엑소좀 수득 조건 최적화 및 퇴행성 신경 질환 치료에의 응용

학위논문(박사)--서울대학교 대학원 :자연과학대학 협동과정 뇌과학전공,2019. 8. 김만호.Exosomes are small extracellular vesicles with 30–100 nm diameter that originate from many type of cells. Exosomes serve as a role of intercellular messenger unit by inward budding and scission of vesicles from the limiting endosomal membranes and released from the MVB lumen into the extracellular environment during exocytosis. Secreted exosomes can be isolated and characterized in blood, urine, saliva in vivo and conditioned medium in vitro. They include various molecular constituents of their cell origin, including proteins, mRNA, and microRNA. Therefore, exosomes have been widely studied as a therapeutic agent, biomarker, and drug delivery vehicle. Despite of their importance in normal physiology and disease progression, the standard criteria of storage conditions, which are generally the pH and the temperature, are indefinite and controversial. Therefore, in this study, we isolated the exosomes after incubation in various pH conditions and analyzed the recovery yield of exosomes, and we examined the stability of exosomes at various storage temperatures. We have discovered that acidic condition was favorable for the high yield for exosome isolation, and the temperature below -70 °C is crucial for the preservation of exosomes in fresh condition for clinical and basic applications. After optimizing the isolation and storage conditions for the exosome, we examined that the human adipose stem cell derived exosome (ASC-exo) has a therapeutic potential for treating Alzheimers disease (AD), Huntingtons disease (HD), Amyotrophic lateral sclerosis (ALS) by modulating representative cellular phenotypes. Adipose stem cells (ASCs) are regarded as the potential source of regenerative medicine, since ASCs express multiple factors for the paracrine effects and have a high proliferation rate with a lower senescence than other tissue-derived stem cells. Although exosomes derived from stem cells has therapeutic potential on many illnesses, precise mechanism of how ASC-exo affect the progression of HD, AD, and ALS has not been studied. We examined the effects of ASC-exo on key phenotypes of HD including cell death, and mitochondrial dysfunction in R6/2-derived neuronal cells, and mHtt aggregates levels. Also, we examined the effects of ASC-exo on key hallmarks of ALS including SOD1 aggregation, elevated SOD1 level and abnormal mitochondrial protein levels in G93A neuronal cells. Furthermore, we developed an in vitro AD model using neuronal stem cells (NSCs) from the transgenic mice line, TG2576. The differentiated NSCs of AD mice, which showed major phenotypes of AD including the Aβ pathology and increased apoptosis, were treated by ASC-exo. In addition, changes in neurite outgrowth were examined, as neurites are involved in the synapse formation of the central nervous system. We proved that ASC-exo ameliorate the progression of Aβ-induced neuronal death in AD, and ASC-exo modulates SOD-1 aggregation and mitochondrial dysfunction, making it a therapeutic candidate for ALS. In HD, we confirmed that the ASC-exo up-regulates PGC1, phospho-CREB and