A Dual‐Chamber Model of the Female Genital Tract to Evaluate Epithelial Toxicity of Candidate Anti‐HIV Microbicides

Kevin K. Ariën1, Guido Vanham1, Youssef Gali1

1 Virology Unit, Department of Microbiology, Institute of Tropical Medicine, Antwerp, Belgium
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 26.13
DOI:  10.1002/0471143030.cb2613s52
Online Posting Date:  September, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Heterosexual transmission of human immunodeficiency virus (HIV) is the predominant mode of infection worldwide. The early steps of transepithelial infection are crucial, but how exactly infection is established in the female genital tract (FGT) is still under debate. Using epithelial cells originating from the FGT and primary cells as subepithelial HIV target cells, an in vitro dual‐chamber model of the FGT was developed. Here we describe how this in vitro model can be used to assess the cellular toxicity and anti‐HIV activity of antiretrovirals and formulations thereof that are intended to be used as microbicides. Curr. Protoc. Cell Biol. 52:26.13.1‐26.13.17. © 2011 by John Wiley & Sons, Inc.

Keywords: HIV; sexual transmission; microbicides; toxicity; female genital tract

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Dual‐Chamber System to Evaluate Candidate Microbicides
  • Alternate Protocol 1: Dual‐Chamber Model for High‐Throughput Toxicity Screening of Anti‐HIV Substances
  • Basic Protocol 2: Epithelial Toxicity Assays for Dual‐Chamber Models
  • Alternate Protocol 2: Assessing Anti‐Retroviral Activity Against HIV Virions and Cell‐Associated Virus
  • Support Protocol 1: Culture of ME‐180, CaSki, SiHa, and HEC‐1A Cells
  • Support Protocol 2: Culture of VK2/E6E7, Ect1/E6E7, and End1/E6E7 Cells
  • Support Protocol 3: Isolation of Peripheral Blood Mononuclear Cells
  • Support Protocol 4: Culture of TZM‐bl Target Cells
  • Support Protocol 5: Isolation and Differentiation of Monocytes into Dendritic Cells and Macrophages, and Isolation of CD4+ T Lymphocytes
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Dual‐Chamber System to Evaluate Candidate Microbicides

  Materials
  • 200 ng/ml laminin (Sigma‐Aldrich) in 1× phosphate‐buffered saline (PBS; appendix 2A)
  • Genital epithelial cells and appropriate culture medium containing 10% FBS (see Support Protocols protocol 51 and protocol 62)
  • Substance of interest
  • 24‐well Transwell system (3.0‐µm pore, 0.3‐cm2 growth area; Corning Costar)

Alternate Protocol 1: Dual‐Chamber Model for High‐Throughput Toxicity Screening of Anti‐HIV Substances

  • HTS Transwell‐96 system (3.0‐µm pore, PC membrane, 0.14 cm2 growth area; Corning Costar)

Basic Protocol 2: Epithelial Toxicity Assays for Dual‐Chamber Models

  Materials
  • Treated cells in 24‐ or 96‐well Transwell inserts (see protocol 1 or protocol 2)
  • Culture medium (see Support Protocol protocol 51 and protocol 62)
  • WST‐1 Cell Proliferation Assay (Roche Applied Science)
  • 0.1‐µm yellow‐green fluorescent (505/515) FluoSpheres (2% solids; Molecular Probes)
  • HuMab‐HIVp24 MAB37‐bio (Biomaric, Gent, Belgium) or equivalent anti‐p24 antibody or commercially available p24 antigen detection kit
  • PHA/IL‐2‐stimulated PBMCs (see protocol 7)
  • Millicell‐ERS Volt‐Ohm Meter (Millipore)
  • ELISA plate reader (Bio‐Rad model 550) or equivalent
  • TriStar LB 941 fluorometer (Berthold Technologies GmbH, Germany) or equivalent

Alternate Protocol 2: Assessing Anti‐Retroviral Activity Against HIV Virions and Cell‐Associated Virus

  • Unstimulated PBMCs (see protocol 7, step 7)

