저자는 이러한 이미징 절차 수립에 대 한 감염 절차와 L. Eoin 카 니와 브라이언 Lopresti 개요에 대 한 마크 로저스를 인정 하고자 합니다. 이 작품에 대 한 자금 제공 되었습니다는 빌과 멜 린다 게이츠 재단 (J.L.F., P.L.L.)에 의해 건강의 국가 학회, 알레르기의 국가 학회 및 감염 질환 R01 AI111871 (P.L.L.), 국가 심 혼 폐 및 혈액 연구소 R01 HL106804 (J . 왼발), R01 HL110811.

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저자는 공개 없다.

PET/CT에서 수집 된 데이터는 이러한 기술 없이 관찰할 것 M. 결핵 감염의 여러 측면에 대 한 대리 측정으로 사용할 수 있습니다. PET/CT는 원숭이 연구에서 자주 사용 되는 x 선 기술 보다 훨씬 더. PET/CT, 공간 구조 및 기능 정보를 제공합니다. 위에서 설명한 분석 있다 질병의 진행을 모니터링, 약물 치료의 효과 평가 하 고 다시 활성화6,,10<sup ...

결핵균 수천년 동안 인간을 괴롭힌 고 오늘 세계에서 다른 단일 전염성이 에이전트 보다 더 많은 사망 원인. 2015 년에 10.5 백만 보고 새로운 경우 했다 결핵 (TB)의 세계적으로 대부분의 경우 인도, 인도네시아, 중국, 나이지리아, 파키스탄, 그리고 남아 프리 카 공화국에서 집중적으로1 . 견적은 그 같은 기간 동안 1.4 백만 사람들에 TB에서 글로벌 사망자를 놓습니다. 이 값은 거의 25% 사망률 보다 낮은 100 년 전. 약물 과민 한 TB 치료 이지만, 처방 긴 여러 약물 요구 이며 준수가 관심사. 다중 약물 내성 (MDR) 긴장의 출현 ~ 580,000 새로운 결핵 케이스의 2015 년에 차지 했다. M. 결핵 의 MDR 변종과 함께 환자의 성공적인 치료 율은 약 50%로만 추정 된다. 더욱 놀라운 거의 모든 사용할 수 있는 약물에 저항력이 있다 M. 결핵, 광범위 하 게 저항 (XDR) 긴장의 출현 이다. 따라서, 새로운 결핵을 진단 하는 능력을 강화 하는 결핵 연구 분야, 질병 과정의 면역학 이해를 증가 하 고 새로운 치료와 항생제를 포함 한 예방 전략의 심사에 대 한 허용 필요 식이요법 하 고 백신 효능 연구입니다.
M. 결핵 은 실제로 매우 복잡 한 외부 세포 벽 및 느린 성장 속도 의해 특징 이다는 에어로빅 acid-fast 간 균 이다. 감염 증상, 감염 된 개인에서 기침, 재채기, 또는 노래 하는 동안 퇴 학 aerosolized 작은 물방울에 포함 된 개별 박테리아의 흡입을 통해 발생 합니다. 감염을 개발 하는 노출 된 개인의 사람들의 유일한 5-10% 활성 임상 결핵을 개발 합니다. 나머지 90%는 모두의 잠재성 결핵 감염 (LTBI)2,3으로 임상 분류는 전혀 없는 병 subclinical 감염에서 범위 무 증상 감염의 다양 한 스펙트럼이 있다. 이 무 증상 감염이 인구 약 10%는 그들의 일생에서 포함 된 감염의 재 활성화에 의해 활성 결핵을 개발할 것입니다. 다시 활성화의 위험 극적으로 증가 하는 경우 무 증상 감염 계약 에이즈를 가진 사람 또는 TNF 억제제4,,56등 면역 억제 약물으로 치료를 겪 습. 또한 활성 결핵 질병 폐 결핵, 폐, 흉부 림프절에 영향을 미치는 데 대부분의 사람들과 스펙트럼으로 제공 합니다. 그러나, M. 결핵 감염 또한 참여의 extrapulmonary 사이트에 표시할 수 있도록 어떤 기관, 감염 수 있습니다.
M. 결핵 감염의 pathologic 특징은 육아 라는 호스트 세포의 조직된 구형 구조 이다. 대 식 세포, T 세포와 B 세포는 호 중구7의 변수 숫자와 육아의 주요 구성 요소. 육아의 중심은 종종 회 저 성. 따라서, 육아 죽 이십시오, 폐의 다른 부분에 확산 방지를 포함 하는 면역 microenvironment로 동작 합니다. 그러나, M. 결핵 , 육아에 의해 살인을 파괴 하 고 수십 년 동안 이러한 구조 내에서 유지 수 있습니다. 새로운 감염 후 활성 결핵 질병의 개발 또는 LTBI의 재 활성화에 대 한 일관 하 고 정기적으로 모니터링 하는 것은 허무, 과학적 도전 하 고 시간이 소요. 공부 이러한 프로세스 경도, 인간 및 인간 같은 동물 모델, M. 결핵 감염 및 질병의 많은 복잡 한 이해를 발전에 과학적인 사회에 매우 유용한 기술.