amelio rates abnormal apoptotic protein level, and reduces mHtt aggregates level. In addition, MitoSOX Red, JC-1 and cell viability assay showed that ASC-exo reduces mitochondrial dysfunction and cell apoptosis. These findings suggest that ASC-exo has a therapeutic potential for treating neurodegenerative diseases by modulating representative cellular phenotypes. Next focus is ameliroration of HD by heterochronic parabiosis (HP) which have been reported by a few paper in AD, presenting that exposure to a young blood circulation through HP reverses cognitive deficits observed with normal aging. To determine the effect of young circulatory factors on HD-like disease in R6/2 mice, we used HP, in which we joined the circulatory systems of young, wildtype animals together with R6/2 mice which express exon1 of the human HD gene with around 150CAG repeats. We analyzed the changes in protein expression in the brains of these mice with established disease after heterochronic parabiosis, and we examined the effects of young serum exosome on key phenotypes of HD including p-CREB-PGC-1 pathway, apoptotic proteins in R6/2-derived neuronal cells. mutant Huntingtin aggregation protein mice with established disease showed a restoration in levels of HD pathology. Moreover, young blood associated with improved mitochondria dysfunction and cognition, decreased cell death after exposure to young blood in heterochronic parabiosis. In addition, in vitro HD model cells treated with young blood serum and serum exosome showed decreased mHtt aggregation, mitochondrial dysfunction, cell death and cell proliferation. This study provides useful information for understanding the optimum conditions for the isolation and storage of exosomes, and by identifying the exosomes as a messenger unit that can transport the positive factors for the diseases, this study opens the possibilities for adopting the exosomes to various neurodegenerative diseases.엑소좀은 직경이 30-100 nm인 작은 세포 외 소포이며 많은 종류의 세포에서 유래한다. 엑소좀은 endosomal membrane으로부터의 소포의 내부 발아 및 절단에 의해 세포 간 전달 단위의 역할을 하며, 세포외 배출 시 MVB 루멘에서 세포 외 환경으로 방출된다. 분비된 엑소좀은 체외에서 혈액, 소변, 생체 내 타액 및 조정 배지에서 분리되고 분석 될 수 있다. 여기에는 단백질, mRNA 및 마이크로 RNA를 비롯한 세포 기원의 다양한 분자 구성 요소가 포함된다. 따라서, 엑소좀은 치료제, 바이오 마커 및 약물 전달체로서 광범위하게 연구되어왔다. 정상 생리 및 질병의 진행에서의 중요성에도 불구하고, 일반적으로 exosome의 수득 및 저장 조건의 표준 기준은 불명확하고 논쟁의 여지가 있어 왔다. 따라서 본 연구에서는 다양한 pH 조건에서 엑소좀을 배양 한 후 분리하고 회수율을 분석하였으며, 다양한 저장 온도에서 엑소좀의 안정성을 조사하였다. 본 연구에서는 산성 조건이 엑소좀 분리에 대하여 높은 수율을 얻는 데에 유리하다는 것을 발견했으며, 임상 및 기본 분야 응용을 위하여 엑소좀을 신선하게 보존하기 위해서는 -20 ℃ 이하의 온도가 중요하다는 것을 발견하였다. 엑소좀에 대한 분리 및 저장 조건을 최적화 한 후 우리는 인간지방줄기세포에서 유래한 엑소좀이 (ASC-exo) 알츠하이머 병 (AD), 헌팅톤 병 (HD), 근 위축성 측삭 경화증 (ALS) 등의 질병의 대표적인 세포 표현형을 조절하는 것을 확인하였다. 지방 줄기 세포 (ASCs)는 재생 의학의 잠재적 원천으로 여겨지고 있는데, 지방 줄기 세포는 파라크린 효과에 대해 여러 인자를 내포하고 있으며, 다른 조직 유래 줄기 세포보다 낮은 노화로 높은 증식률을 나타낸다고 알려져 있다. 비록 줄기 세포에서 유래된 엑소좀은 많은 질병에서 치료적 잠재력을 가지고 있지만, ASC-exo가 HD, AD 및 ALS의 진행에 미치는 정확한 기전은 연구되지 않았다. 이에 본 연구에서는 R6/2 유래 신경 세포의 세포 사멸 및 미토콘드리아 기능 장애 및 mHtt 응집체 수준을 비롯한 주요 HD 표현형에 ASC-exo가 미치는 영향을 조사하였다. 또한, 본 연구는 G93A 신경 세포에서 SOD1 집합체, 증가된 SOD1 수준 및 비정상적인 미토콘드리아 단백질 수준을 포함하는 ALS의 주요 특징에 대해 ASC-exo의 효과를 조사 하였다. 