유전체 및 전사체 분석을 활용한 항암제(MTX) 내성 HT-29 세포주의 tandem DHFR 유전자 증폭 특성 및 기전 연구

학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의과학과, 2019. 2. 김종일.The massively parallel sequencing technology known as next-generation sequencing (NGS) has been currently developed and evolved for cancer genome research to obtain the molecular microscope findings and treatment of disease. The time and cost for NGS analysis have been greatly reduced, so the mechanisms from the basic mechanism of human evolution to the complicated mechanism underlying how genetic changes have driven the resistance of cancer cells under anti-cancer drugs have been comprehensively investigated through advancements in NGS technologies. Therefore, the combination of these NGS technologies has contributed to cancer research such as diagnosis, management, and treatment by identifying and elucidating the molecular tumor profiling and it would play an important role in the future of cancer treatment and of personalized medicine in cancer research. DHFR gene amplification is present in methotrexate (MTX) resistant colon cancer cells and in acute lymphoblastic leukemia. The region of chromosome 5q14 contains many genes as well as DHFR gene, and little is known about DHFR gene amplification at this position since quantifying amplification size and recognizing the involved repetitive rearrangements in gene amplification position require extra time and efforts with limited technologies and bioinformatics. Also, there is no clear way to assemble the complete structure of the amplified region with short read (read length repeat length), which provide exceptionally long read lengths, have the potential to overcome these limitations and allow for complete assembly of the region. Here I have proposed an integrative framework to quantify the amplified region and detect structural variations, which are large, complex DNA segments involving repeats by using a combination of technologies, including single molecule real-times sequencing, next generation optical mapping, and high throughput chromosome conformation capture (Hi-C). The amplification units of 11 genes from DHFR gene to ATP6AP1L gene position on chromosome 5 (~2.2Mbp) and tandem gene amplification about twentyfold longer amplified region than control have been identified by several NGS technologies such as optical mapping and single molecule real-times sequencing, and its abnormally increased expression and complicated splicing patterns were characterized by RNA sequencing data. The novel inversion (chr5:80,618,750-80,631,409) at the DHFR gene of amplified region was detected which might stimulate chromosomal breakage for gene amplification Using Hi-C technology, the high adjusted interaction frequencies which indicated the inter-chromosomal contact and significant adjusted p-value were detected on the amplified unit and unsuspected position on 5q in MTX resistant HT-29 sample compared to control. It might explain that chromosomal structure from the start position of the amplified unit (80.6Mb - 82.8Mb) to end of 5q (109Mb-138Mb) could have the complex network of spatial contacts to harbor the gene amplification. Also, the increased relative copy number, the several newly identified topologically associating domains (TADs), and extrachromosomal double minutes (DMs) on this amplified region, which were not detected by other technologies, were identified and described for finding the association with the gene amplification mechanism. Interestingly, the novel frameshift insertions in most of MSH and MLH genes were identified, which could cause the dysregulation of mismatch repair pathway under MTX condition and play an important role on the rapid progression of gene