PMID- 12161281
OWN - NLM
STAT- MEDLINE
DCOM- 20030317
LR  - 20190826
IS  - 0165-2478 (Print)
IS  - 0165-2478 (Linking)
VI  - 84
IP  - 1
DP  - 2002 Oct 21
TI  - Optimisation of the CT h4S bioassay for detection of human interleukin-4 secreted 
      by mononuclear cells stimulated by phytohaemaglutinin or by human leukocyte 
      antigen mismatched mixed lymphocyte culture.
PG  - 29-39
AB  - Limiting dilution analysis has been used in the context of allogeneic bone marrow 
      transplantation to determine anti-recipient interleukin-2 (IL-2) producing helper 
      T lymphocyte precursor (HTLp) frequencies, which in several studies have been 
      predictive of graft-versus-host disease (GVHD). Recently high anti-recipient IL-4 
      producing HTLp frequencies have been reported and associated with a decreased 
      risk of GVHD. The aim of the present study was to define the optimal conditions 
      for combined determination of IL-2 and IL-4 producing anti-recipient HTLp 
      frequencies. We have optimised the CT.h4S bioassay with regards to specificity, 
      sensitivity, detection limit, and reproducibility. We have found the optimal 
      assay conditions to be 1 x 10 (4) CT.h4S cells/well deprived of IL-4 for 24 h and 
      preincubated for 7 h followed by 18 h of incubation with tritiated 
      methyl-thymidine. In this setting the CT.h4S bioassay detects 5 pg/ml of human 
      recombinant IL-4 with no detection of IL-2 in concentrations below 500 pg/ml. We 
      have found 72 h of culture optimal for detection of IL-2 and IL-4 produced by 
      human mononuclear cells (MNC) in response to stimulation with phytohaemaglutinin 
      and for detection of IL-2 in human leukocyte antigen (HLA)-mismatched mixed 
      leukocyte culture (MLC). An interindividual variation in cytokine accumulation 
      was demonstrated for IL-4 but not for IL-2. With the use of 5x10(4) responder 
      cells/well no IL-4 could be detected in HLA-mismatched MLC between days 1 and 16. 
      The lack of IL-4 detection was not due to high amounts of soluble IL-4 receptor. 
      With the use of 1x10(6) responder cells/well in HLA-mismatched MLC, we found 
      limited IL-4 accumulation still increasing at day 12. We conclude that the CT.h4S 
      bioassay is a reliable and specific method for quantification of IL-4 
      accumulation in cultures of human MNC. The difference in optimal timing for IL-2 
      (day 3) and IL-4 (>/=day 12) detection and evidence of very low IL-4 producing 
      HTLp frequencies makes the relevance of a combined IL-2/IL-4 HTLp assay 
      questionable.
FAU - Petersen, Soren Lykke
AU  - Petersen SL
AD  - Department of Haematology, Rigshospitalet, Blegdamsvej 9, DK-2100, Copenhagen, 
      Denmark. s.l.peterson@dadlnet.dk
FAU - Russell, Charlotte Astrid
AU  - Russell CA
FAU - Bendtzen, Klaus
AU  - Bendtzen K
FAU - Vindelov, Lars Lindhardt
AU  - Vindelov LL
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Netherlands
TA  - Immunol Lett
JT  - Immunology letters
JID - 7910006
RN  - 0 (HLA Antigens)
RN  - 0 (Interleukin-2)
RN  - 0 (Phytohemagglutinins)
RN  - 0 (Receptors, Interleukin-4)
RN  - 0 (Recombinant Proteins)
RN  - 207137-56-2 (Interleukin-4)
SB  - IM
MH  - Biological Assay/*methods/statistics & numerical data
MH  - Cell Line
MH  - Enzyme-Linked Immunosorbent Assay
MH  - HLA Antigens
MH  - Humans
MH  - In Vitro Techniques
MH  - Interleukin-2/analysis/biosynthesis
MH  - Interleukin-4/*analysis/biosynthesis
MH  - Kinetics
MH  - Leukocytes, Mononuclear/drug effects/immunology
MH  - Lymphocyte Culture Test, Mixed
MH  - Phytohemagglutinins/pharmacology
MH  - Receptors, Interleukin-4/analysis
MH  - Recombinant Proteins/analysis
MH  - Reproducibility of Results
MH  - Sensitivity and Specificity
EDAT- 2002/08/06 10:00
MHDA- 2003/03/18 04:00
CRDT- 2002/08/06 10:00
PHST- 2002/08/06 10:00 [pubmed]
PHST- 2003/03/18 04:00 [medline]
PHST- 2002/08/06 10:00 [entrez]
AID - S0165247802001293 [pii]
AID - 10.1016/s0165-2478(02)00129-3 [doi]
PST - ppublish
SO  - Immunol Lett. 2002 Oct 21;84(1):29-39. doi: 10.1016/s0165-2478(02)00129-3.