또한, AD 질병 모델에 대표적으로 알려진 트랜스 제닉 생쥐 라인인 TG2576에서 유래한 줄기 세포 (NSCs)를 사용하여 시험관내 AD 모델을 개발하였다. Aβ 병리 및 증가된 세포 사멸을 포함하는 AD의 주요 표현형을 나타내는 AD 마우스에서 분화된 NSC는 ASC-exo에 의해 질병이 개선됨을 확인하였다. 또한, 신경 돌기가 중추 신경계의 시냅스 형성에 관여하기 때문에, 신경 돌기 성장의 변화를 조사하였다. 이를 통해 ASC-exo가 AD에서 Aβ 유도성 신경 세포의 진행을 완화시키고 ASC-exo가 SOD-1 응집과 미토콘드리아 기능 장애를 조절하여 AD의 치료적 후보 물질이 될 수 있다는 것을 증명하였다. HD에서는 ASC-exo가 PGC1, phospho-CREB를 상향 조절하고 비정상적인 세포 사멸 단백질 수준을 개선하고 mHtt 응집체 수준을 감소 시킨다는 것을 확인하였다. 또한, MitoSOX Red, JC-1 및 세포 생존능 분석 결과, ASC-exo는 미토콘드리아 기능 장애 및 세포 사멸을 감소시키는 것을 확인하였다. 이러한 결과는 ASC-exo가 대표적인 세포 표현형을 조절함으로써 신경 퇴행성 질환을 치료할 수 있는 치료적 잠재력을 가지고 있음을 시사한다. 다음 주제는 Heterochronic parabiosis (HP)를 통한 젊은 혈액 순환을 통한 노출은 정상적인 노화로 관찰되는 인지 장애를 개선시킴을 확인한 결과입니다. R6 / 2 마우스의 HD 유사 질환에 대한 젊은 순환기 요인의 영향을 알아보기 위해 본 연구에서는 R6 / 2 마우스와 함께 젊은 야생형 동물의 순환계에 HP방법을 통하여 합류시켰다. Heterochronic parabiosis 후 질병이 확립된 R6 / 2 마우스의 뇌에서 단백질 발현의 변화를 분석하였고, p-CREB-PGC-1a 경로, 세포 사멸 단백질 및 세포 사멸을 포함한 HD의 주요 표현형에 젊은 혈장 exosome의 영향을 조사하였다. 그 결과, 헌팅톤 병의 대표적인 돌연변이형 헌팅틴 응집 단백질의 병리 수준이 회복됨을 확인하였고, 미토콘드리아 기능 장애가 개선되었으며, 인지 부분이 개선 됨을 확인할 수 있었다. 또한, 젊은 혈액에 노출된 후에 세포 사멸 감소를 확인하였다. 마지막으로 젊은 혈청 및 혈청 엑소좀으로 처리한 시험관 내 HD 모델 세포는 mHtt 응집, 미토콘드리아 기능 장애 회복 및 세포 증식을 확인하였으며, 세포 사멸을 감소 시킴을 확인하였다. 이를 통해 헌팅톤 병 개선을 시키는 물질이 젊은 혈장 내 exosome 내 물질일 가능성을 제시하였다. 이 연구는 엑소좀의 분리 및 저장을 위한 최적 조건을 이해하고 엑소좀을 질병 개선에 긍정적인 요소를 전달할 수 있는 메신저 단위로 확인함으로써 유용한 정보를 제공하고, 다양한 신경 퇴행성 질환에 대한 엑소좀을 치료제로 채택 할 수 있는 가능성을 열어준다.Chapter 1. Introduction 1 1.1. Neurodegeneration process . 1 1.2. Exosomes 9 1.2.1. Role of exosomes as an intercellular cargo unit 9 1.2.2. Application of exosoems for diseases 12 1.3. Objective of the thesis 17 1.4. Organization of the thesis . 19 Chapter 2. Theoretical Background 21 2.1. Neurodegenerative diseases . 21 2.1.1. Hungintons disease (HD) . 21 2.1.2. Amyotrophic Lateral Sclerosis (ALS) . 29 2.1.3. Alzheimers disease (AD) . 32 2.2. Parabiosis 39 2.2.1. Surgical process of parabiosis 39 2.2.2. Application of parabiosis for diseases . 47 2.3. In vivo Analysis technique . 51 2.3.1. Animal model (R6/2 and ZQ175) . 51 2.3.2. Animal modeling for parabiosis . 54 2.3.3. BrdU injection . 54 2.4. In vitro Analysis technique . 56 2.4.1. Neural stem cell 56 2.4.2. Exosome isolation 56 2.4.3. Immunohistochemistry . 57 2.4.4. Immunocytochemistry . 58 2.4.5. Western blot 60 2.4.6. Fluorescence Activated Cell Sorting (FACS) . 62 2.4.7. Enzyme Linked Immunosorbent Assay (ELISA) 63 Chapter 3. Effect of pH on the Yield of Exosome Isolation 65 3.1. Introduction 65 3.2. Methods . 67 3.2.1. HEK 293 cell culture . 67 3.2.2. Adjustment of pH in conditioned medium . 67 3.2.3. Exosomes isolation . 67 3.2.4. Protein isolation and western blot . 68 3.2.5. RNA isolation 68 3.2.6. Statistical analysis 69 3.3. Isolation and characterization of exosomes . 70 3.4. Effect of pH on the representative exosome markers 71 3.5. Effect of pH on the exosomal protein and RNA concentration . 73 3.6. Summary . 76 Chapter 4. Influence of Storage Condition on Exosome Recovery 78 4.1. Introduction 78 4.2. Methods . 80 4.2.1. HEK 293 cell culture . 80 4.2.2. Isolation of exosomes . 80 4.2.3. Storage of exosomes . 81 4.2.4. Protein concentration measurement and western blot 81 4.2.5. Flow cytometry . 