PMID- 11350664
OWN - NLM
STAT- MEDLINE
DCOM- 20010712
LR  - 20131121
IS  - 0889-2229 (Print)
IS  - 0889-2229 (Linking)
VI  - 17
IP  - 6
DP  - 2001 Apr 10
TI  - Automated quantitation of cell-mediated HIV type 1 infection of human 
      syncytiotrophoblast cells by fluorescence in situ hybridization and laser 
      scanning cytometry.
PG  - 507-16
AB  - Infection of human placental syncytiotrophoblast cells with HIV requires direct 
      contact with infected leukocytes. In vitro investigations into mechanisms 
      regulating placental HIV transmission and into the development of therapeutic 
      interventions have been hampered by difficulties inherent in quantitating HIV 
      levels in cocultures of infected lymphocytes and adherent multinucleated 
      syncytiotrophoblast cells. Here, we have used fluorescence in situ hybridization 
      (FISH) for the direct detection of HIV-1 RNA within syncytiotrophoblast cells 
      combined with laser scanning cytometry (LSC) to quantitate HIV levels exclusively 
      in the syncytiotrophoblast cells. HIV-1-infected lymphocytic MOLT-4 cells were 
      cocultured with primary human syncytiotrophoblast cells. Lymphocytic cells were 
      identified with an anti-vimentin antibody and Cy5. HIV RNA was localized by in 
      situ hybridization, using a digoxigenin-labeled riboprobe detected by Oregon 
      Green, and nuclei were stained with 7-aminoactinomycin D. The three-color 
      cocultures were analyzed by LSC to remove unwanted cell populations and 
      quantitate HIV expression levels. The total HIV RNA level (green fluorescence 
      integral) in each colony was normalized for cell size by dividing by the total 
      DNA content (red fluorescence integral). The nuclear-normalized fluorescence 
      integral was 2.3 times higher in infected cocultures than in uninfected cultures. 
      When cocultures were incubated with 10 microM AZT, the green/red fluorescence 
      integral value was significantly lower than that of cocultures incubated in the 
      absence of AZT, corresponding to a 78% reduction in fluorescence. Laser scanning 
      cytometry can be used to quantitate cell-mediated HIV infection in 
      syncytiotrophoblast cells and should allow drug assessment studies and studies 
      aimed at understanding the mechanism of virus entry into trophoblast cells to be 
      carried out.
FAU - Douglas, G C
AU  - Douglas GC
AD  - Department of Cell Biology and Human Anatomy, School of Medicine, University of 
      California, Davis, California 95616, USA. gcdouglas@ucdavis.edu
FAU - Thirkill, T L
AU  - Thirkill TL
FAU - LaSalle, J
AU  - LaSalle J
LA  - eng
GR  - HD11658/HD/NICHD NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - AIDS Res Hum Retroviruses
JT  - AIDS research and human retroviruses
JID - 8709376
RN  - 0 (Anti-HIV Agents)
RN  - 0 (RNA, Viral)
RN  - 0 (Reverse Transcriptase Inhibitors)
RN  - 4B9XT59T7S (Zidovudine)
SB  - IM
MH  - Anti-HIV Agents/pharmacology
MH  - Automation
MH  - Cells, Cultured
MH  - Coculture Techniques
MH  - Female
MH  - Flow Cytometry/methods
MH  - Fluorescent Antibody Technique, Indirect
MH  - HIV-1/drug effects/genetics/*isolation & purification
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/methods
MH  - Lasers
MH  - Lymphocytes/cytology
MH  - RNA, Viral/*analysis
MH  - Reverse Transcriptase Inhibitors/pharmacology
MH  - Trophoblasts/cytology/*virology
MH  - Zidovudine/pharmacology
EDAT- 2001/05/15 10:00
MHDA- 2001/07/13 10:01
CRDT- 2001/05/15 10:00
PHST- 2001/05/15 10:00 [pubmed]
PHST- 2001/07/13 10:01 [medline]
PHST- 2001/05/15 10:00 [entrez]
AID - 10.1089/08892220151126562 [doi]
PST - ppublish
SO  - AIDS Res Hum Retroviruses. 2001 Apr 10;17(6):507-16. doi: 
      10.1089/08892220151126562.