Support Protocol 1: Culture of ME‐180, CaSki, SiHa, and HEC‐1A Cells

  Materials
  • ME‐180, HEC‐1A, SiHa, or CaSki cells (ATCC HTB‐33, HTB‐112, HTB‐35, and CRL‐1550, respectively)
  • Culture medium:
    • ME‐180 cells: RPMI‐1640 medium (Invitrogen) with 10 mM HEPES, 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany), 2 mM l‐glutamine, 100 U/ml penicillin (Bio‐Whittaker), and 100 µg/ml streptomycin (Bio‐Whittaker)
    • HEC‐1A cells: McCoy's 5A modified medium (Invitrogen) with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin
    • SiHa and CaSki cells: DMEM/F12 medium (Invitrogen) with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin
  • Trypsin/EDTA solution (Invitrogen)
  • 50‐ml centrifuge tubes
  • T‐75 cell culture flasks (BD Falcon)

Support Protocol 2: Culture of VK2/E6E7, Ect1/E6E7, and End1/E6E7 Cells

  Materials
  • Ect1/E6E7, End1/E6E7, or VK2/E6E7 cells (ATCC CRL‐2614, CRL‐2615, and CRL‐2616, respectively)
  • K‐SFM culture medium: serum‐free keratinocyte DMEM/F12 medium (Invitrogen) with 0.05 mg/ml bovine pituitary extract (BPE; Invitrogen), 0.1 ng/ml epidermal growth factor (EGF; Invitrogen), 100 U/ml penicillin (Bio‐Whittaker), and 100 µg/ml streptomycin (Bio‐Whittaker)
  • Fetal bovine serum (FBS; Biochrom, Berlin, Germany)
  • Trypsin/EDTA solution (Invitrogen)
  • T‐75 cell culture flasks (BD Falcon)

Support Protocol 3: Isolation of Peripheral Blood Mononuclear Cells

  Materials
  • Buffy coat blood
  • Sterile 1× PBS without Ca2+ (e.g., Lonza, Verviers, Belgium)
  • Lymphoprep (Axis‐Shield, Oslo, Norway)
  • RPMI‐1640 medium (Invitrogen)
  • Heat‐inactivated fetal bovine serum (FBS; Biochrom, Berlin, Germany)
  • Gentamicin (Lonza, Verviers, Belgium)
  • Phytohemagglutinin (PHA; Remel, Kent, UK)
  • Interleukin‐2 (IL‐2; Gentaur, Brussels, Belgium)
  • Polybrene (Sigma‐Aldrich)
  • Hydrocortisone (Calbiochem)
  • 50‐ml centrifuge tubes (BD Falcon)

Support Protocol 4: Culture of TZM‐bl Target Cells

  Materials
  • TZM‐bl cells (NIH AIDS Research and Reference Reagent Program, Germantown, USA; cat. no. 8129)
  • Dulbecco's Minimum Essential Medium (DMEM; Invitrogen)
  • Fetal bovine serum (Biochrom, Berlin, Germany), heat‐inactivated
  • Gentamicin (Lonza, Verviers, Belgium)
  • Trypsin/EDTA solution (0.25% trypsin/1 mM EDTA; Invitrogen)
  • T‐75 cell culture flasks (BD Falcon)

Support Protocol 5: Isolation and Differentiation of Monocytes into Dendritic Cells and Macrophages, and Isolation of CD4+ T Lymphocytes

