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Simultaneous Ultrasensitive Subpopulation Staining/Hybridization In Situ (SUSHI) in HIV-1 Disease Monitoring

  • Bruce K. PattersonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 659)

Abstract

The field of virology is undergoing a revolution as diagnostic tests and new therapies are allowing clinicians to treat, monitor, and predict outcomes of viral diseases. The majority of these techniques, however, destroy the factory of viral production and the information inherent in the reservoir – the cell. In this chapter, we describe a technique that combines cell surface immunophenotyping (to unequivocally identify cell types) and ultrasensitive fluorescence in situ hybridization (U-FISH) for HIV-1 to detect productively infected cells. Identification of virus and host (cells) allows earlier detection of changes in viral production and viral suppression but most importantly allows clinicians to monitor response to anti-viral therapy on a cell-by-cell and tissue-by-tissue basis taking into account the fact that the human body consists of very different, distinct compartments with unique selection pressures exerted on the viral life cycle.

Key words

Virus HIV Tropism T-cells Monocytes Flow cytometry Laser confocal image analysis Oligonucleotides 

Notes

Acknowledgements

The author would like to acknowledge and thank Keith Shults for his collaborative efforts and unyielding support in the development of these techniques.

References

  1. 1.
    Patterson, B.K., Czerniewski, M.A., Pottage, J., Agnoli, M., Kessler, H., Landay, A. (1999) Monitoring HIV therapy in immune cell subsets using ultrasensitive fluorescence in situ hybridization. Lancet 353, 211–212.PubMedCrossRefGoogle Scholar
  2. 2.
    Furtado, M.R., Callaway, D.S., Phair, J.P., Kunstman, K.J., Stanton, J.L., Macken, C.A., Perelson, A.S., Wolinsky, S.M. (1999) Persistence of HIV-1 transcription in peripheral-blood mononuclear cells in patients receiving potent antiretroviral therapy. N Engl J Med 340, 1614–1622PubMedCrossRefGoogle Scholar
  3. 3.
    Dronda, F., Moreno, S., Moreno A., Casado, JL., Perez-Elias, M.J., and Antela, A. (2002) Long-term outcomes among antiretroviral-naïve human immunodeficiency virus-infected patients with small increases in CD4+ counts after successful virologic suppression. Clin Infect Dis 35, 1005–1009.PubMedCrossRefGoogle Scholar
  4. 4.
    Kedzierska, K. and Crowe, S. (2002) The role of monocytes and macrophages in the pathogenesis of HIV-1 infection. Curr Med Chem 9, 1893–1903.PubMedCrossRefGoogle Scholar
  5. 5.
    Schutten, M., van Baalen, C., Guillon, C., Huisman, R., Boers P., Sintnicolaas, K., Gruters, R., and Osterhaus, A. (2001) Macrophage tropism of human immunodeficiency virus type 1 facilitates in vivo escape from cytotoxic T-lymphocyte pressure. J Virol 75, 2706–2709.PubMedCrossRefGoogle Scholar
  6. 6.
    Li, S., Juarez, J., Alali, M., Dwyer, D., Collman, R., Cunningham, A., and Naif, H. (1999) Persistent CCR5 utilization and enhanced macrophage tropism by primary blood human immunodeficiency virus type 1 isolates from advanced stages of disease and comparison to tissue-derived isolates. J Virol 73, 9741–9755.PubMedGoogle Scholar
  7. 7.
    Kulkosky, J., Nunnari, G., Otero, M., Calarota, S., Dornadula, G., Zhang, H., Malin, A., Sullivan, J., Xu, Y., DeSimone, J., Babinchak, T., Stern, J., Cavert, W., Haase, A., Pomerantz, R. (2002) Intensification and stimulation therapy for human immunodeficiency virus type 1 reservoirs in infected persons receiving virally suppressive highly active antiretroviral therapy. J Infect Dis 186, 1403–1411PubMedCrossRefGoogle Scholar
  8. 8.
    Balzarini, J., Van Herrewege, Y, and Vanham G. (2002) Metabolic activation of nucleoside and nucleotide reverse transcriptase inhibitors in dendritic and Langerhans cells. AIDS 16, 2159–2163.PubMedCrossRefGoogle Scholar
  9. 9.
    Folks T, Benn S, Rabson A, Theodore T, Hoggan MD, Martin M, Lightfoote M, Sell K. (1985). Characterization of a continuous T-cell line susceptible to the cytopathic effects of the acquired immunodeficiency syndrome (AIDS)-associated retrovirus. Proc Natl Acad Sci U S A 82, 4539–4543.PubMedCrossRefGoogle Scholar
  10. 10.
    Folks TM, Clouse KA, Justement J, Rabson A, Duh E, Kehrl JH, Fauci AS. (1989) Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc Natl Acad Sci U S A 86, 2365–2368.PubMedCrossRefGoogle Scholar
  11. 11.
    Purvis, N. and Stelzer, G. (1998) Multi-platform, multi-site instrumentation and reagent standardization. Cytometry 33, 156–165.PubMedCrossRefGoogle Scholar
  12. 12.
    Patterson, B.K., McCallister, S., Schutz, M., Siegel, J.N., Shults, K., Flener, Z., Landay, A. (2001) Persistence of intracellular HIV-1 mRNA correlates with HIV-1-specific immune responses in HIV-1-infected subjects on stable HAART therapy. AIDS 15, 1635–1641.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of PathologyStanford University Medical SchoolStanfordUSA

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