المؤلفون أن نشيد رودجرز مارك لتحدد إجراءات العدوى ولام أوين كارني و Lopresti ريان للتوجيه في وضع هذه الإجراءات التصوير. التمويل اللازم لهذا العمل قد قدمت قبل بيل وميليندا غيتس (J.L.F.، P.L.L.)، المعاهد الوطنية للصحة، والمعاهد الوطنية للحساسية و AI111871 R01 الأمراض المعدية (P.L.L.)، والوطنية القلب الرئة والدم المعهد R01 HL106804 (ي . L.F.)، R01 HL110811.

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الكتاب ليس لها علاقة بالكشف عن.

يمكن استخدام البيانات التي تم الحصول عليها من الحيوانات الأليفة/CT القياسات البديلة لجوانب كثيرة من عدوى السل م أن ينتبه دون مثل هذه التكنولوجيا. PET/CT أكثر حساسية من تكنولوجيا الأشعة السينية، التي غالباً ما تستخدم في الدراسات ماكاكويس. PET/CT يوفر المعلومات الهيكلية والمكانية والوظيفية....

متفطره ابتلي البشر لآلاف السنين ويسبب وفيات أكثر من أي عامل المعدية الأخرى الوحيدة في العالم اليوم. في عام 2015، هناك 10.5 مليون حالات جديدة لمرض السل (TB) على الصعيد العالمي1 مع معظم الحالات النابعة من الهند، وإندونيسيا، والصين، ونيجيريا، وباكستان وجنوب أفريقيا. وضع تقديرات الوفيات العالمية من السل في 1.4 مليون شخص خلال تلك الفترة الزمنية نفسها. هذه القيمة ما يقرب من 25 ٪ أقل من معدل الوفاة قبل 100 سنة. على الرغم من أن المخدرات السل حساسة يمكن علاجها، النظام المطولة التي تتطلب أدوية متعددة والامتثال مصدر قلق. واستأثرت ظهور سلالات (المقاوم) المقاوم للعقاقير المتعددة ~ 580,000 حالات السل الجديدة في عام 2015. ويقدر معدل نجاح العلاج للمرضى مع سلالات المقاوم السل م فقط إلى حوالي 50%. حتى أكثر مثيرة للقلق هو ظهور سلالات (XDR) على نطاق واسع مقاومة السل م، ومقاومة للعقاقير المتاحة تقريبا كل. وبالتالي، هناك حاجة إلى تقنيات جديدة داخل مجال بحوث السل التي تعزز القدرة على تشخيص السل، زيادة فهم عملية الأمراض المناعية، والسماح لفحص استراتيجيات الوقاية، بما في ذلك المضادات الحيوية وعلاجات جديدة أنظمة ودراسات نجاعة اللقاح.
هو السل م عصيات acid-fast هوائية تتسم فعلياً بجدار الخلية الخارجي معقدة للغاية وحركية النمو البطيء. عموما تحدث العدوى عن طريق استنشاق البكتيريا الفردية الواردة في قطرات الضبوبيه التي يطردون من فرد بالأعراض أو المصابة أثناء السعال والعطس، أو الغناء. من الأفراد المعرضين التي تتطور العدوى، تطوير فقط 5-10% من الناس السل النشط السريرية. 90% المتبقية لديها طائفة متفاوتة من أعراض العدوى التي تتراوح من الإصابة السريرية للا مرض على الإطلاق، التي تصنف سريرياً العدوى (لتبي) السل الكامنة2،3. من السكان أن هذه العدوى أعراض، ستضع 10% تقريبا السل النشط بإعادة تنشيط العدوى الواردة في حياتهم. خطر إعادة تنشيط هائلة تزداد إذا كان الشخص مع العقود لا تظهر عليهن أعراض الإصابة بفيروس نقص المناعة البشرية أو يخضع للعلاج العقاقير المثبطة للمناعة، مثل تنف مثبطات4،،من56. ويعرض أيضا مرض السل النشط كطائفة، مع معظم الناس بعد أن السل الرئوي، الذي يؤثر على الرئتين والغدد الليمفاوية الصدرية. ومع ذلك، يمكن أن تصيب السل م. أي جهاز، حيث أن العدوى يمكن أن يقدم أيضا في مواقع خارج الرئة من المشاركة.
السمة المميزة مرضية للإصابة السل م بنية منظمة كروية من الخلايا المضيفة، تسمى الحبيبي. الضامة، والخلايا T والخلايا ب هي المكونات الرئيسية الحبيبي، مع أرقام متغير من العَدلات7. غالباً ما مركز الحبيبي نخرية. وهكذا، حبيبية تعمل المكروية محصنة بقتل أو تحتوي على العصيات، منع الانتشار إلى أجزاء أخرى من الرئتين. ومع ذلك، السل م. يمكن تخريب قتل قبل الحبيبي، وتستمر داخل هذه الهياكل لعقود. رصد متسقة ومنتظمة لتطوير مرض السل النشط بعد عدوى جديدة أو إعادة تنشيط لتبي غير عملي وتحديا علمياً ومضيعة للوقت. تقنيات دراسة هذه العمليات طوليا، في البشر ونماذج الحيوانات الشبيهة بالإنسان، وهي مفيدة للغاية للمجتمع العلمي في تعزيز فهم تعقيدات كثيرة الإصابة السل م والمرض.