82 4.2.6. Statistical analysis 82 4.3. Exosome isolation from conditioned medium 83 4.4. Temperature dependence on short-term storage of exosomes . 84 4.5. Temperature dependence on short-term storage of exosomes . 86 4.6. Summary 90 Chapter 5. in vitro Modeling of Exosomes from ASCs for Huntingtons disease 92 5.1. Introduction . 92 5.2. Methods . 94 5.2.1. Preparation of human ASCs . 94 5.2.2. Preparation of in vitro HD model 94 5.2.3. Isolation and treatment of ASC derived exosome . 95 5.2.4. Analysis of mHtt aggregation in cells . 96 5.2.5. Protein extraction and western blot analysis . 96 5.2.6. Mitochondrial Dysfunction analysis . 97 5.2.7. Cell survival assay . 97 5.2.8. Statistical analysis . 98 5.3. Reduction of accumulation of mHtt aggregates by ASC-exo 98 5.4. Activation of mitochondria and apoptotic proteins by ASC-exo . 101 5.5. Mitochondrial protection and cell survival roles of ASC-exo 102 5.6. Summary 104 Chapter 6. Alleviation of Amyotrophic Lateral Sclerosis by ASC-exo 108 6.1. Introduction . 108 6.2. Methods . 110 6.2.1. Human ASC culture . 110 6.2.2. G93A primary neuronal cell culture 110 6.2.3. Isolation of ASC-exo . 111 6.2.4. Immunocytochemistry . 112 6.2.5. Protein extraction and western blot 112 6.2.6. Preparation of cytoplasmic extracts 113 6.2.7. Dot blot assay . 113 6.2.8. Statistical analysis 114 6.3. Primary cell culture and exosome isolation 114 6.4. Reduction of mutant SOD1 aggregation in G93A by ASC-exo . 118 6.5. Normalization of p-CREB-PGC1α pathway in G93A by ASC-exo . 121 6.6. Summary . 124 Chapter 7. Amelioration of pathology of Alzheimers disease by ASC-exo 126 7.1. Introduction 126 7.2. Methods . 129 7.2.1. In vitro AD model . 129 7.2.2. Culture of ASCs . 130 7.2.3. Isolation and treatment of ASC-exo 131 7.2.4. Aβ ELISA . 133 7.2.5. Western blot analysis . 134 7.2.6. Flow cytometry . 135 7.2.7. Statistical analysis 135 7.3. Reduction of Aβ levels and Aβ 42/40 by ASC-exo . 136 7.4. Increase in apoptotic molecules by ASC-exo . 140 7.5. Attenuation of the apoptosis of AD model cells by ASC-exo 142 7.6. Neurite outgrowth of Neuronal Stem Cells by ASC-exo . 144 7.7. Summary . 146 Chapter 8. Alleviation of Huntingtons disease by Heterochronic Parabiosis 149 8.1. Introduction . 149 8.2. Methods 152 8.2.1. Animal modeling . 152 8.2.2. Parabiosis 153 8.2.3. Administration of BrdU and immunohistochemistry . 154 8.2.4. Preparation of in vitro Huntingtons disease model . 155 8.2.5. Isolation of exosomes from serum and treatment 156 8.2.6. Analysis of mHtt aggregation in cells . 156 8.2.7. Preparation of tissue and fluorescent immunohistochemistry . 157 8.2.8. DAB Immunohistochemistry 158 8.2.9. Protein extraction and western blot analysis 159 8.2.10. Cell survival assay 160 8.2.11. Flow cytometry 161 8.2.12. Statistical analysis 161 8.3. HP modeling and reduction of mHtt aggregation 162 8.4. Activation of mitochondria and modulation of apoptotic proteins . 174 8.5. Reduction of mHtt aggregation by serum exosomes . 183 8.6. Increase of cell survival by serum exosomes . 190 8.7. Summary . 199 Chapter 9. Conclusion 204 9.1. Summary of Results 204 References . 206 Abstract (In Korean) 256 Curriculum Vitae . 259Docto