PET/CT 인간과 동물 모델8주에서 질병의 광대 한 범위를 공부를 고용 하는 매우 유용한 이미징 기술입니다. 애완 동물 기자로 서 양전자 방출 방사성 화합물을 사용 하는 기능 기술입니다. 이러한 radioisotopes는 일반적으로 기능성 포도 당, 같은 대사 화합물 또는 관심의 수용 체에 바인딩할 설계 대상 그룹. 애완 동물 동위 원소에서 방출 되는 방사 조직에 침투 하기에 충분히 강력 이기 때문에, 매우 낮은 농도 사용할 수 있게 수용 체를 대상으로 화합물에 충분히 낮은 농도에서 채도 수준 연구 신진 대사에 영향을 주지는 처리 하는 2 같은 에이전트를 사용 하 여--2-(18F) 의하여 불 화-D-포도 당 (FDG). CT9신체 내 장기의 물리적 특성을 식별 하기 위해 x 선 감쇠의 다양 한 수준의 사용 하는 3 차원 x 선 영상 기술입니다. 와 결합 하는 때 애완 동물, 코네티컷 특정 위치 및 애완 동물 radiotracer의 표시 구조를 결정 하는 지도로 사용 됩니다. PET/CT는 vivo에서 인간 및 감염 병, 약물 치료, 질병 스펙트럼에 많은 중요 한 통찰력을 주도하 고 있다 M. 결핵 감염 된 동물 모델의 이미지에 대 한 강력한 도구 등6 ,10,,1112. 이 작품에서는 경도 육아 크기, 개별 병 변, 전체 폐와 림프절 FDG 갈망, 그리고 extrapulmonary의 탐지에 FDG 글귀 같은 매개 변수를 사용 하 여 비 인간 영장류 모델에 TB 공부 하 특정 PET/CT 분석 방법을 설명 합니다. 질병6,10,,1112.
이 원고 이미징 비 인간 대주교 (NHPs), 특히 cynomolgus 원숭이, 경도 M. 결핵 감염에 따라 약물 치료 및 질병의 진행을 평가 하는 데 사용 되는 분석의 방법론을 설명 합니다. . NHPs는 귀중 한 동물 모델 M. 결핵 Erdman 스트레인의 낮은 복용량으로 주사 될 때 동물 ~ 50% 활성 결핵 및 무 증상 감염 발생 나머지 동물 개발 다양 한 결과 표시 하기 때문에 (, 즉 , LTBI의 감염 제어), 인간3,13,14,,1516본 임상 질병 스펙트럼에 가까운 모델을 제공 하. LTBI의 재 활성화는 원숭이에서 인간을 하 등 인간 면역 결핍 바이러스 (HIV, 에이즈의 원숭이 버전으로 유인원 면역 결핍 바이러스 (SIV)를 사용 하 여), CD4 고갈 또는 종양에서 다시 활성화 시키는 동일한 대리인에 의해 트리거됩니다. 괴 사 (TNF) 중화13,16요소. 또한, 원숭이 매우 유사한 인간, 폐 또는 다른 기관17형성 조직된 육아를 포함 하 여 본 병 리와 함께 제시. 따라서,이 모델에 M. 결핵 감염으로 약물 regimens과 결핵14,18 에 대 한 백신에 대 한 귀중 한 지식을 기본 호스트 병원 체 상호 작용에 중요 한 통찰력을 제공 하고있다 , 19 , 20 , 21.
PET/CT 영상 모양, 분포, 및 개별 육아의 진행에 따라 기능을 제공 합니다.. 이 작품은 주로 포도 당 아날로그로 metabolically 활성 호스트 세포, 대 식 세포, 호 중구, 림프 톨8, 등이 모두 육아에로 통합 하는 프로브로 FDG를 사용. 따라서, FDG 호스트 염증에 대 한 프록시입니다. 상세한 분석 절차는 여기 OsiriX, 구입 및 사용 하는 데 사용할 수 있는 널리 DICOM 뷰어를 사용 합니다. 설명 하는 이미지 분석 방법을 시간이 지남에 모양, 크기, 및 개별 육아의 신진 대사 활동 (통해 FDG 통풍 관)을 추적 하 고 지도 동물 검 시에 특정 병 변 식별에 대 한 이미징 사용. 또한, 별도 방법 개발 되었습니다 특정 임계값 (SUV ≥ 2.3) 이상 폐에 FDG 통풍 관의 변론을 수량화 하 고 백신에서 배열 하는 연구를 통해 제어 및 실험 그룹 사이의 차이를이 값을 사용 하 여 공동 감염 모델에 실험입니다. 이러한 데이터는 폐에 FDG 이해의이 전반적인 측정 세균성 부담, 질병 상태에 대 한 정보 제공과 상관 하는 지원 합니다. 공부 뿐만 아니라 질병의 진행을 흉부 림프절의 FDG 통풍 관에 유사한 분석을 수행할 수 있습니다. 다음 프로토콜에서 이미지 분석을 통해 동물 감염 실험 과정을 설명합니다.