وهي PET/CT تقنية مفيدة للغاية تصوير التي استخدمت لدراسة طائفة واسعة من الدول المرض في البشر ونماذج حيوانية8. الحيوانات الأليفة هي تقنية وظيفية تستخدم المركبات المشعة التي تنبعث منها البوزيترون كمراسل. يتم عادة فونكتيوناليزيد هذه النظائر المشعة إلى مجمع ايضية، مثل السكر، أو إلى مجموعة استهداف الذي يهدف إلى ربط مستقبلات للفائدة. ولما قوية بما فيه الكفاية لاختراق الأنسجة الإشعاعات المنبعثة من النظائر المشعة في الحيوانات الأليفة، تركيزات منخفضة للغاية يمكن استخدامها الذي يسمح للدراسة دون مستويات التشبع في استهداف مستقبلات المركبات وفي تركيز منخفضة بما يكفي ليكون له تأثير على التمثيل الغذائي عمليات عند استخدام عوامل مثل 2-ديوكسي-2-(18و) مخفضات-د-الجلوكوز (فدج). التصوير المقطعي هو تقنية تصوير ثلاثية الأبعاد بالأشعة سينية التي تستخدم مستويات متفاوتة من توهين الأشعة السينية لتحديد الخصائص الفيزيائية للأجهزة داخل الجسم9. عندما يقترن مع الحيوانات الأليفة، والتصوير المقطعي يستخدم كمخطط لتحديد مواقع محددة والهياكل التي تظهر الإقبال راديوتراسير الحيوانات الأليفة. PET/CT هو أداة قوية للتصوير في فيفو البشر ونماذج حيوانية مصاب بعدوى السل م التي أدت إلى العديد من الأفكار الهامة في الآلية المرضية، استجابة للعلاج من تعاطي المخدرات، مرض الطيف، إلخ6 ،10،،من1112. ويصف هذا العمل محددة PET/CT الأساليب التحليلية لدراسة مرض السل في نماذج الرئيسيات غير البشرية طوليا باستخدام معلمات مثل حجم الورم، وامتصاص فدج في الآفات الفردية وكله اللذة فدج العقدة الليمفاوية والرئة، وكشف خارج الرئة المرض6،10،،من1112.
ويصف هذه المخطوطة منهجيات للتصوير التحليل في المقدمات غير البشرية (NHPs)، على وجه التحديد macaques سينومولوغوس، التي تستخدم لتقييم طوليا تطور المرض والعلاج من تعاطي المخدرات عقب العدوى مع السل م. . نهبس نموذج حيوانية قيمة لأنه عند تلقيح مع جرعة منخفضة من سلالة اردمان السل م ، الحيوانات تظهر مجموعة متنوعة من نتائج المرض مع ~ 50% وضع مرض السل النشط والحيوانات المتبقية بعد الإصابة بأعراض (أي السيطرة على العدوى، لتبي)، تقديم النموذج الأقرب إلى الطيف الأمراض السريرية، ينظر في البشر3،،من1314،،من1516. يتم تشغيل تنشيط لتبي في ماكاكويس بنفس العوامل التي تسبب إعادة التنشيط في البشر، والأمثلة التي تشمل فيروس نقص المناعة البشرية (فيروس نقص المناعة البشرية، فيروس نقص المناعة القردي (سيف) باستخدام الإصدار المكاك من فيروس نقص المناعة البشرية)، ونضوب CD4 أو الورم عامل نخر (تنف) تحييد13،16. وبالإضافة إلى ذلك، يقدم macaques مع أمراض مشابهة جداً لأن ينظر في البشر، بما في ذلك حبيبية المنظمة التي تشكل في الرئتين أو أجهزة أخرى17. وهكذا، قدم هذا النموذج معلومات هامة بشأن التفاعلات الممرض المضيف الأساسية في الإصابة السل م ، فضلا عن معرفة قيمة حول أنظمة العقاقير واللقاحات المضادة للدرن14،18 , 19 , 20 , 21.
تصوير PET/CT يوفر القدرة على تتبع مظهر، والتوزيع، وتطور حبيبية الفردية. واستخدمت هذا العمل أساسا فدج كتحقيق، التي، التماثلية جلوكوز، ودمج الخلايا المضيفة نشاط أيضي، مثل الضامة والعدلات واللمفاويات8، كلها في حبيبية. وهكذا، فدج وكيل لالتهاب المضيف. ويستخدم تحليل الإجراءات المفصلة هنا أوسيريكس، عارض DICOM مستخدمة على نطاق واسع متاحة لشراء واستخدام. أساليب تحليل الصورة ووصف تتبع الشكل والحجم، والنشاط الأيضي (عن طريق امتصاص فدج) من حبيبية الفردية على مر الزمن، ويستخدم التصوير كمخطط لتحديد آفات محددة عند نيكروبسي الحيوانية. بالإضافة إلى ذلك، أسلوب منفصل وضعت يوضحها الجمع امتصاص فدج في الرئة عتبة محددة (≥ 2.3 من سيارات الدفع الرباعي) ويستخدم هذه القيمة لتقييم الفروق بين المجموعات التجريبية والتحكم عبر الدراسات التي تتراوح من اللقاح المحاكمات لنماذج الإصابة المشتركة. تدعم هذه البيانات أن يرتبط هذا المقياس العام لامتصاص فدج في الرئتين بعبء البكتيرية، مما يوفر معلومات حول حالة المرض. يمكن إجراء تحليلات مماثلة على استيعاب فدج الليمفاوية الصدرية لدراسة تطور المرض، وكذلك. البروتوكول التالي يصف عملية تجريبية من العدوى الحيوانية من خلال تحليل الصور.

