نود أن نشكر باتريك هايدن (ماتيك) لإجراء مناقشات مفيدة ، وروبرت سميث وبيري ليبي للعلوم الصحية للجامعة جورجيا EM المهارات الخاصة بهم. وقد تم تمويل هذه الدراسة من خلال منحة NIDCD DC010187 إلى DAD

<ol>
	<li>Murphy, T. F., Apicella, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nontypeable+Haemophilus+influenzae:+a+review+of+clinical+aspects,+surface+antigens,+and+the+human+immune+response+to+infection.">Nontypeable Haemophilus influenzae: a review of clinical aspects, surface antigens, and the human immune response to infection.</a> Rev. Infect. Dis. 9, 1-15 (1987).</li><li>Murphy, T. F., Faden, H., Bakaletz, L. O., Kyd, J. M., Forsgren, A., Campos, J., Virji, M., Pelton, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nontypeable+Haemophilus+influenzae+as+a+pathogen+in+children.">Nontypeable Haemophilus influenzae as a pathogen in children.</a> Ped. Infect. Dis. J. 28, 43-48 (2009).</li><li>Hotomi, M., Arai, J., Billal, D. S., Takei, S., KIkeda, Y., Ogami, M., Kono, M., Beder, L. B., Toya, K., Kimura, M., Yamanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nontypeable+Haemophilus+influenzae+isolated+from+intractable+acute+otitis+media+internalized+into+cultured+human+epithelial+cells.">Nontypeable Haemophilus influenzae isolated from intractable acute otitis media internalized into cultured human epithelial cells.</a> Auris Nasus Larynx. 37, 137-144 (2010).</li><li>Chemuturi, N. V., Hayden, P., Kalausner, M., Donovan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+tracheal/bronchial+epithelial+cell+culture+and+bovine+nasal+respiratory+explants+for+nasal+drug+transport+studies.">Comparison of human tracheal/bronchial epithelial cell culture and bovine nasal respiratory explants for nasal drug transport studies.</a> J. Pharm. Sci. 94, 1976-1985 (2005).</li><li>Sharma, M., Schoop, R., Hudson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+Echinacea+in+a+3-D+tissue+model+of+human+airway+epithelium.">The efficacy of Echinacea in a 3-D tissue model of human airway epithelium.</a> Phytother. Res. 24, 900-904 (2010).</li><li>Sexton, K., Balharry, D., BeruBe, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+biomarkers+of+pulmonary+exposure+to+tobacco+smoke+components.">Genomic biomarkers of pulmonary exposure to tobacco smoke components.</a> Pharmacogenet Genomics. 10, 853-860 (2008).</li><li>Babu, R. J., Dayal, P., Singh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cyclodextrins+on+the+complexation+and+nasal+permeation+of+melatonin.">Effect of cyclodextrins on the complexation and nasal permeation of melatonin.</a> Drug Deliv. 6, 381-388 (2008).</li><li>Balharry, D., Sexton, K., BeruBe, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+approach+to+assess+the+toxicity+of+inhaled+tobacco+smoke+components:+nicotine,+cadmium,+formaldehyde+and+urethane.">An in vitro approach to assess the toxicity of inhaled tobacco smoke components: nicotine, cadmium, formaldehyde and urethane.</a> Toxicology. 244, 66-76 (2008).</li><li>Fahy, J. V., Dickey, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+mucus+function+and+dysfunction.">Airway mucus function and dysfunction.</a> N. Engl. J. Med. 363, 2233-2247 (2010).</li><li>Huang, Y., Mikami, F., Jono, H., Zhang, W., Weng, X., Koga, T., Xu, H., Yan, C., Kai, H., Li, J. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+roles+of+PAK2+and+PAK4+in+synergistic+induction+of+MUC5AC+mucin+by+bacterium+NTHi.">Opposing roles of PAK2 and PAK4 in synergistic induction of MUC5AC mucin by bacterium NTHi.</a> 359, 691-696 (2007).</li></ol>

الإعلان عن أي تضارب في المصالح.