Organizing pneumonia as the initial presentation of systemic lupus erythematosus in a Korean adolescent

Organizing pneumonia is characterized histologically by the formation of granulation-tissue plugs within the lumens of small airways. It was reported in association with various disorders including infection, drug reactions and collagen vascular diseases. However, there have been only a few reports on organizing pneumonia accompanied by systemic lupus erythematosus (SLE), especially in the pediatric population. Herein, we report a case of an adolescent with SLE who initially developed respiratory illnesses due to organizing pneumonia. A 14-year-old girl was referred to our clinic for protracted cough with fever, dyspnea, and hemoptysis. Her chest x-ray revealed predominant multifocal consolidations in bilateral lung fields with pleural effusion. Computed tomography scan showed patchy consolidations with surrounding ground-glass opacities and a crazy paving appearance with multiple centrilobular nodules. Laboratory tests exhibited pancytopenia, elevated blood urea nitrogen and creatinine, proteinuria, low serum levels of complements, and positivity for antinuclear antibody and anti-double-stranded DNA antibody, which were suggestive of SLE. Lung biopsy was performed to exclude the possibility of vasculitis and other mixed connective tissue diseases, which confirmed focal organizing pneumonia. Systemic steroid therapy, including high-dose methylprednisolone, was started. After the treatment, her respiratory symptoms and radiologic findings showed significant improvements. The patient has been followed up so far, and she has remined disease-free. This pediatric case of organizing pneumonia as the initial presentation of SLE alerts clinicians to consider thorough assessment of pulmonary manifestations of SLE in children.ope

Liver stiffness and perfusion changes for hepatic sinusoidal obstruction syndrome in rabbit model

BACKGROUND: Hepatic sinusoidal obstruction syndrome (SOS) is caused by damage to hepatic sinusoidal endothelial cells that results in fibrous obliteration of intrahepatic venules and necrosis of hepatocytes. Currently the diagnosis is primarily based on nonspecific clinical features and invasive liver biopsy. Therefore, noninvasive imaging methods are required for the early diagnosis and severity assessment of hepatic SOS. AIM: To determine the effectiveness of supersonic shear wave imaging (SSI) and dual energy computed tomography (DECT) for diagnosing hepatic SOS using a rabbit model. METHODS: Among nine New Zealand white rabbits (3-4 kg, male), three in control group ingested normal saline for 20 d and six in the SOS group ingested 6-thioguanine (5 mg/kg/d) for 20 d. Liver stiffness was measured using SSI on days 0, 3, 10, and 20. On the same days, liver perfusion was evaluated from virtual monochromatic images of 55 keV and iodine map using DECT. Morphologic changes in the liver were assessed using CT. Final pathology scores were compared between the two groups. Liver stiffness and perfusion parameters were compared according to the groups, days, and pathology scores. RESULTS: Final pathology scores were significantly higher in the SOS than the control group (median 22 vs 2, P = 0.024). No gross morphologic changes were seen in livers. Liver stiffness, Hounsfield Unit values, and iodine concentrations were higher in the SOS compared to the control group on days 10 and 20 (all, P ≤ 0.007). Compared to day 0, liver stiffness and perfusion parameters were higher on day 20 in the SOS group (all, P ≤ 0.001). Correlation coefficients for liver stiffness (r = 0.635), Hounsfield Unit values (r = 0.587), and iodine concentration (r = 0.611) with final pathology scores were positive without significance (all, P > 0.05). CONCLUSION: Liver stiffness and perfusion parameters were significantly increased in the livers of a rabbit SOS model. SSI and DECT might aid in early diagnosis of hepatic SOS.ope