<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

بكتريا السل لا يزال عامل المعدية رقم واحد في العالم اليوم. مع ظهور سلالات مقاومة للمضادات الحيوية، طرق جديدة ذات الصلة سريرياً وهناك حاجة إلى تقييم عملية المرض وشاشة لعلاجات المضادات الحيوية واللقاحات المحتملة. وقد أنشئت Computed التصوير المقطعي بالبوزيترون/"التصوير المقطعي" (PET/CT) كأداة قيمة لدراسة عدد من الأمراض مثل السرطان ومرض الزهايمر، والإصابة بالتهاب/. المبينة هنا هي عدد من الاستراتيجيات التي استخدمت لتقييم الصور PET/CT في macaques cynomolgus التي يصاب إينترابرونتشيالي بجرعات منخفضة من السل م. من خلال تقييم حجم الآفة على الأشعة المقطعية والإقبال على 18وفلوروديوكسيجلوكوسي (فدج) في الآفات والغدد الليمفاوية في صور الحيوانات الأليفة، تظهر هذه الأساليب المذكورة أن التصوير PET/CT يمكن التنبؤ بالتطور المستقبلي لنشاط مقابل المرض الكامنة الميل لإعادة تنشيط من حالة العدوى كامنة. بالإضافة إلى ذلك، من خلال تحليل المستوى الإجمالي لالتهاب الرئة، تحدد هذه الأساليب فعالية المضادات الحيوية من العقاقير المضادة السل م في نموذج الحيوانية الموجودة ذات الصلة سريرياً أكثر. أساليب تحليل الصورة هذه بعض من أقوى الأدوات في الترسانة مكافحة هذا المرض كما لا يمكن لها أن تقيم عددا من الخصائص للإصابة والعلاج من تعاطي المخدرات فحسب، بل أيضا الترجمة مباشرة إلى إعداد سريرية لاستخدامها في الإنسان الدراسات.