amplification as well as being resistant to MTX. Considering the several characteristics of variable size of tandem gene amplification patterns with homogeneously staining chromosome regions (HSRs), extrachromosomal DM suggested that the gene amplification might be produced from the Breakage-fusion-bridge (BFB) cycles. Overall, the characterized tandem gene amplified unit, more complicated interaction on intra-chromosome 5, inversion of the amplification unit as well as the mutations in MSH and MLH genes can be the critical factor for identifying the mechanism of genomic rearrangements, and these findings may give new insight into the mechanism underlying the amplification process and evolution of resistance to drugs. Therefore, the comprehensive approach of combined advanced technologies is a powerful tool for interpretation of cancer genomes, and this will provide the depth of insight to identify the most important therapeutic mechanism and new targets of the anti-cancer drug.차세대 시퀀싱 (next generation sequencingNGS)으로 알려진 대량 병렬 시퀀싱 기술은 암 유전체 내의 질병의 분자 현미경 수준의 새로운 발견 및 치료법을 얻기 위해 개발되고 발전해 왔다. 현재 차세대 시퀀싱 분석을 위한 시간과 비용이 크게 줄어들었으며, 인간 진화의 기본 메커니즘에서 항암제 내성을 보이는 암 세포의 유전자 변형에 관련된 복잡한 메커니즘에 이르기까지 차세대 시퀀싱 분석의 발전을 통하여 종합적으로 분석되어왔다. 따라서 이러한 차세대 시퀀싱 분석 기술들의 조합은 분자 수준의 종양 프로파일을 규명하고 밝혀줌으로써 진단, 관리 및 치료를 위한 암 연구에 기여했으며, 암 치료 및 암 연구에서의 맞춤 의학의 미래에 중요한 역할을 할 것이다. DHFR 유전자 증폭 현상은 항암제 매토트렉세이트(methotrexateMTX)에 내성을 보이는 결장암 세포에 존재하며 또한 급성 림프 구성 백혈병에 존재한다. 5q14 염색체의 영역은 많은 유전자를 포함하고 있으며 대장 암 세포가 매토트렉세이트 상태에서 저항을 보일 때 유전자 증폭 현상의 근원이 되는 것으로 알려져 있으나, 실제 유전체의 변화에 대해서는 거의 알려져 있지 않았다. 이전에는 짧은 염기 서열 분석 기술을 사용해서 분석하였지만, 제공된 짧은 서열은 반복서열 영역 (repetitive region)을 분석 할 수 없고 접합 서열 (junction reads)를 식별 할 수 없기 때문에 증폭 된 영역의 전체 구조를 조합 (assemble) 할 명확한 방법이 없었다. 예외적으로 긴 서열을 제공하는 단일 분자 실시간 (PacBio SMRT) 시퀀싱은 이러한 한계를 극복하고 반복 영역의 유전체 서열의 완벽한 조립 (assembly) 을 가능하게 한다. 본 연구에서는 단일 분자 실시간 시퀀싱, 차세대 제한효소 광학 지도 (next generation optical mapping) 및 DNA의 3차원(3D) 구성을 측정하는 분석법 (high throughput chromosome conformation captureHi-C )과 같은 새로운 유전자 분석 기술을 사용하여 메토트렉세이트에 내성을 보이는 결장암 세포주(HT-29)내의 유전체 복제 과정을 파악하였고, 크고 복잡한 DNA 단편을 갖는 반복 서열의 구조적 변이(structural variations)를 검출하는 통합적인 프레임워크를 제안하였다. 단일 분자 실시간 시퀀싱과 광학 지도를 활용하여, 유전체 반복서열을 완벽하게 조립하고자 하였고, 5번 염색체의 DHFR 유전자에서 ATP6AP1L 유전자까지 2.2Mbp에 이르는 11 개의 유전자가 복제 단위이자 그 유전자들이 그 일렬 순서대로 대조군에 비해 20배 정도 길게 복제됨을 확인하였다. 또한, 유전자 발현량 및 RNA 유전자 접합 패턴(splicing pattern)을 대조군과 비교 분석한 결과, 유전체 복제 단위에서 작게는 5배에서 크게는 122배까지 비정상적인 유전자 발현량이 측정되었으며, 복잡한 RNA접합 패턴이 동반되는 것을 확인하였다. 또한, 염색체 구조를 파악하는 DNA의 3차원(3D) 구성을 측정한 분석 결과를 토대로, 염색체 내의 유전자가 얼마만큼 상호 작용을 하는가 확인하였을 때, 대조군에 비하여 몇몇의 위상 학적 연관 도메인 (topologically associating domainsTADs)이 매토트렉세이트에 내성을 지신 결장암 세포주(HT-29)의 유전자가 증폭된 영역의 중앙 및 종단점에서 새롭게 발견되었으며, 이 부분에서는 조정된 상호 작용 정도 값이 높고, 그 값이 통계학적으로 유의함(p<0.05)을 확인하였다. 더불어, 발견하기 힘든 이중극미염색체(double minute)가 발견되었다. 흥미롭게도, MSH와 MLH 유전자의 틀이동 삽입 돌연변이 (frameshift insertion)가 매토트렉세이트 (methotrexate) 조건 하에서 염기 쌍의 잘못 짝지움을 수복하는 분자기전(mismatch repair pathway)의 유전적 불안정성과 조절 장애를 일으켰으며, DHFR 유전자 위치에서 역위되어 중복된 경우(inverted duplication)으로 인해 5번 염색체 상의 DHFR 유전자 위치에서 염색체 절단(chromosome breakage)이 발생하였고, 다양한 크기의 유전자가 증폭된 균질염색부위(homogeneously staining regionHSR)가 절단융합가교환(breakage-fusion-bridge cycleBFB cycle)로 생산됨을 유추할 수 있었다. 종합적으로, 본 연구는 5번 염색체 내에서의 보다 복잡한 염색체 상호 작용 및 복제 단위 내의 역위는 유전체 재배열 (genomic rearrangement) 의 기전을 확인하는 중요한 요소가 될 수 있으며, 이러한 발견은 유전자 증폭 과정의 기초가 되는 메커니즘뿐만 아니라 암세포의 항암제 내성 원리에 대한 새로운 통찰력을 제공 할 수 있을 것이라 판단하였다. 따라서 차세대 염기 분석법과 다양한 새로운 첨단 기술을 결합한 분석법은 암 유전체의 해석을 위한 강력한 도구이며, 암 치료의 핵심적인 치료 메커니즘을 파악하여 항암제의 새로운 목표를 설정할 수 있다는 점에서 정밀의학의 발전에 큰 영향을 미칠 것으로 기대한다.Abstract i Contents vi List of Tables vii List of Figures ix List of Abbreviations xiii Introduction 1 Material and Methods 6 Results 28 Discussion 87 References 96 Abstract in Korean 106Docto