  Materials
  • PBMCs (see protocol 7)
  • CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany)
  • Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF; Gentaur, Brussels, Belgium)
  • Interleukin‐4 (IL‐4; Invitrogen)
  • CD4 Dynabeads (Invitrogen)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Adler, K., Erickson, T., and Bobrow, M. 1997. High sensitivity detection of HPV‐16 in SiHa and CaSki cells utilizing FISH enhanced by TSA. Histochem. Cell Biol. 108:321‐324.
   Ariën, K.K., Jespers, V., and Vanham, G. 2011. HIV sexual transmission and microbicides. Rev. Med. Virol. 21:110‐133.
   Baker, C.C., Phelps, W.C., Lindgren, V., Braun, M.J., Gonda, M.A., and Howley, P.M. 1987. Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J. Virol. 61:962‐971.
   Beer, B.E., Doncel, G.F., Krebs, F.C., Shattock, R.J., Fletcher, P.S., Buckheit, R.W. Jr., Watson, K., Dezzutti, C.S., Cummins, J.E., Bromley, E., Richardson‐Harman, N., Pallansch, L.A., Lackman‐Smith, C., Osterling, C., Mankowski, M., Miller, S.R., Catalone, B.J., Welsh, P.A., Howett, M.K., Wigdahl, B., Turpin, J.A., and Reichelderfer, P. 2006. In vitro preclinical testing of nonoxynol‐9 as potential anti‐human immunodeficiency virus microbicide: A retrospective analysis of results from five laboratories. Antimicrob. Agents Chemother. 50:713‐723.
   Blocker, M.E. and Cohen, M.S., 2000. Biologic approaches to the prevention of sexual transmission of human immunodeficiency virus. Infect. Dis. Clin. N. Am. 14:983‐999.
   Coombs, R.W., Reichelderfer, P.S., and Landay, A.L. 2003. Recent observations on HIV type‐1 infection in the genital tract of men and women. AIDS 17:455‐480.
   Cutler, B. and Justman, J. 2008. Vaginal microbicides and the prevention of HIV transmission. Lancet Infect. Dis. 8:685‐697.
   Dezzutti, C.S., Guenthner, P.C., Cummins, J.E. Jr., Cabrera, T., Marshall, J.H., Dillberger, A., and Lal, R.B. 2001. Cervical and prostate primary epithelial cells are not productively infected but sequester human immunodeficiency virus type 1. J. Infect. Dis. 183:1204‐1213.
   Elias, C.J. and Coggins, C. 1996. Female‐controlled methods to prevent sexual transmission of HIV. AIDS 10:S43‐S51.
   Fichorova, R.N., Rheinwald, J.G., and Anderson, D.J. 1997. Generation of papillomavirus immortalized cell lines from normal human ectocervical, endocervical, and vaginal epithelium that maintain expression of tissue‐specific differentiation proteins. Biol. Reprod. 57:847‐855.
   Fichorova, R.N., Tucker, L.D., and Anderson, D.J. 2001. The molecular basis of nonoxynol‐9‐induced vaginal inflammation and its possible relevance to human immunodeficiency virus type 1 transmission. J. Infect. Dis. 184:418‐428.
   Fichorova, R.N., Bajpai, M., Chandra, N., Hsiu, J.G., Spangler, M., Ratnam, V., and Doncel, G.F. 2004. Interleukin (IL)‐1, IL‐6, and IL‐8 predict mucosal toxicity of vaginal microbicidal contraceptives. Biol. Reprod. 71:761‐769.
   Fichorova, R.N., Zhou, F., Ratnam, V., Atanassova, V., Jiang, S., Strick, N., and Neurath, A.R. 2005. Anti‐human immunodeficiency virus type 1 microbicide cellulose acetate 1,2‐benzenedicarboxylate in a human in vitro model of vaginal inflammation. Antimicrob. Agents Chemother. 49:323‐335.
   Furuta, Y., Eriksson, K., Svennerholm, B., Fredman, P., Horal, P., Jeansson, S., Vahlne, A., Holmgren, J., and Czerkinsky, C. 1994. Infection of vaginal and colonic epithelial cells by the human immunodeficiency virus type 1 is neutralized by antibodies raised against conserved epitopes in the envelope glycoprotein gp120. Proc. Natl. Acad. Sci. U.S.A. 91:12559‐12563.
   Gali, Y., Ariën, K.K., Praet, M., Van den Bergh, R., Temmerman, M., Delezay, O., and Vanham, G. 2010a. Development of an in vitro dual‐chamber model of the female genital tract as a screening tool for epithelial toxicity. J. Virol. Methods 165:186‐97.
   Gali, Y., Delezay, O., Brouwers, J., Addad, N., Augustijns, P., Bourlet, T., Hamzeh‐Cognasse, H., Ariën, K.K., Pozzetto, B., and Vanham, G. 2010b. In vitro evaluation of viability, integrity, and inflammation in genital epithelia upon exposure to pharmaceutical excipients and candidate microbicides. Antimicrob. Agents Chemother. 54:5105‐5114.
