Advantages of multiplex proteomics in clinical immunology: the case of rheumatoid arthritis: novel IgXPLEX™: planar microarray diagnosis.

Clinical multiplex diagnostic proteomics is the application of proteomic technologies to improve a patient's clinical outcomes. The future holds impact potential for testing prognosis, diagnosis, and drug therapy, while monitoring efficacious treatment with qualitative and quantitative data. Multiplex clinical diagnostic use of novel biomarkers in body fluids to confirm presence and severity of clinical disease states, holds great promise for clinical use. Challenges for diagnostic clinics include awareness of proteome complexity in clinical samples, the effects of high-abundance proteins, such as albumin, that could mask detection of other and low abundance disease proteins or biomarkers. Standardized approaches to sample collection and preparation, new analytical techniques and novel algorithms for bio-statistical analysis will facilitate release of the great potential of clinical multiplex diagnostic proteomics. A sensitive RA assay has been developed for the simultaneous measurement of the three rheumatoid factors (RFs), RF-IgA, IgG, and IgM, with the option to simultaneously measure anti-cyclic citrullinated peptide (anti-CCP) IgG antibodies using IgXPLEX™: technology. Testing 10-µL serum samples, SQI's multiplex microarray rheumatoid arthritis assay provides both positive/negative as well as qualitative/semi-quantitative results for anti-CCP IgG, RF-IgA, IgG, and IgM in each sample well on a 96-well microtiter-formatted microarray plate. Signal detection uses sensitive fluorescent-tagged markers captured onto planar microarray spots and read in a microarray scanner. Each result is verified with confidence confirmation technology and validating quality controls in every sample well. For an 80-RA positive patient cohort, the 4-PLEX profile sensitivity was determined at 82.5%. The specificity for the 44 RA healthy control cohort was determined at 97.7%. The multiplex data also demonstrated that a patients' severity of disease profile, mild to severe, correlates the status of RA biomarkers to disease status.

A combination anti-HIV-1 gene therapy approach using a single transcription unit that expresses antisense, decoy, and sense RNAs, and trans-dominant negative mutant Gag and Env proteins.

Oncoretroviral vectors were engineered to allow constitutive expression of an antisense RNA and the trans-activator of transcription (Tat)-inducible expression of a mRNA containing the trans-activation response (TAR) element, the Rev response element (RRE), and the efficient packaging signal (Psi(e) of human immunodeficiency virus-1 (HIV-1) RNA. Nuclear export of this mRNA by the regulator of expression of virion proteins (Rev) would allow its translation into wild type (WT) (MoTN-Ti-GE-Ri- Ter) or trans-dominant negative mutant (TDM) (MoTN-Ti-GmEm-Ri-Ter) Gag and Env proteins. Thus, the antisense RNA produced in a constitutive manner would ensure that even if there is leaky expression, no WT/TDM Gag or Env protein would be produced in the uninfected cells. If cells become infected by HIV-1, the antisense RNA would inhibit HIV-1 replication. Failure on the part of antisense RNA to inhibit virus replication would allow GE/GmEm mRNA production. The GE/GmEm mRNA would cause partial inhibition of HIV-1 replication as it contains the TAR, RRE, and Psi(e) signal sequences. Translation of GmEm mRNA would give rise to TDM Gag and Env proteins, which would further decrease progeny virus infectivity. Tat- and Rev-inducibility was demonstrated in transfected HeLa and HeLa-Tev cells. Full-length WT/TDM Gag production was confirmed by Western blot analysis. Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid cell line, and the stable transductants were challenged with HIV-1. Virus replication was better inhibited by the MoTN-Ti-GE-Ri-Ter vector than by the MoTN-Ti-GmEm-Ri-Ter vector. Inhibition of HIV-1 replication was also demonstrated in transduced CD4+ human peripheral blood T lymphocytes (PBLs). Moreover, our results suggest that cloning in the reverse transcriptional orientation must be avoided to prevent antisense RNA-mediated inhibition of transgene and endogenous gene expression.