A Study on the Operational Improvement of Busan N-anchorage

Busan Port opened since 1876 under the name of "Busanpo". It is the first- class trading port in the Port Act and also considered the oldest and the largest port in Republic of Korea. Busan port serves as the gateway to trade and has been growing steadily as the East Asia's hub-port. Busan Port is comprised of the North port, South port, Kamcheon port, Dadeapo port, Busan new Port and Anchorage. N-Anchorage is being used to wait for schedule or receive oil or change crews. N-anchorage is divided into 5 sub-areas: N-1 ~ N-5. A total of 90 vessels can be anchored according to port laws. Sometimes Anchorage is congested by vessels anchored at the same time. This study analyzes the present states and characteristics in N-anchorage. Anchorage usage is analyzed in duration of 3 years. Weather conditions and marine accidents in Busan are analyzed for 5 years. According to this analysis, we found operational risks of the N-anchorage. The first potential risk is long-term anchoring vessels in N-anchorage. Long-term anchoring vessels include seized vessels, waiting vessels for repair and departure, etc. These vessels lack her operational ability. So, it increased the risk of N-anchorage. The second potential risk is traffic flow crossing N-anchorage. Traffic flow assessment is implemented by using ES(Environmental Stress) model which is formal assessment model for marine flow situation in the diagnosis system. This study suggests that N-2 anchorage should be designed a circle designation system for Long-term anchoring vessels, and traffic flow crossing N-anchorage should also be changed traffic flow bypassing to improve the operational risks in N-anchorage. This suggestions are supported by questionnaire survey and ES model in terms of safety assessment. Therefore, the study will be improving the navigational safety and reducing the risks in N-anchorage.List of Tables ⅲ List of Figures ⅴ Abstract ⅶ 1. 서 론 1.1 연구의 배경 및 목적 1 1.2 연구의 범위 및 방법 2 2. 남외항 대기 정박지 현황 2.1 정박지 운영 4 2.1.1 정박지 정의 4 2.1.2 정박지 지정 방식 및 특성 4 2.1.3 남외항 대기 정박지 현황 11 2.1.4 남외항 대기 정박지 운영 15 2.2 남외항 대기 정박지 이용 현황 19 2.2.1 VHF 교신량(이용선박 척수) 19 2.2.2 톤급별 이용 현황 21 2.2.3 시간별 이용 현황 22 2.2.4 선종별 이용 현황 23 2.3 남외항 대기 정박지 기상 현상 24 2.3.1 풍향 및 풍속 24 2.3.2 안개 27 2.3.3 태풍 28 2.3.4 기상특보 32 3. 남외항 대기 정박지 위험 요소 3.1 해양사고 분석 35 3.1.1 부산항 해양사고 분석 36 3.1.2 남외항 정박지 해양사고 분석 39 3.2 장기대기 선박 50 3.3 남외항 대기 정박지 교통 흐름 59 4. 부산 남외항 대기 정박지 운영 개선 방안 4.1 남외항 대기 정박지 지정방식 운영개선 70 4.1.1 남외항 대기 정박지 지정방식 70 4.1.2 N-2 정박지 장기대기 선박 지정 운영 72 4.2 남외항 대기 정박지 교통 흐름 운영개선 75 4.2.1 남외항 대기 정박지 교통 흐름 변경 75 4.2.2 남외항 대기 정박지 교통 흐름 변경 평가 79 5. 결 론 81 감사의 글 85 참고문헌 8