تحديد وتحليل للآفات الفردية
يمكن تصور حبيبية الفردية لعدد وحجم، وامتصاص فدج نوعيا لفهم النطاق العام لعملية العدوى (الشكل 1). باستخدام هذه الصور، عد حبيبية مع مرور الزمن مقياس كمي لانتشار المرض. ويبين الشكل...

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

نقدم هنا، بروتوكولا لوصف تحليل التصوير PET/CT فدج و 18في المقدمات غير البشرية التي قد أصيبوا السل م لدراسة عملية المرض، والعلاج من تعاطي المخدرات، وإعادة تنشيط المرض.

تحليل 18"فدج التصوير" PET/CT كأداة لدراسة بكتريا السل العدوى والعلاج في الإنسان عدم الرئيسات

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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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 Views.  University of Pittsburgh School of Medicine. نقدم هنا، بروتوكولا لوصف تحليل التصوير PET/CT فدج و 18في المقدمات غير البشرية التي قد أصيبوا السل م لدراسة عملية المرض، والعلاج من تعاطي المخدرات، وإعادة تنشيط المرض.

Video: 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.

إدخال إجهاد القص في دراسة الالتصاق الجرثومي

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

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.

تحضير العينة من<em&gt; المتفطرة السلية</em&gt; خلاصات لدراسات النووية المغناطيسي الرنين Metabolomic

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.

نمو المتفطرة السلية الأغشية الحيوية

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.

مما يدل على متعدد المخدرات مقاومة<em&gt; السل المتفطرة</em&gt; التضخيم ميكروأري

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.

نموذج الأنسجة 3D الرئة الإنسان للدراسات وظيفية على<em&gt; المتفطرة السلية</em&gt; العدوى

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).

الفئران اللمفاويات وسمها من قبل<sup&gt; 64</sup&gt; النحاس والأجسام المضادة مستقبلات استهداف ل<em&gt; في فيفو</em&gt; الاتجار خلية بواسطة PET / ط م

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.

نظام لفعالية والسمسة فحص مثبطات استهداف بين الخلايا<em&gt; المتفطرة السلية</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.

تصوير بكتريا السل في الفئران مع "مراسل إنزيم الأسفار"

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.

السل النمذجة في "الزرد الكبار المصابين" بكتريا مارينوم

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.

استخدام اللافقاريات جاليريا ميلونيلا كنموذج للعدوى لدراسة مجمع السل الميكوباكتيريوم

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 ...

تحليل ¹⁸"فدج التصوير" PET/CT كأداة لدراسة بكتريا السل العدوى والعلاج في الإنسان عدم الرئيسات

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إدخال إجهاد القص في دراسة الالتصاق الجرثومي

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نموذج الأنسجة 3D الرئة الإنسان للدراسات وظيفية على

الفئران اللمفاويات وسمها من قبل

نظام لفعالية والسمسة فحص مثبطات استهداف بين الخلايا

تصوير بكتريا السل في الفئران مع "مراسل إنزيم الأسفار"

السل النمذجة في "الزرد الكبار المصابين" بكتريا مارينوم

استخدام اللافقاريات جاليريا ميلونيلا كنموذج للعدوى لدراسة مجمع السل الميكوباكتيريوم