불린 (immunoglobulin) 염색 기법을 통해, 많은 수의 E 타입 항체를 분비하는 형질 세포가 BALB/c 생쥐의 흉선에 특이적으로 존재하고, 자연 살해 T 세포가 결핍된 생쥐에서 해당 세포의 수가 인터류킨-4가 결핍된 생쥐만큼 감소함을 확인하였다. 또한, 흉선을 제거한 생쥐와 자연 살해 T 세포가 결핍된 생쥐에서 혈청 내 E 타입 항체의 농도가 유의미하게 감소한 것을 확인하였다. In vitro 흉선 배양과 다양한 in vivo 생쥐 모델을 통해 E 타입 항체를 생산하는 형질 세포는 어린 시기에 골수 유래 전구체를 통해 발달한 흉선 거주 세포라는 것을 확인했으며, E 타입 항체 생성에 있어서 장내 항원과 상관없이 자연 살해 T 세포가 분비하는 인터류킨-4가 필수적이라는 것을 확인하였다. 다음으로, 단일세포유전체 분석을 통해 흉선에는 형질 세포를 포함하는 다양한 B 세포가 존재하고, 형질 세포는 극도의 다양한 레파토리 (Repertoire)를 지닌 다클론성 (polyclonal) 항체를 분비함을 확인하였다. 유전자 변형 생쥐를 통한 유세포 분석 실험 결과를 바탕으로, 흉선 형질 세포는 배중심 (germinal center)을 거치지 않으며, 다클론성 CD4 도움 T 세포를 통해 발달하여 흉선 내 수질 (medulla) 부분에 위치하는 것을 확인하였다. 마지막으로, 대부분 흉선 형질 세포에서 분비되는 E 타입 항체는 소장과 피부에 존재하는 비만 세포의 수를 증가시켜 아나필락시스 반응을 심화시킨다는 것을 관찰함으로써 E 타입 항체를 생산하는 흉선 내 형질 세포의 생리학적 역할을 규명하였다. 결론적으로, 본 연구는 정상 상태에서 혈중 내 E 타입 항체를 분비하는 형질 세포의 발달 메커니즘과 역할을 규명했다는 것에 의의가 있다. 이는, 사람들마다 왜 아나필락시스와 같은 알르레기 질환의 감수성과 반응성이 다르게 나타나는지에 대한 새로운 기초 생물학적 지식을 제공할 뿐만 아니라, 관련 질병의 예방과 치료법 개발에 대한 이해를 넓힐 수 있을 것으로 기대된다.Immunoglobulin E (IgE) antibodies (Abs) are the main mediators of allergic disorders, but also provide protection against helminth infections in both mouse and human. Serum IgE concentrations are tightly regulated in healthy individuals and elevated in various disease conditions, including autoimmunity, immunodeficiency, and perturbed commensal microbiota. However, the regulatory mechanisms of homeostatic serum IgE levels are largely unknown. Under steady state condition, BALB/c mice displayed 100-fold higher serum IgE levels together with a higher frequency of IL-4-producing NKT2 cells than C57BL/6 mice. However, the source of homeostatic IgE-producing B cells in BALB/c mice has been enigmatic. As IgE class-switching requires interleukin-4 (IL-4) and the frequency of NKT2 cells is highest in the thymus, I hypothesized that they regulate the development of IgE-producing B cells. Here, I aimed to identify source and ontogeny of Ab-secreting plasma cells (PCs) responsible for homeostatic serum IgE and their physiological role. In the first part of this study, I demonstrated that CD138+Blimp1+ PCs develop in the thymus and potently secrete IgE and other natural immunoglobulins including IgM, IgA, and IgG. The development of IgE-secreting thymic PCs was induced by IL-4 produced from invariant natural killer T (iNKT) cells, independent of a CD1d-mediated interaction and intestinal antigens. In the second part of this study, I found that thymus supports the developmental landscape of thymic B cells using single cell RNA-sequencing (scRNA-seq). B cell receptor analysis also revealed that thymic PCs produce polyclonal Abs without somatic hypermutations. In the third of part of this study, I established mouse model of anaphylaxis to investigate the physiological role of IgE-secreting PCs. Under homeostatic condition, thymus-derived IgE Abs increase the number of mast cells (MCs) in the gut and skin, which consequently aggravate anaphylactic response. Collectively, these findings suggest a previously unrecognized immune axis between thymus and peripheral tissues mediated by IgE Abs and open up new avenues for studying the genetic causes of allergic disorders

2XXX Al-SiC 복합재료의 동적파괴거동

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주조 A356 Al-SiCp 복합재료의 파괴인성 해석 연구

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열연강판 Cupping시의 수직균열 현상 연구

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The Application of Interventional Radiology in Living-Donor Liver Transplantation

Owing to improvements in surgical techniques and medical care, living-donor liver transplantation has become an established treatment modality in patients with end-stage liver disease. However, various vascular or non-vascular complications may occur during or after transplantation. Herein, we review how interventional radiologic techniques can be used to treat these complications

Transjugular Liver Biopsy in Patients with Liver Transplantation: Comparison of Quick-Core Biopsy and Forceps Biopsy

Purpose To compare the safety and efficacy of transjugular liver biopsy (TJLB) using a Quick-Core biopsy needle or a forceps biopsy in patients with liver transplantation (LT) who were suspected of having rejection. Materials and Methods From June 2015 to January 2017, 98 TJLBs (60 patients) with the Quick-Core biopsy needle and 95 TJLBs (58 patients) with a forceps biopsy system were attempted in patients with LT suspected of having rejection. Technical success, adequacy for diagnosis, number of biopsy instrument passes, the maximum and mean length of the obtained samples, and the complications were retrospectively analyzed. Results TJLB was technically successful in all patients. Adequate specimens were obtained in 95.9% of the biopsy needle group and 91.6% in the forceps group (p = 0.246). The mean number of biopsies was 4.8 +/- 1.8 in the biopsy needle group and 6.2 +/- 1.7 in the forceps group. The mean size of the biopsy sample was 11.1 +/- 3.0 mm in the biopsy needle group and 2.5 +/- 1.2 mm in the forceps group. Only one minor complication (a subcapsular hematoma) occurred in the biopsy needle group. No major complication was observed in any patient. Conclusion TJLB using a Quick-Core biopsy needle or forceps can be safely and effectively performed in LT patients. The adequacy of sampling for diagnosis was equivalent between the groups