   Herold, B.C., Mesquita, P.M., Madan, R.P., and Keller, M.J. 2011. Female genital tract secretions and semen impact the development of microbicides for the prevention of HIV and other sexually transmitted infections. Am. J. Reprod. Immunol. 65:325‐333.
   Hillier, S.L., Moench, T., Shattock, R., Black, R., Reichelderfer, P., and Veronese, F. 2005. In vitro and in vivo: The story of nonoxynol 9. J. Acquir. Immune Defic. Syndr. 39:1‐8.
   Kuramoto, H., Tamura, S., and Notake, Y. 1972. Establishment of a cell line of human endometrial adenocarcinoma in vitro. Am. J. Obstet. Gynecol. 114:1012‐1019.
   Li, Q., Estes, J.D., Schlievert, P.M., Duan, L., Brosnahan, A.J., Southern, P.J., Reilly, C.S., Peterson, M.L., Schultz‐Darken, N., Brunner, K.G., Nephew, K.R., Pambuccian, S., Lifson, J.D., Carlis, J.V., and Haase, A.T. 2009. Glycerol monolaurate prevents mucosal SIV transmission. Nature 458:1034‐1038.
   Losikoff, P., Fichorova, R., Snyder, B., Rodriguez, I., Cu‐Uvin, S., Harwell, J., and Mayer, K.H. 2007. Genital tract interleukin‐8 but not interleukin‐1β or interleukin‐6 concentration is associated with bacterial vaginosis and its clearance in HIV‐infected and HIV‐uninfected women. Infect. Dis. Obstet. Gynecol. 2007:92307.
   Narimatsu, R., Wolday, D., and Patterson, B.K. 2005. IL‐8 increases transmission of HIV type 1 in cervical explant tissue. AIDS Res. Hum. Retrovir. 21:228‐233.
   Nicolosi, A., Corrêa Leite, M.L., Musicco, M., Arici, C., Gavazzeni, G., and Lazzarin, A. 1994. The efficiency of male‐to‐female and female‐to‐male sexual transmission of the human immunodeficiency virus: A study of 730 stable couples. Italian Study Group on HIV Heterosexual Transmission. Epidemiology 5:570‐575.
   Poonia, B., Wang, X., and Veazey, R.S. 2006. Distribution of simian immunodeficiency virus target cells in vaginal tissues of normal rhesus macaques: Implications for virus transmission. J. Reprod. Immunol. 72:74‐84.
   Pope, M. 2003. Dendritic cells as a conduit to improve HIV vaccines. Curr. Mol. Med. 3:229‐242.
   Pudney, J., Quayle, A.J., and Anderson, D.J., 2005. Immunological microenvironments in the human vagina and cervix: Mediators of cellular immunity are concentrated in the cervical transformation zone. Biol. Reprod. 73:1253‐1263.
   Reuter, S., Delius, H., Kahn, T., Hofmann, B., zur Hausen, H., and Schwarz, E. 1991. Characterization of a novel human papillomavirus DNA in the cervical carcinoma cell line ME180. J. Virol. 65:5564‐5568.
   Shen, R., Richter, H.E., Clements, R.H., Novak, L., Huff, K., Bimczok, D., Sankaran‐Walters, S., Dandekar, S., Clapham, P.R., Smythies, L.E., and Smith, P.D. 2009. Macrophages in vaginal but not intestinal mucosa are monocyte‐like and permissive to human immunodeficiency virus type 1 infection. J. Virol. 83:3258‐3267.
   Sugaya, M., Lore, K., Koup, R.A., Douek, D.C., and Blauvelt, A. 2004. HIV‐infected Langerhans cells preferentially transmit virus to proliferating autologous CD4+ memory T cells located within Langerhans cell‐T cell clusters. J. Immunol. 172:2219‐2224.
   Tan, X. and Phillips, D.M. 1996. Cell‐mediated infection of cervix derived epithelial cells with primary isolates of human immunodeficiency virus. Arch. Virol. 141:1177‐1189.
   Shattock, R.J. and Moore, J.P. 2003. Inhibiting sexual transmission of HIV‐1 infection. Nat. Rev. Microbiol. 1:25‐34.
   Van Damme, L., Ramjee, G., Alary, M., Vuylsteke, B., Chandeying, V., Rees, H., Sirivongrangson, P., Tshibaka, L.M., Ettiègne‐Traoré,, V., Uaheowitchai, C., Karim, S.S.A., Mâsse, B., Perriëns, J., and Laga, M. 2002. Effectiveness of COL‐1492, a nonoxynol‐9 vaginal gel, on HIV‐1 transmission in female sex workers: A randomised controlled trial. Lancet 360:971‐977.
   Wei, X., Decker, J.M., Wang, S., Hui, H., Kappes, J.C., Wu, X., Salazar‐Gonzalez, J.F., Salazar, M.G., Kilby, J.M., Saag, M.S., Komarova, N.L., Nowak, M.A., Hahn, B.H., Kwong, P.D., and Shaw, G.M. 2003. Antibody neutralization and escape by HIV‐1. Nature 422:307‐312.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library