Role of Chest Computed Tomography in Children with Pneumonia Associated with Coronavirus Disease 2019

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Mesenteric venous thrombosis as a complication of appendicitis in an adolescent: A case report and literature review

RATIONALE: Mesenteric venous thrombosis is an uncommon but potentially fatal condition that can cause bowel ischemia. It results from a systemic hypercoagulable state or abdominal infection draining into the portal venous system. Several cases regarding portomesenteric venous thrombosis as a complication of appendicitis were reported in adults, but there are far fewer reports in pediatric patients. The mortality rate of the condition is high if untreated, especially in children, reaching up to 50%. PATIENT CONCERNS: A healthy 15-year-old male with no significant past medical history presented with right lower quadrant pain, lethargy, and fever. The computed tomography scan showed a focal thrombosis at the superior mesenteric vein branch and an inflamed appendix. DIAGNOSES: Mesenteric venous thrombosis complicating acute appendicitis. INTERVENTIONS: Intravenous antibiotics along with anticoagulants and laparoscopic appendectomy OUTCOMES:: After 1 month, a follow-up ultrasonography revealed full resolution of the thrombosis. LESSONS: Appendicitis is one of the most frequently encountered causes of pediatric surgical emergencies; therefore, physicians should be conscious of mesenteric venous thrombosis as a possible complication of acute appendicitis, irrespective of whether patients have thrombophilic conditions or not.ope

Guideline for Fluoroscopy of Low Gastrointestinal Tract in Pediatrics

Although the availability of CT, MRI and endoscopy has resulted in a marked decline in fluoroscopic procedures in adult patients, fluoroscopy remains an important and frequently used procedure in pediatric patients because there is no appropriate choice of diagnostic imaging or treatment modality for certain diseases. The Korean Society of Pediatric Radiology has formulated evidence-based guidelines for fluoroscopy of the lower intestinal tract in the pediatric population (under age 18 including neonates) in order to assist physicians in clinical practice. The guidelines offer standards of examination practice including radiation doses that are as low as reasonably achievable for children under 18 years old, including neonates, for fluoroscopy of the lower intestinal tract, which has typically used relatively high doses. The recommendations of these guidelines should not be used as an absolute standard, and physicians should always refer to methods that do not adhere to the guidelines when those methods are considered more reasonable and beneficial to an individual patient’s medical situation.ope

Ultrasonography of Congenital Hydronephrosis

Hydronephrosis is the most common urogenital anomaly with the potential for obstructive process. And ultrasonography, as a first-line imaging modality, is a useful tool for evaluation of prenatal and postnatal hydronephrosis, even though evaluation of renal function is still needed. In this article, we review the common causes of congenital hydronephrosis and the sonographic findings of those diseases.ope

Diagnostic Imaging of Biliary Atresia

Biliary atresia is a rare but significant cause of neonatal cholestasis. An early and accurate diagnosis is important for proper management and prognosis. To diagnose biliary atresia, various imaging studies using ultrasonography, MRI, hepatobiliary scans, and cholangiography can be performed, although ultrasonography is more important for initial imaging studies. In this article, we review the findings of biliary atresia from various imaging modalities, including ultrasonography, MRI, hepatobiliary scans, and cholangiography. The known key imaging features include abnormal gallbladder size and shape, periportal thickening visible as a 'triangular cord' sign, invisible common bile duct, increased hepatic arterial flow, and combined anomalies. Aside from the imaging findings of biliary atresia, we also reviewed the diagnostic difficulty in the early neonatal period and the role of imaging in predicting hepatic fibrosis. We hope that this review will aid in the diagnosis of biliary atresia.ope