모음조화의 변화 양상 연구

2011년 2월 국어학 석사학위 논문, 지도교수 김성규 선생

Presence of mEGFR ctDNA predicts a poor clinical outcome in lung adenocarcinoma

BACKGROUND: Circulating tumor DNA (ctDNA) is a biomarker for the selection of target agents in various malignancies. In this study, we examined the effect of ctDNA presence on the response to EGFR-tyrosine kinase inhibitor (TKI) and on the prognosis in lung adenocarcinoma. METHODS: ctDNA of EGFR-TKI sensitizing mutations (mEGFR), L858R substitution and Exon 19 deletion (E19d) mutation, was evaluated using droplet digital PCR (ddPCR) in 81 patients with lung adenocarcinoma which harbored mEGFR in the corresponding tumor tissues. RESULTS: The study recruited lung cancer patients at various stages, and the sensitivity, specificity, and area under the curve (AUC) of mEGFR ctDNA detection by ddPCR were 40.0%, 88.5%, and 0.68, respectively. It showed higher sensitivity (75.0% vs. 10.0%) and AUC (0.83 vs. 0.49) in the advanced stages of lung adenocarcinoma compared with the early stages and the number of metastases and the fractional abundance of mEGFR ctDNA showed a strong correlation (σ = 0.516; P < 0.001, Spearman correlation test). There was a significantly shorter progression-free survival and duration of disease control by EGFR-TKIs in the ctDNA-positive group than the negative group (14.0 vs. 41.0 months, P = 0.02 and 12.0 vs. 23.0 months, P = 0.02, log-rank test, respectively). There was a trend for overall survival time to be shorter in patients with mEGFR ctDNA than for patients without mEGFR ctDNA (35.6 vs. 67.1 months, P = 0.06, log-rank test). CONCLUSIONS: These data showed that mEGFR ctDNA detection using ddPCR is useful in the advanced stages and its presence predicted distant metastasis and poor clinical outcome in lung adenocarcinoma.ope

(The) factors influencing on the attitude toward service quality evaluation : long-term care hospital employees

