Biti RA, Williamson P, Stewart GJ; International Conference on AIDS.

Int Conf AIDS. 1996 Jul 7-12; 11: 65 (abstract no. We.A.3052).

Dept. Clinical Immunology, ICPMR, Westmead Hospital, Westmead, NSW, Australia. Fax: 61-2-891 3889. E-mail: ROBYNB@westmed.wh.su.edu.au.

Objective: To develop quantitative reverse transcription-polymerase chain reaction (QRT-PCR) assays for measurement of interleukin-2 (IL-2) and IL-2 receptor (IL-2R)alpha mRNA, to study (i) the loss of T helper cells ability from HIV+ individuals to respond to antigen and (ii) the effect of Nef and Gag on CD28 signalling pathways. Methods: RNA templates were transcribed from novel plasmid constructs containing either the IL-2 or IL-2Ralpha gene, altered by site-directed mutagenesis to incorporate a unique restriction enzyme site. QRT-PCR standard curves were constructed using serial dilutions of unmutated IL-2/IL-2Ralpha RNA combined with a constant amount of the mutant. RT-PCR products were digested with the restriction enzyme and laser densitometry performed on the PAGE gels. Jurkat cells plus or minus Nef/Gag with PMA (5-200ng/mL) and alpha-CD28 antibody (Ab) (1/1600-1/4000 dilution) are incubated for 0-24 hours and RT-PCR performed to detect the presence of IL-2, IL-2R, bcl-2 and c-fos mRNA. Results: The 223bp of the IL-2 gene fragment (458bp) was mutated to produce a unique Bgl II site in the RNA. Digestion with Bgl II results in 223 and 236bp fragments. Likewise, the IL-2Ralpha gene fragment (398bp) is mutated at the 191 bp to give a novel Nhe I site, resulting in a 189 and a 209bp fragment. The addition of 106 mutated RNA molecules to 104-108 non-muated RNA molecules provides a control for both the RT and PCR steps of the reaction. Thus, highly reproducible standard curves for both IL-2 and IL-2Ralpha were constructed and validated. Similarly, 106 molecules of mutant RNA controls is added to each test specimen; ensuring accuracy and reproducibility within and between experiments. We are currently measuring IL-2 and IL-2Ralpha mRNA from HIV+/- PBMC in response to PHA and tetanus toxoid. Peak production of IL-2 and IL-2R mRNA was detected in Jurkat cells stimulated with 5ng/mL PMA and 1/4000 alpha-CD28 Ab at 6 hours, with consistently high levels of bcl-2 and c-fos throughout. Preliminary results suggest Nef and Gag may effect CD28 signalling pathways. Conclusions: We have designed and constructed a novel internal control, which when added to each specimen allows for a highly reproducible QRT-PCR. Quantitation of T cell early gene expression will provide a useful method for measuring the effects of HIV proteins on infected individuals.

Publication Types:

• Meeting Abstracts

Keywords:

• AIDS Vaccines
• Antigens, CD28
• DNA Primers
• Gene Expression
• Humans
• Interleukin-2
• Jurkat Cells
• Phytohemagglutinins
• Polymerase Chain Reaction
• RNA, Messenger
• Receptors, Interleukin-2
• Reverse Transcriptase Polymerase Chain Reaction
• T-Lymphocytes
• Transcription, Genetic
• genetics
• immunology

Other ID:

• 96923414

UI: 102219313

From Meeting Abstracts