Differentiation between Clear Cell Sarcoma of the Kidney and Wilms' Tumor with CT

Objective: Clear cell sarcoma of the kidney (CCSK) is the second-most common but extremely rare primary renal malignancy in children after Wilms' tumor. The aims of this study were to evaluate the imaging features that could distinguish between CCSK and Wilms' tumor and to assess the features with diagnostic value for identifying CCSK. Materials and methods: We reviewed the initial contrast-enhanced abdominal-pelvic CT scans of children with CCSK and Wilms' tumor between 2010 to 2019. Fifty-eight children (32 males and 26 females; age, 0.3-10 years), 7 with CCSK, and 51 with Wilms' tumor, were included. The maximum tumor diameter, presence of engorged perinephric vessels, maximum density of the tumor (Tmax) of the enhancing solid portion, paraspinal muscle, contralateral renal vein density, and density ratios (Tmax/muscle and Tmax/vein) were analyzed on the renal parenchymal phase of contrast-enhanced CT. Fisher's exact tests and Mann-Whitney U tests were conducted to analyze the categorical and continuous variables, respectively. Logistic regression and receiver operating characteristic curve analyses were also performed. Results: The age, sex, and tumor diameter did not differ between the two groups. Engorged perinephric vessels were more common in patients in the CCSK group (71% [5/7] vs. 16% [8/51], p = 0.005). Tmax (median, 148.0 vs. 111.0 Hounsfield unit, p = 0.004), Tmax/muscle (median, 2.64 vs. 1.67, p = 0.002), and Tmax/vein (median, 0.94 vs. 0.59, p = 0.002) were higher in the CCSK compared to the Wilms' group. Multiple logistic regression revealed that engorged vessels (odds ratio 13.615; 95% confidence interval [CI], 1.770-104.730) and Tmax/muscle (odds ratio 5.881; 95% CI, 1.337-25.871) were significant predictors of CCSK. The cutoff values of Tmax/muscle (86% sensitivity, 77% specificity) and Tmax/vein (71% sensitivity, 86% specificity) for the diagnosis of CCSK were 1.97 and 0.76, respectively. Conclusion: Perinephric vessel engorgement and greater tumor enhancement (Tmax/muscle > 1.97 or Tmax/vein > 0.76) are helpful for differentiating between CCSK and Wilms' tumor in children aged below 10 years.ope

Use of Animated Cartoons with Children's Songs to Increase Compliance with Ultrasonography in Young Children

PURPOSE: To evaluate the effect of animated cartoons with children's songs to increase compliance with ultrasonography (US) examination in young children. MATERIALS AND METHODS: Animated cartoons with children's songs viewed on a cell phone were played just before the start of US examination when pediatric patients were agitated or irritable. The effect of this method was evaluated for initial responses and sustained responses (grade 0, no response; 1, partial response; and 2, good response). Site of US examination, scan duration, and the helpfulness of this method (0, useless; 1, partially helpful; and 2, very helpful) were also recorded. RESULTS: Among 464 pediatric patients who underwent US during the study period, 88 children (19%) needed to be calmed (67 abdominal and 21 other parts of the body). All subjects were less than five years of age (mean 1.5 years), except for four patients with mental retardation. Scan duration was less than 5 minutes in almost all examinations. Five children refused to watch the cartoon. Initial responses were good in 75 and partial in eight children. Sustained responses were good in 70 and partial in 12 children. The cartoons were very helpful in 73 (83%) and partially helpful in nine (10%) children. The effect of watching the cartoon did not change with sex, age (less or more than one year), or site of examination. CONCLUSION: Animated cartoons with children's songs viewed on a cell phone were helpful (93%) in increasing compliance with US examination in young children of both the abdomen and other parts.ope