<table>
	<tbody>
		<tr>
			<td>Ketamine</td>
			<td>Henry Schein</td>
			<td>23061</td>
			<td>Henry Schein</td>
		</tr>
		<tr>
			<td>Telazol</td>
			<td>Zoetis</td>
			<td>4866</td>
			<td>Henry Schein</td>
		</tr>
		<tr>
			<td>Cetacaine</td>
			<td>Patterson Vet Generics</td>
			<td>07-892-6862</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Hospira</td>
			<td>07-800-9721</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>7H11 agar</td>
			<td>BD</td>
			<td>283810</td>
			<td>BD Biosciences</td>
		</tr>
		<tr>
			<td>IV catheter</td>
			<td>Surflash</td>
			<td>07-806-7659</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>18F-FDG</td>
			<td>Zevacor</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotracheal tube</td>
			<td>Jorgensen Labs Inc</td>
			<td>07-887-0284</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Artificial tears</td>
			<td>Patterson Vet Generics</td>
			<td>07-888-1663</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis</td>
			<td>07-806-3204</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Neurologica Ceretom CT</td>
			<td>Samsung Neurologica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Siemens Focus 220 microPET</td>
			<td>Siemens Molecular Imaging Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Inveon Research Software</td>
			<td>Siemens Molecular Imaging Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>OsiriX</td>
			<td>Pixmeo</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of 18fdg pet/ct imaging as a tool for studying mycobacterium tuberculosis infection and treatment in non-human primates

