PMID- 20070903
OWN - NLM
STAT- MEDLINE
DCOM- 20100707
LR  - 20211020
IS  - 1479-5876 (Electronic)
IS  - 1479-5876 (Linking)
VI  - 8
DP  - 2010 Jan 13
TI  - A quantitative real time PCR method to analyze T cell receptor Vbeta subgroup 
      expansion by staphylococcal superantigens.
PG  - 2
LID - 10.1186/1479-5876-8-2 [doi]
AB  - BACKGROUND: Staphylococcal enterotoxins (SEs), SE-like (SEl) toxins, and toxic 
      shock syndrome toxin-1 (TSST-1), produced by Staphylococcus aureus, belong to the 
      subgroup of microbial superantigens (SAgs). SAgs induce clonal proliferation of T 
      cells bearing specific variable regions of the T cell receptor beta chain 
      (Vbeta). Quantitative real time PCR (qRT-PCR) has become widely accepted for 
      rapid and reproducible mRNA quantification. Although the quantification of Vbeta 
      subgroups using qRT-PCR has been reported, qRT-PCR using both primers annealing 
      to selected Vbeta nucleotide sequences and SYBR Green I reporter has not been 
      applied to assess Vbeta-dependent expansion of T cells by SAgs. METHODS: Human 
      peripheral blood mononuclear cells were stimulated with various SAgs or a 
      monoclonal antibody specific to human CD3. Highly specific expansion of Vbeta 
      subgroups was assessed by qRT-PCR using SYBR Green I reporter and primers 
      corresponding to selected Vbeta nucleotide sequences. RESULTS: qRT-PCR 
      specificities were confirmed by sequencing amplified PCR products and melting 
      curve analysis. To assess qRT-PCR efficiencies, standard curves were generated 
      for each primer set. The average slope and R2 of standard curves were -3.3764 +/- 
      0.0245 and 0.99856 +/- 0.000478, respectively, demonstrating that the qRT-PCR 
      established in this study is highly efficient. With some exceptions, SAg Vbeta 
      specificities observed in this study were similar to those reported in previous 
      studies. CONCLUSIONS: The qRT-PCR method established in this study produced an 
      accurate and reproducible assessment of Vbeta-dependent expansion of human T 
      cells by staphylococcal SAgs. This method could be a useful tool in the 
      characterization T cell proliferation by newly discovered SAg and in the 
      investigation of biological effects of SAgs linked to pathogenesis.
FAU - Seo, Keun Seok
AU  - Seo KS
AD  - Department of Microbiology, Molecular Biology and Biochemistry, University of 
      Idaho, Moscow, ID 83844, USA. baram717@uidaho.edu
FAU - Park, Joo Youn
AU  - Park JY
FAU - Terman, David S
AU  - Terman DS
FAU - Bohach, Gregory A
AU  - Bohach GA
LA  - eng
GR  - P20 RR016454/RR/NCRR NIH HHS/United States
GR  - P20 RR15587/RR/NCRR NIH HHS/United States
GR  - U54AI57141/AI/NIAID NIH HHS/United States
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20100113
PL  - England
TA  - J Transl Med
JT  - Journal of translational medicine
JID - 101190741
RN  - 0 (Antigens, Bacterial)
RN  - 0 (Enterotoxins)
RN  - 0 (Receptors, Antigen, T-Cell, alpha-beta)
RN  - 0 (Superantigens)
SB  - IM
MH  - Antigens, Bacterial/immunology
MH  - Enterotoxins/*immunology
MH  - Humans
MH  - Leukocytes, Mononuclear/immunology
MH  - Molecular Sequence Data
MH  - Polymerase Chain Reaction/*methods
MH  - Receptors, Antigen, T-Cell, alpha-beta/genetics/*immunology
MH  - Reproducibility of Results
MH  - Sensitivity and Specificity
MH  - Staphylococcus aureus/genetics/*immunology
MH  - Superantigens/*immunology
MH  - T-Lymphocyte Subsets/immunology
MH  - T-Lymphocytes/immunology
PMC - PMC2841588
EDAT- 2010/01/15 06:00
MHDA- 2010/07/08 06:00
PMCR- 2010/01/13
CRDT- 2010/01/15 06:00
PHST- 2009/07/01 00:00 [received]
PHST- 2010/01/13 00:00 [accepted]
PHST- 2010/01/15 06:00 [entrez]
PHST- 2010/01/15 06:00 [pubmed]
PHST- 2010/07/08 06:00 [medline]
PHST- 2010/01/13 00:00 [pmc-release]
AID - 1479-5876-8-2 [pii]
AID - 10.1186/1479-5876-8-2 [doi]
PST - epublish
SO  - J Transl Med. 2010 Jan 13;8:2. doi: 10.1186/1479-5876-8-2.