Waiting
Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove
Click here for the English version

Immunology and Infection

Uma doi: 10.3791/50766 Published: December 10, 2013

Abstract

O vírus sincicial respiratório humano (HRSV) infecções apresentar um largo espectro de gravidade da doença, variando desde as infecções suaves a bronquiolite com risco de vida. Uma parte importante da patogênese da doença grave é uma resposta imune melhorada levando a imunopatologia.

Aqui, nós descrevemos um protocolo usado para investigar a resposta imune de células do sistema imunológico humano a uma infecção HRSV. Em primeiro lugar, descrevemos métodos utilizados para a cultura, purificação e quantificação de HRSV. Subsequentemente, foi descrito um modelo humano in vitro, em que as células mononucleares do sangue periférico (PBMC) são estimuladas com HRSV vivo. Este sistema modelo pode ser usado para estudar vários parâmetros que podem contribuir para a gravidade da doença, incluindo a resposta imune inata e adaptativa. Estas respostas podem ser medido ao nível da transcrição e da tradução. Além disso, a infecção virai de células pode ser facilmente medida utilizando citometria de fluxo. Tomadojuntos, a estimulação de PBMC com HRSV vivo fornece um sistema modelo rápido e reprodutível para examinar os mecanismos envolvidos na doença induzida por HRSV.

Materials

Name Company Catalog Number Comments
Reagent
PBS Lonza BE17-516F PBS without Ca2+ Mg2+ or phenol red
HBSS Invitrogen 14025-100 HBSS, Calcium, Magnesium, no Phenol Red
DMEM Invitrogen 31966-047 DMEM, High Glucose, GlutaMAX, Pyruvate
RPMI Invitrogen 72400054 RPMI 1640 Medium, GlutaMAX, HEPES
Reduced Serum Medium (Opti-MEM) Invitrogen 51985-026 Opti-MEM I Reduced Serum Medium, GlutaMAX
FCS Greiner Bio-one FBS (Fetal Bovine Serum)
BSA Sigma Aldrich A7030 Albumin from bovine serum
P/S Invitrogen 15140-122 Penicillin-Streptomycin, liquid
Trypsin Invitrogen 25300-054 0.05% Trypsin-EDTA (1x), Phenol Red
HeLa cells ATCC CCL-2
A549 cells ATCC CCL-185
Sucrose Sigma Aldrich S7903 Sucrose BioXtra, ≥99.5%
IgG2 anti-mouse-FITC BD Pharmingen 555057 Ms IgG2b K FITC isotype control
Anti-NP-RSV-FITC Abcam ab25849 Anti-Respiratory Syncytial Virus antibody [671] (FITC)
Density gradient medium (Lymphoprep) Axis-Shield 1114545
EDTA tubes BD Biosciences 367525
Acetone Merck Millipore 1000141000 Acetone for analysis
Material
Centrifuge Tubes Beckman Coulter 326823 Thinwall, Polyallomer, 38.5 ml, 25 mm x 89 mm
SureSpin 630 Rotor with 36 ml buckets Sorvall 79368 Swinging bucket titanium rotor
Sorvall WX80 Ultracentrifuge Sorvall 46900
CB 150l CO2 Incubator Binder