결핵균 은 오늘날 세계에서 번호 하나 전염성이 에이전트를 남아 있습니다. 항생제 내성 변종의 출현으로 새로운 임상 관련 메서드는 질병 과정 및 잠재적인 항생제 및 백신 치료에 대 한 화면을 평가 하는 필요 합니다. 양전자 방출 단층 촬영/계산 단층 촬영 (PET/CT)는 다양 한 암, Alzheimer의 질병, 염증/감염 등 질병을 공부 하기 위한 유용한 도구로 설립 되었습니다. 여기에 설명 된 전략을 intrabronchially M. 결핵의 낮은 복용량으로 감염 되는 cynomolgus 원숭이에 PET/CT 이미지를 평가 하기 위해 고용 수가 있습니다. CT에서 병 변 크기의 평가 및 18F-fluorodeoxyglucose (FDG) 병 변 및 애완 동물 이미지에 림프절에서의 통풍 관을 통해 이러한 설명된 방법을 보여 PET/CT 영상 잠재성 질병 대 활성의 미래 발전을 예측할 수 있는 고 감염의 잠재적인 상태에서 재 활성화의 성향 또한, 폐 염증의 전반적인 수준을 분석 하 여 이러한 방법을 가장 임상 관련 기존 동물 모델에 M. 결핵 에 대 한 약의 항생제 효능을 결정 합니다. 이러한 이미지 분석 메서드는이 질병에 대 한 아 스 날에서 가장 강력한 도구 중의 일부 뿐만 아니라 그들은 감염 및 약물 치료의 특성의 수를 평가할 수 있습니다 하지만 그들은 또한 인간에서 사용 하기 위해 임상 설정으로 직접 번역 연구입니다.

식별 및 개별 장애의 분석
개별 육아 숫자, 크기, 및 FDG 통풍 관 감염 프로세스 (그림 1)의 일반 범위를 이해 하는 것은 질적으로 구상 될 수 있다. 이러한 이미지를 사용 하 여 질병 확산의 양이 많은 측정은 시간이 지남에 육아를 계산. 그림 2 10 동물의 그룹에서 시간이 지남에...

Watch this Scientific Journal Video about Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates at JoVE.com

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

여기, 우리가 질병 과정, 약물 치료, 및 질병의 재 활성화에 M. 결핵 감염 된 비 인간 영장류에서 18F FDG PET/CT 영상의 분석을 설명 하는 프로토콜 제시.

18FDG PET/CT 영상 공부 결핵균 감염 및 비 인간 영장류에서 치료 도구로 서의 분석

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

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JoVE Journal

Immunology and Infection

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

18.7K 조회수.  University of Pittsburgh School of Medicine. 여기, 우리가 질병 과정, 약물 치료, 및 질병의 재 활성화에 M. 결핵 감염 된 비 인간 영장류에서 18F FDG PET/CT 영상의 분석을 설명 하는 프로토콜 제시.

비디오: Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

연구

면역학 및 감염병학

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

의 샘플 준비<em&gt; Mycobacterium 결핵</em핵 자기 공명 Metabolomic의 연구&gt; 압축을 풉니 다

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

의 성장 결핵은 Mycobacterium Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

다중 약물 내성을 보여<em&gt; 결핵균</em&gt; 증폭 마이크로 어레이

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

에 대한 기능성 연구를위한 3D 인간의 폐 조직 모델<em&gt; 결핵균</em&gt; 감염

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

에 의해 쥐 림프구 라벨링<sup&gt; (64)</sup&gt; CU-항체 수용체는 대한 타겟팅<em&gt; 생체</em&gt; PET / CT에 의한 세포 트래 피킹

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

세포 내 타겟팅 억제제의 효능 및 세포 독성 검사를위한 시스템<em&gt; 결핵균</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

기자 효소 형광와 쥐에서 이미징 결핵균

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

진 균 marinum 감염 성인 Zebrafish에 모델링 결핵

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

결핵 균복합체를 연구하기 위한 감염 모델로 무척추동물 갤러리아 멜로넬라 사용

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...