هذا الأسلوب يسمح للتحقيق في التفاعلات المضيف الممرض على المدى الطويل في خلفية ذات الصلة بيولوجيا من الأنسجة الابتدائية التنفسي للإنسان في واجهة الهواء السائل. هنا استخدمنا NTHi كما يصيب الكائن ، ولكن يمكن كميا أي تفاعل البكتيريا التي لا يدخل السمسة غير مقبولة على مر ا...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم كاشف </th><th> شركة </th><th> كتالوج # </th><th> تعليقات </th></tr><tr><td> سابونين </td><td> Calbiochem </td><td> 558255 - 25GM </td><td> 1 ٪ في برنامج تلفزيوني من دون مد الكالسيوم أو المغنسيوم ، وتصفية تعقيم </td></tr><tr><td> 1 X Dulbecco والفوسفات مخزنة المالحة مع الكالسيوم والمغنيسيوم </td><td> Lonza </td><td> 17 - 513Q </td><td></td></tr><tr><td> 1 X Dulbecco والفوسفات مخزنة المالحة دون الكالسيوم أو المغنسيوم </td><td> Lonza </td><td> 17 - 515Q </td><td></td></tr><tr><td> EpiAirway المضادات الحيوية خالية من الأنسجة </td><td> ماتيك </td><td> AIR - 100 - ABF </td><td></td></tr><tr><td> وسائل الاعلام EpiAirway صيانة خالية من المضادات الحيوية </td><td> ماتيك </td><td> AIR - 100 - MM - ABF </td><td> الموردة مع طقم </td></tr><tr><td> الفوسفات مخزنة 10X محلول ملحي </td><td> EMD </td><td> 6506 </td><td> لتمييع 1X قبل الاستخدام </td></tr><tr><td> الجيلاتين </td><td> JT بيكر </td><td> 2124-01 </td><td> إضافة إلى التركيز النهائي من 0.1 ٪ في برنامج تلفزيوني X 1 و الأوتوكلاف </td></tr><tr><td> Difco قاعدة متوسطة GC (آجار الشوكولاته) </td><td> VWR </td><td> 90002-016 </td><td> الأوتوكلاف 36 ز في 500 مل O 2 DDH وباردة إلى 60 درجة مئوية </td></tr><tr><td> BBL الهيموغلوبين (آجار الشوكولاته) </td><td> VWR </td><td> 90000-662 </td><td> الأوتوكلاف 10 ز في 500 مل O 2 DDH بارد إلى 60 درجة مئوية ، والاختلاط مع قاعدة المتوسطة أعلاه GC </td></tr><tr><td> دينار بحريني تخصيب IsoVitaleX BBL (أجار الشوكولاته) </td><td> VWR </td><td> 90000-414 </td><td> تبريد خليط من قاعدة GC المتوسطة والهيموجلوبين إلى 55 درجة مئوية ، وإضافة 10 مل من IsoVitaleX ممهى ، صب لوحات أجار الشوكولاتة </td></tr><tr><td> ملقط تشريح ، غيض غرامة ، ومنحني </td><td> VWR </td><td> 82027-406 </td><td></td></tr><tr><td> الحقائب التعقيم الختم الذاتي </td><td> VWR </td><td> 89140-802 </td><td></td></tr><tr><td> جنتاميسين سلفات ، 10 ملغ / مل </td><td> Lonza </td><td> 17 - 519Z </td><td> إضافة microliters 10 / مل لMM EpiAirway لقتل جنتاميسين </td></tr></tbody></table>

use of the epiairway model for characterizing long-term host-pathogen interactions

