PMID- 12957395
OWN - NLM
STAT- MEDLINE
DCOM- 20031030
LR  - 20190723
IS  - 0022-1759 (Print)
IS  - 0022-1759 (Linking)
VI  - 278
IP  - 1-2
DP  - 2003 Jul
TI  - Centrifugation facilitates transduction of green fluorescent protein in human 
      monocytes and macrophages by adenovirus at low multiplicity of infection.
PG  - 45-56
AB  - Due to their phagocytic and poorly proliferative nature, it has been difficult to 
      transfect human monocytes and macrophages. Adenoviral vectors have recently 
      allowed transduction of a high percentage of human macrophages, but only after 
      CSF upregulation of the integrins, alphavbeta3 or alphavbeta5, during culture for 
      48 h, a time allowing significant monocyte to macrophage differentiation. In our 
      hands, after 24-h incubation with M-CSF (20 ng/ml) and a further 24-h incubation 
      with an adenoviral vector encoding green fluorescent protein (AdV-GFP) 
      [multiplicity of infection (MOI)=50:1], only 35% of CD14-positive cells express 
      GFP. We report that centrifugation of these cells with AdV-GFP at 2000 x g for 1 
      h at 37 degrees C significantly enhanced the number of cells expressing GFP (to 
      65%) and the level of GFP expression per transduced cell (fivefold). The 
      viability of the cells was not compromised (<5 % CD14-positive cells were 
      7-aminoactinomycin D (7AAD)-positive after 24 h AdV-GFP exposure at MOI=50:1). 
      Centrifugation allowed efficient transduction of monocytes and macrophages with 
      an MOI at least tenfold lower than otherwise required and did not activate the 
      transduced cells or affect their ability to produce TNFalpha or IL-1beta in 
      response to lipopolysaccharide (LPS). This methodology was also suitable for 
      transducing large numbers of in vitro monocyte-derived macrophages (MDMac) and 
      macrophages isolated from synovial fluids with up to 75-80% of CD14-positive 
      cells transduced after 24-h exposure to AdV-GFP (50:1) and centrifugation (2000 x 
      g). This methodology should provide significant expression of transgenes in human 
      monocytes and macrophages.
FAU - Mayne, George C
AU  - Mayne GC
AD  - Department of Microbiology and Infectious Diseases, School of Medicine, Flinders 
      University, GPO Box 2100, Adelaide 5001, Australia.
FAU - Borowicz, Romana A
AU  - Borowicz RA
FAU - Greeneklee, Kate V L
AU  - Greeneklee KV
FAU - Finlay-Jones, John J
AU  - Finlay-Jones JJ
FAU - Williams, Keryn A
AU  - Williams KA
FAU - Hart, Prue H
AU  - Hart PH
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Netherlands
TA  - J Immunol Methods
JT  - Journal of immunological methods
JID - 1305440
RN  - 0 (Integrin alphaVbeta3)
RN  - 0 (Integrins)
RN  - 0 (Interleukin-1)
RN  - 0 (Luminescent Proteins)
RN  - 0 (Receptors, Vitronectin)
RN  - 0 (Tumor Necrosis Factor-alpha)
RN  - 0 (integrin alphaVbeta5)
RN  - 147336-22-9 (Green Fluorescent Proteins)
SB  - IM
MH  - Adenoviridae/genetics
MH  - Animals
MH  - Cell Survival
MH  - *Centrifugation/methods
MH  - Extracellular Space/chemistry
MH  - Flow Cytometry
MH  - Genetic Vectors
MH  - Green Fluorescent Proteins
MH  - Humans
MH  - Integrin alphaVbeta3/biosynthesis
MH  - Integrins/biosynthesis
MH  - Interleukin-1/analysis
MH  - Luminescent Proteins/genetics
MH  - Macrophages/*cytology
MH  - Monocytes/*cytology
MH  - Receptors, Vitronectin/biosynthesis
MH  - Synovial Fluid/cytology
MH  - Transduction, Genetic/*methods
MH  - Tumor Necrosis Factor-alpha/analysis
EDAT- 2003/09/06 05:00
MHDA- 2003/10/31 05:00
CRDT- 2003/09/06 05:00
PHST- 2003/09/06 05:00 [pubmed]
PHST- 2003/10/31 05:00 [medline]
PHST- 2003/09/06 05:00 [entrez]
AID - S0022175903002291 [pii]
AID - 10.1016/s0022-1759(03)00229-1 [doi]
PST - ppublish
SO  - J Immunol Methods. 2003 Jul;278(1-2):45-56. doi: 10.1016/s0022-1759(03)00229-1.