보건행정학과/석사[한글]본 연구는 요양병원 종사자들이 국내 요양병원의 현황과 요양병원 서비스 질 평 가에 대해 어떻게 인식하고 있는지 알아보고, 서비스 질 평가의 필요성 인식에 영향을 미치는 요인을 파악하기 위해 실시되었다. 조사 대상자는 건강보험심사평가원에서 『요양병원 질 지표 개발 연구』를 위해 표본기관으로 선정한 20개 요양병원의 종사자로, 총 539명이다. 조사 기간은 2008. 8. 11 ~ 8. 25 이고, 수집된 자료는 SPSS 12.0 program을 사용하여 분석하였다. 본 연구의 주요 결과는 다음과 같다. 첫째, 국내 요양병원 현황과 서비스 질 평가에 대한 요양병원 종사자의 인식을 알아보았다. 많은 응답자들이 요양병원의 서비스 질 향상을 위한 가장 효과적인 방법은 일정 기준을 충족하는 기관에 대해 서비스의 질을 보증하는 인증제를 실시하는 것이라고 하였다. 서비스 질 평가의 기준은 업무와 관련한 일부만 알고 있거나, 평가 기준을 모르고 있는 응답자가 많았다. 그리고 평가 결과에 따라 일정수준 이하인 기관의 경우, 서비스 질 향상을 위해 외부적 개입과 지원이 필요하다는 응답이 많았다. 둘째, 평가의 효과에 대한 인식에 영향을 미친 요인을 알아보기 위하여 위계적 중다회귀분석을 실시하였다. 분석 결과, 연령, 직종(의사), 질 향상 활동 여부, 임상진료지침 유무 변수가 유의한 영향을 미치는 것으로 나타났다. 즉 연령이 높을수록, 의사가 아닌 직종이 의사에 비해, 질 향상 활동을 하고 있는 병원의 응답자가, 그렇지 않은 경우에 비해, 임상진료지침이 준비되지 않은 병원의 응답자가 준비된 병원의 응답자에 비해 평가의 효과에 대해 긍정적으로 인식하고 있었다. 셋째, 요양병원 서비스 질 평가의 필요성 인식에 영향을 미치는 요인을 알아보기 위하여 로지스틱 회귀분석을 실시하였다. 분석 결과, 성별, 교육수준, 환자평가도구 유무, 서비스의 질, 병원 경쟁력, 병원 환경 변수가 유의한 영향을 미치는 것으로 나타났다. 즉, 여성이 남성에 비해, 대졸 이상이 대졸 이하에 비해, 환자평가도구가 준비되지 않은 병원의 응답자가 준비되어있는 병원의 응답자에 비해, 서비스의 질이 향상될 것으로 인식할수록, 병원 경쟁력이 강화될 것으로 인식할수록, 병원 환경이 좋아질 것으로 인식할수록 서비스 질 평가가 필요하다고 응답할 가능성이 큰 것으로 나타났다. 본 연구결과를 토대로 몇 가지 제안을 하고자 한다. 첫째, 요양병원의 서비스 질 향상을 위해 인증제를 실시하는 것이 가장 효과적일 것이라는 의견을 바탕으로, 일정 기준을 충족하는 기관에 대해 서비스의 질을 보증하는 인증제를 고려해볼 필요가 있다. 그리고 일정수준 이하인 기관에 대해서는 외부적 개입과 지원이 요구된다. 둘째, 응답자들이 평가기준에 대해 일부만 알고 있거나, 모르고 있는 경우가 많았다. 요양병원 서비스 질 평가를 수행하기에 앞서, 평가의 목적이나 기준에 대해 요양병원 종사자들에게 충분한 설명이 필요하다. 셋째, 요양병원 서비스 질 평가의 필요성 인식에 가장 유의한 영향을 미치는 변수는 평가의 효과에 대한 인식 중 서비스의 질 이었다. 종사자들이 서비스의 질이 향상될 것으로 인식할 수 있도록 평가의 목적과 방향, 평가 후 임상의 질이 향상된 사례에 대한 교육이 필요할 것이다. 이상의 연구에서는 요양병원과 서비스 질 평가에 대해 요양병원 종사자들이 어떻게 인식하고 있는지 알아본 것으로, 요양병원을 이용하는 환자와 보호자들이 어떻게 인식하고 있는지에 대한 추가연구가 필요하다. 그리고 평가 시행 전과 후, 평가 결과에 따라, 임상의 질에 어떠한 차이가 있는지에 대한 연구도 함께 이루어져야 할 것이다. [영문]This study was carried out to see how long-term care hospital employees perceive the service quality evaluation and to identify factors influencing the perception of necessities for the service quality evaluation. Subjects for research included 539 in total who were employed for 20 long-term care hospitals which were selected as sample institutions for 『a study on the development of quality indicator for long-term care hospitals』 in Health Insurance Review & Assessment Service. The term of research was a period from 11~25 August 2008 and the collected data were analyzed using SPSS 12.0 program. Major findings are as follows in this study. First, the perception of long-term care hospital employees toward service quality evaluation was identified. Many respondents expressed that the most effective method to increase the service quality of long-term care hospitals was to implement a system to certify and provide a guarantee for the service quality to institutions which meet certain standards. There were many respondents who knew only part of business related to the standards on the service quality evaluation or nothing about such evaluation standards. In addition, there were many respondents who said it was necessary to have any external involvements and supports to increase the service quality in case of institutions under certain standards according to the evaluation results. Second, hierarchical regression analyses were done to find the factors influencing the perception. As a result of such analyses, more positive perception was expressed from those with higher age, with non-doctor jobs comparing to doctor jobs, with activities for the quality improvements comparing to other activities, and without preparation for the clinical diagnosis and treatment guidelines comparing to with those preparations. Third, logistic regression analyses were done to see factors influencing the perception of necessities for the service quality evaluation of long-term care hospitals. As a result of such analyses, there were more possibilities of positive responses from women, those with college degree or higher, hospitals without preparations for the patients evaluation tools, those with positive expectation for the service quality, those expecting the increase of competitiveness for hospitals, and those expecting the improvement of hospital environments. This study attempts to make a few suggestions based on the findings. First, it is necessary to consider bringing forward a certification system to guarantee the service quality for institutions which meet certain standards. In addition, it is required to have any external involvements and supports for institutions under certain standards. Second, it is necessary to provide long-term care hospital employees with sufficient explanations on the objectives or standards of evaluation before the service quality evaluation for long-term care hospitals is implemented. Third, it is necessary to provide education regarding the improved examples of clinical quality after such evaluation along with the purposes and directions of evaluation, so that those employees might perceive that the service quality would be improved.ope