DOWNLOAD MATERIALS LIST

References

  1. Nair, H., Nokes, D. J., et al. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet. 375, 1545-1555 (2010).
  2. Falsey, A. R., Hennessey, P. A., Formica, M. A., Cox, C., Walsh, E. E. Respiratory syncytial virus infection in elderly and high-risk adults. N. Eng. J. Med. 352-1749 (2005).
  3. Openshaw, P. J. Antiviral Immune Responses and Lung Inflammation after Respiratory Syncytial Virus Infection. Proc. Am. Thorac. Soc. 2, (2), 121-125 (2005).
  4. Openshaw, P. J., Tregoning, J. S. Immune responses and disease enhancement during respiratory syncytial virus infection. Clin. Microbio. Rev. 18, (3), 541-555 (2005).
  5. Villenave, R., Thavagnanam, S., et al. In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo. Proc. Natl. Acad. Sci. U.S.A. 109, (13), 5040-5045 (2012).
  6. Tulic, M. K., Hurrelbrink, R. J., Prêle, C. M., Laing, I. A., Upham, J. W., Le Souef, P., Sly, P. D., Holt, P. G. TLR4 polymorphisms mediate impaired responses to respiratory syncytial virus and lipopolysaccharide. J. Immunol. 179, (1), 132-140 (2007).
  7. Vissers, M., Remijn, T., et al. Respiratory syncytial virus infection augments NOD2 signaling in an IFN-β-dependent manner in human primary cells. Eur. J. Immunol. 42, (10), 2727-2735 (2012).
  8. Cherukuri, A., Patton, K., Gasser, R. A. Jr, Zuo, F., Woo, J., Esser, M. T., Tang, R. S. Adults 65 years old and older have reduced number of functional memory T cells to respiratory syncytial virus fusion protein. Clin. Vacc. Immunol. 20, (2), 239-247 (2013).
  9. Shafique, M., Wilschut, J., de Haan, A. Induction of mucosal and systemic immunity against respiratory syncytial virus by inactivated virus supplemented with TLR9 and NOD2 ligands. Vaccine. 30, 597-606 (2012).
  10. Kwilas, S., Liesman, R. M., Zhang, L., Walsh, E., Pickles, R. J., Peeples, M. E. Respiratory syncytial virus grown in Vero cells contains a truncated attachment protein that alters its infectivity and dependence on glycosaminoglycans. J. Virol. 83, (20), 10710-10718 (2009).
  11. Treuhaft, M. W., Beem, M. O. Defective interfering particles of respiratory syncytial virus. Infect. Immun. 37, (2), 439-444 (1982).
  12. Korns Johnson, D., Homann, D. Accelerated and improved quantification of lymphocytic choriomeningitis virus (LCMV) titers by flow cytometry. PLoS One. 7, (5), e37337 (2012).
  13. Wurfel, M. M., Park, W. Y., et al. Identification of high and low responders to lipopolysaccharide in normal subjects: an unbiased approach to identify modulators of innate immunity. J. Immunol. 175, (4), 2570-2578 (2005).
  14. Kimpen, J. L. Respiratory syncytial virus and asthma. The role of monocytes. Am. J. Respir. Crit. Care Med. 163, 7-9 (2001).
  15. Wang, S. Z., Forsyth, K. D. The interaction of neutrophils with respiratory epithelial cells in viral infection. Respirology. 5, (1), 1-10 (2000).
  16. San-Juan-Vergara, H., Sampayo-Escobar, V., Reyes, N., Cha, B., Pacheco-Lugo, L., Wong, T., Peeples, M. E., Collins, P. L., Castano, M. E., Mohapatra, S. S. Cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells. J. Virol. 86, (3), 1832-1843 (2012).
  17. Davidson, D., Zaytseva, A., Miskolci, V., Castro-Alcaraz, S., Vancurova, I., Patel, H. Gene expression profile of endotoxin-stimulated leukocytes of term new born: control of cytokine gene expression by interleukin-10. Plos One. 8, (1), e53641 (2013).
Uma<em&gt; In vitro</em&gt; Modelo para estudar respostas imunes de sangue periférico humano células mononucleares para Vírus Respiratório Sincicial Humano Infecção
Play Video
PDF DOI DOWNLOAD MATERIALS LIST

Cite this Article

Vissers, M., Habets, M. N., Ahout, I. M. L., Jans, J., de Jonge, M. I., Diavatopoulos, D. A., Ferwerda, G. An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection. J. Vis. Exp. (82), e50766, doi:10.3791/50766 (2013).More

Vissers, M., Habets, M. N., Ahout, I. M. L., Jans, J., de Jonge, M. I., Diavatopoulos, D. A., Ferwerda, G. An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection. J. Vis. Exp. (82), e50766, doi:10.3791/50766 (2013).

Less
Copy Citation Download Citation Reprints and Permissions
View Video

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
Simple Hit Counter