लंबे समय तक मेजबान रोगज़नक़ सहभागिता निस्र्पक के लिए EpiAirway मॉडल का उपयोग
Summary
इस विधि EpiAirways, प्राथमिक मानव हवा तरल अंतरफलक पर हो श्वसन उपकला ऊतक, एक biologically प्रासंगिक के साथ विस्तारित सह संस्कृति बैक्टीरिया के लक्षण वर्णन की अनुमति देता है<em> इन विट्रो में</em> मॉडल. दृष्टिकोण किसी भी सूक्ष्म जीव है कि सह संस्कृति लंबी अवधि के लिए उत्तरदायी है के साथ इस्तेमाल किया जा सकता है.
Abstract
Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection.
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.
Protocol
Discussion
इस विधि हवा तरल इंटरफ़ेस में प्राथमिक मानव श्वसन ऊतकों की एक biologically प्रासंगिक पृष्ठभूमि में लंबे समय तक मेजबान रोगज़नक़ बातचीत की जांच की अनुमति देता है. यहाँ हम संक्रमण जीव के रूप में NTHi का इस्तेमाल किय?…
Declarações
The authors have nothing to disclose.
Acknowledgements
हम उपयोगी विचार – विमर्श के लिए पैट्रिक हेडन (MatTek) का शुक्रिया अदा करना होगा, और रॉबर्ट स्मिथ और उनके EM कौशल के लिए जॉर्जिया स्वास्थ्य विज्ञान विश्वविद्यालय के Libby पेरी. इस अध्ययन NIDCD अनुदान DC010187 द्वारा पिताजी को वित्त पोषित किया गया था
Materials
| Name of reagent | Company | Catalogue# | Comments |
|---|---|---|---|
| Saponin | Calbiochem | 558255-25GM | 1% in D-PBS without calcium or magnesium, filter sterilize |
| 1 X Dulbecco’s phosphate-buffered saline with calcium and magnesium | Lonza | 17-513Q | |
| 1 X Dulbecco’s phosphate-buffered saline without calcium or magnesium | Lonza | 17-515Q | |
| EpiAirway antibiotic-free tissues | MatTek | AIR-100-ABF | |
| EpiAirway antibiotic-free maintenance media | MatTek | AIR-100-MM-ABF | Supplied with kit |
| 10X phosphate-buffered saline solution | EMD | 6506 | Dilute to 1X before use |
| Gelatin | JT Baker | 2124-01 | Add to a final concentration of 0.1% in 1 X PBS and autoclave |
| Difco GC Medium Base (chocolate agar) | VWR | 90002-016 | Autoclave 36 g in 500 ml ddH2O and cool to 60°C |
| BBL Hemoglobin (chocolate agar) | VWR | 90000-662 | Autoclave 10 g in 500 ml ddH2O, cool to 60°C and mix with the GC medium base above |
| BD BBL IsoVitaleX enrichment (chocolate agar) | VWR | 90000-414 | Cool the mixture of GC medium base and hemoglobin to 55°C and add 10 ml of rehydrated IsoVitaleX, pour chocolate agar plates |
| Dissecting forceps, fine tip, curved | VWR | 82027-406 | |
| Self-sealing sterilization pouches | VWR | 89140-802 | |
| Gentamicin sulfate, 10 mg/ml | Lonza | 17-519Z | Add 10 microliters/ml to EpiAirway MM for the gentamicin kill |
Referências
- Murphy, T. F., Apicella, M. A. Nontypeable Haemophilus influenzae: a review of clinical aspects, surface antigens, and the human immune response to infection. Rev. Infect. Dis. 9, 1-15 (1987).
- Murphy, T. F., Faden, H., Bakaletz, L. O., Kyd, J. M., Forsgren, A., Campos, J., Virji, M., Pelton, S. I. Nontypeable Haemophilus influenzae as a pathogen in children. Ped. Infect. Dis. J. 28, 43-48 (2009).
- Hotomi, M., Arai, J., Billal, D. S., Takei, S., KIkeda, Y., Ogami, M., Kono, M., Beder, L. B., Toya, K., Kimura, M., Yamanaka, N. Nontypeable Haemophilus influenzae isolated from intractable acute otitis media internalized into cultured human epithelial cells. Auris Nasus Larynx. 37, 137-144 (2010).
- Chemuturi, N. V., Hayden, P., Kalausner, M., Donovan, M. D. Comparison of human tracheal/bronchial epithelial cell culture and bovine nasal respiratory explants for nasal drug transport studies. J. Pharm. Sci. 94, 1976-1985 (2005).
- Sharma, M., Schoop, R., Hudson, J. B. The efficacy of Echinacea in a 3-D tissue model of human airway epithelium. Phytother. Res. 24, 900-904 (2010).
- Sexton, K., Balharry, D., BeruBe, K. A. Genomic biomarkers of pulmonary exposure to tobacco smoke components. Pharmacogenet Genomics. 10, 853-860 (2008).
- Babu, R. J., Dayal, P., Singh, M. Effect of cyclodextrins on the complexation and nasal permeation of melatonin. Drug Deliv. 6, 381-388 (2008).
- Balharry, D., Sexton, K., BeruBe, K. A. An in vitro approach to assess the toxicity of inhaled tobacco smoke components: nicotine, cadmium, formaldehyde and urethane. Toxicology. 244, 66-76 (2008).
- Fahy, J. V., Dickey, M. D. Airway mucus function and dysfunction. N. Engl. J. Med. 363, 2233-2247 (2010).
- Huang, Y., Mikami, F., Jono, H., Zhang, W., Weng, X., Koga, T., Xu, H., Yan, C., Kai, H., Li, J. -. D. Opposing roles of PAK2 and PAK4 in synergistic induction of MUC5AC mucin by bacterium NTHi. 359, 691-696 (2007).