Comparison of QFT-Plus and QFT-GIT tests for diagnosis of M. tuberculosis infection in immunocompetent Korean subjects

Background: QFT-Plus is a recently developed next-generation QuantiFERON-TB Gold In-Tube (QFT-GIT) test. Unlike the QFT-GIT test, it includes a TB2 antigen tube with peptides that may stimulate CD8+ T cells. This study evaluated the diagnostic performance of QFT-Plus and compared it with that of QFT-GIT. Methods: QFT-Plus and QFT-GIT tests were performed in 33 patients with active tuberculosis (TB) and 57 healthy controls including subjects with latent TB infection (LTBI). Positivity and negativity of IFN-γ responses were compared between tests, and total concordance of the outcome was analyzed. Results: Positive and negative outcomes of QFT-Plus and QFT-GIT tests showed substantial agreement (91.1%, kappa=0.8). The sensitivity and the specificity of QFT-Plus (93.9% sensitivity, 92.6% specificity) were similar with those of QFT-GIT (93.9% sensitivity, 100% specificity). Of eight discordant results, five (5.56%) and three (3.3%) were positive in QFT-GIT alone and QFT-plus alone, respectively. Reactivity in the TB2 tube contributes to the difference between QFT-GIT and QFT-Plus. Median IFN-γ production in TB2 (10.0 IU/mL in TB, 3.850 IU/mL in LTBI, P=0.001) is significantly higher in the TB group than the LTBI group. The QFT-Plus did not clearly discriminate between active TB and latent TB, although it showed significantly lower IFN-γ concentrations compared with the QFT-GIT in individuals with LTBI (3.850 vs. 7.205 IU/mL). Conclusions: Similar accuracy of detecting Mycobacterium tuberculosis infection was observed for QFT-Plus and QFT-GIT tests in immunocompetent patients and healthy controls, including those with LTBI. Improved efficacy for identifying M. tuberculosis infection was not found with the QFT-Plus, but further studies in a larger population may confirm the clinical significance of positive response in the TB2 tube of QFT-Plus.ope

Unique Chemokine Profiles of Lung Tissues Distinguish Post-chemotherapeutic Persistent and Chronic Tuberculosis in a Mouse Model

There is a substantial need for biomarkers to distinguish latent stage from active Mycobacterium tuberculosis infections, for predicting disease progression. To induce the reactivation of tuberculosis, we present a new experimental animal model modified based on the previous model established by our group. In the new model, the reactivation of tuberculosis is induced without administration of immunosuppressive agents, which might disturb immune responses. To identify the immunological status of the persistent and chronic stages, we analyzed immunological genes in lung tissues from mice infected with M. tuberculosis. Gene expression was screened using cDNA microarray analysis and confirmed by quantitative RT-PCR. Based on the cDNA microarray results, 11 candidate cytokines genes, which were obviously up-regulated during the chronic stage compared with those during the persistent stage, were selected and clustered into three groups: (1) chemokine genes, except those of monocyte chemoattractant proteins (MCPs; CXCL9, CXCL10, CXCL11, CCL5, CCL19); (2) MCP genes (CCL2, CCL7, CCL8, CCL12); and (3) TNF and IFN-γ genes. Results from the cDNA microarray and quantitative RT-PCR analyses revealed that the mRNA expression of the selected cytokine genes was significantly higher in lung tissues of the chronic stage than of the persistent stage. Three chemokines (CCL5, CCL19, and CXCL9) and three MCPs (CCL7, CCL2, and CCL12) were noticeably increased in the chronic stage compared with the persistent stage by cDNA microarray (p < 0.01, except CCL12) or RT-PCR (p < 0.01). Therefore, these six significantly increased cytokines in lung tissue from the mouse tuberculosis model might be candidates for biomarkers to distinguish the two disease stages. This information can be combined with already reported potential biomarkers to construct a network of more efficient tuberculosis markers.ope

