Document 0280
 DOCN  M9590280
 TI    Assessment of disease progression and T helper cell responsiveness to
       HIV peptides by quantitative RT-PCR of IL-2 & IL-2R mRNA.
 DT    9509
 AU    Biti R; Stewart G; Dept. Clinical Immunology, Westmead Hospital.
 SO    Annu Conf Australas Soc HIV Med. 1994 Nov 3-6;6:242 (unnumbered poster).
       Unique Identifier : AIDSLINE ASHM6/95291819
 AB    An important requirement for both the development and assessment of
       immune-based therapy is the accurate measurement of HIV-specific T cell
       immunity, with respect to T helper cell function. However, standard
       methodologies fail to address the difficulties associated with
       assessment of the immunocompromised individuals. We propose that some of
       these shortcomings may be overcome by detecting responses to HIV at an
       earlier stage in the T cell activation pathways than proliferation and
       via more sensitive assays. We have developed quantitative RT-PCR assays
       for the measurement of mRNA production of interleukin-2 (IL-2) and
       interleukin-2 receptor (IL-2R) alpha-chain. In addition, we have used
       these assays to measure responsiveness to recall antigens, such as
       tetanus toxoid, to provide a more detailed assessment of the immune
       status of HIV infected individuals. To ensure a high level of accuracy
       and reproducibility by minimising inter-sample variation occurring
       during processing and amplification, an internal RNA control was
       developed. Site-directed mutagenesis was performed to create a mutant
       IL-2 and IL-2R RNA, containing a novel restriction enzyme site. A
       constant amount of this is added to each of the samples, which are then
       co-amplified. After solution hybridization of radio-labelled, specific
       probes (probe and PCR product are denatured at 94 degrees C for 5
       minutes, followed by annealing at 50 degrees C for a further 5 minutes),
       PCR product levels can be determined by densitometry of autorads or
       radioactive counting of excised bands.
 DE    Gene Expression Regulation, Viral/IMMUNOLOGY  Human  HIV
       Antigens/*IMMUNOLOGY  HIV Infections/*IMMUNOLOGY  Immunologic
       Memory/IMMUNOLOGY  Interleukin-2/*GENETICS  Lymphocyte
       Transformation/GENETICS/IMMUNOLOGY  Peptides/*IMMUNOLOGY  Polymerase
       Chain Reaction/*METHODS  Receptors, Interleukin-2/*GENETICS  RNA,
       Messenger/*GENETICS  T-Lymphocytes, Helper-Inducer/*IMMUNOLOGY  MEETING
       ABSTRACT

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).