Nontypeable المستدمية النزلية (NTHi) هي تكييف الإنسان الجراثيم سلبية الغرام التي يمكن أن تسبب التهابات متكررة ومزمنة في الأغشية المخاطية التنفسية 1 ، 2. لدراسة الآليات التي من خلالها هذه الكائنات على البقاء على قيد الحياة وداخل أنسجة الجهاز التنفسي ، وهذا نموذج التي يمكن أن يؤديها بنجاح على المدى الطويل المشترك ثقافة البكتيريا والخلايا البشرية هو مطلوب. نستخدم الإنسان التنفسي الأنسجة الطلائية الأولية التي أثيرت في واجهة الهواء السائل ، نموذج EpiAirway (ماتيك ، آشلاند ، MA). هذه هي غير خلدت جيدا ومتباينة ، 3 الابعاد الأنسجة التي تحتوي على منعطفات ضيقة ، وخلايا مهدبة وnonciliated ، والخلايا التي تنتج القدح الميوسين ، والابقاء على القدرة على إنتاج السيتوكينات في الاستجابة للعدوى. ويمكن استخدام هذا النموذج ذات الصلة بيولوجيا في المختبر من مجرى الهواء العلوي الإنسان في عدد من الطرق ، والهدف العام من هذا الأسلوب هو لأداء على المدى الطويل المشترك ثقافة الأنسجة EpiAirway مع NTHi وquantitate الخلية المرتبطة البكتيريا والمنضوية مع مرور الوقت . كذلك ، يمكن تحديد الإنتاج والميوسين ملف خلوى من المصابين المشترك الثقافات. يحسن هذا النهج على الأساليب الموجودة في العديد من البروتوكولات التي تستخدم الحالية مغمورة أحادي الطبقة أو Transwell الثقافات من الخلايا البشرية ، وهي ليست قادرة على دعم الالتهابات البكتيرية على مدى فترات طويلة 3. على سبيل المثال ، إذا كان الكائن الحي يمكن أن تتكرر في وسائل الإعلام الفوقية ، وهذا يمكن أن يؤدي إلى مستويات غير مقبولة من السمسة وفقدان الخلايا المضيفة والقت القبض على التجربة. نموذج EpiAirway يسمح توصيف التفاعلات المضيف الممرض على المدى الطويل. علاوة على ذلك ، منذ المصدر للEpiAirway أمر طبيعي الإنسان الرغامى خلايا الشعب الهوائية بدلا من خط خلده ، كل واحدة على تمثيل ممتاز من الإنسان التنفسي العلوي الفعلية أنسجة المسالك ، سواء من حيث الهيكل وظيفة 4. لهذا الأسلوب ، الأنسجة EpiAirway مفطوم الخروج من المركبات المضادة للميكروبات ومكافحة الفطريات لمدة 2 أيام قبل التسليم ، ويتم تنفيذ كافة الإجراءات في إطار المضادات الحيوية خالية من الشروط. هذا يتطلب اعتبارات خاصة ، حيث يتم استخدام كل من البكتيريا والأنسجة البشرية الأساسية للسلامة الأحيائية في الحكومة نفسها ، وشارك في تربيتها لفترات طويلة.

Watch this Scientific Journal Video about Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions at JoVE.com

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

هذا الأسلوب يسمح توصيف البكتيرية تمديد التعاون مع EpiAirways الثقافة البشرية والجهاز التنفسي الأولية الأنسجة الطلائية نمت في واجهة الهواء السائل ، وهو ذات الصلة بيولوجيا<em&gt; في المختبر</em&gt; النموذج. ويمكن استخدام هذا النهج مع أي ميكروب التي قابلة للالمدى الطويل المشترك الثقافة.

استخدام نموذج لوصف EpiAirway طويل الأجل المضيف الممرض التفاعلات

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

use-epiairway-model-for-characterizing-long-term-host-pathogen

Research

JoVE Journal

Immunology and Infection

11.6K Views.  Mercer University School of Medicine. هذا الأسلوب يسمح توصيف البكتيرية تمديد التعاون مع EpiAirways الثقافة البشرية والجهاز التنفسي الأولية الأنسجة الطلائية نمت في واجهة الهواء السائل ، وهو ذات الصلة بيولوجيا في المختبر النموذج. ويمكن استخدام هذا النهج مع أي ميكروب التي قابلة للالمدى الطويل الم...

Video: Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

استخدام اعتراض البصرية لدراسة التفاعلات المرضي المضيف الحيوي للخلية الحية التصوير

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

A المنهج المقارن لتوصيف المشهد من المضيف الممرض البروتين البروتين التفاعلات

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

على المدى الطويل المزمن<em&gt; الزائفة الزنجارية</em&gt; عدوى مجرى الهواء في الفئران

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

استخدام<em&gt; الشيجلا الفلكسنرية</em&gt; لدراسة التفاعلات الالتهام الذاتي الهيكل الخلوي

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

المدى الطويل حيوي داخلي Multiphoton المجهر التصوير من الخلايا المناعية في السليمة والمريضة الكبد عن طريق الفئران CXCR6.Gfp مراسل

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

تقييم فعالية وسمية الرنا استهداف HIV-1 الإنتاج للاستخدام في الجينات أو العلاج بالعقاقير

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

و إيل-8 عابر نموذج الماوس المعدلة وراثيا ل<em&gt; في فيفو</em&gt; رصد طويل الأمد للردود الالتهابية

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

طويلة الأمد في فيفو تتبع ديناميات الخلايا التحريضية داخل الخوادر المورفولوجية

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

الاستخدام التكميلي للتقنيات المجهرية والقراءة الفلورية في دراسة تفاعلات Cryptococcus-Amoeba

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

تقييم استجابات المضيف الممرض ونجاعة اللقاح في الفئران

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...