Protective Vaccine Efficacy of the Complete Form of PPE39 Protein from Mycobacterium tuberculosis Beijing/K Strain in Mice

The aim of this study was to evaluate the protective efficacy of MTBK_24820, a complete form of PPE39 protein derived from a predominant Beijing/K strain of Mycobacterium tuberculosis in South Korea. Mice were immunized with MTKB_24820, M. bovis Bacilli Calmette-Guérin (BCG), or adjuvant prior to a high-dosed Beijing/K strain aerosol infection. After 4 and 9 weeks, bacterial loads were determined and histopathologic and immunologic features in the lungs and spleens of the M. tuberculosis-infected mice were analyzed. Putative immunogenic T-cell epitopes were examined using synthetic overlapping peptides. Successful immunization of MTBK_24820 in mice was confirmed by increased IgG responses (P < 0.05) and recalled gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-6, and IL-17 responses (P < 0.05 or P < 0.01) to MTBK_24820. After challenge with the Beijing/K strain, an approximately 0.5 to 1.0 log10 reduction in CFU in lungs and fewer lung inflammation lesions were observed in MTBK_24820-immunized mice compared to those for control mice. Moreover, MTBK_24820 immunization elicited significantly higher numbers of CD4+ T cells producing protective cytokines, such as IFN-γ and IL-17, in lungs and spleens (P < 0.01) and CD4+ multifunctional T cells producing IFN-γ, tumor necrosis factor alpha (TNF-α), and/or IL-17 (P < 0.01) than in control mice, suggesting protection comparable to that of BCG against the hypervirulent Beijing/K strain. The dominant immunogenic T-cell epitopes that induced IFN-γ production were at the N terminus (amino acids 85 to 102 and 217 to 234). Its vaccine potential, along with protective immune responses in vivo, may be informative for vaccine development, particularly in regions where the M. tuberculosis Beijing/K-strain is frequently isolated from TB patients.ope

ABT-737 Synergizes with Cisplatin Bypassing Aberration of Apoptotic Pathway in Non-small Cell Lung Cancer

A subset of non-small cell lung cancer (NSCLC), which does not have a druggable driver mutation, is treated with platinum-based cytotoxic chemotherapy, but it develops resistance triggered by DNA damage responses. Here, we investigated the effect of activation of STAT3 by cisplatin on anti-apoptotic proteins and the effectiveness of a co-treatment with cisplatin and a BH3 mimetic, ABT-737. We analyzed the relationship between cisplatin and STAT3 pathway and effect of ABT-737, when combined with cisplatin in NSCLC cells and K-ras mutant mouse models. The synergism of this combination was evaluated by the Chou-Talalay Combination Index method. In vivo activity was evaluated by micro-CT. In NSCLC cells, there was a time and dose-dependent phosphorylation of SRC-JAK2-STAT3 by cisplatin, followed by increased expression of anti-apoptotic molecules. When the expression of the BCL-2 protein family members was evaluated in clinical samples, BCL-xL was most frequently overexpressed. Dominant negative STAT3 suppressed their expression, suggesting that STAT3 mediates cisplatin mediated overexpression of the anti-apoptotic molecules. ABT-737 displaced BCL-xL from mitochondria and induced oligomerization of BAK. ABT-737 itself showed cytotoxic effects and a combination of ABT-737 with cisplatin showed strong synergistic cytotoxicity. In a murine lung cancer model, co-treatment with ABT-737 and cisplatin induced significant tumor regression. These findings reveal a synergistic cytotoxic and anti-tumor activity of ABT-737 and cisplatin co-treatment in preclinical models, and suggest that clinical trials using this strategy may be beneficial in advanced NSCLC.ope