Quantitative longitudinal analysis of T cell receptor repertoire expression in HIV-infected patients on antiretroviral and interleukin-2 therapy.
ABSTRACT We have developed a single-step reverse transcriptase kinetic PCR assay (kRT-PCR) to accurately determine the expression of each of the 24 TCRbetaV gene families in CD8(+) cells. We analyzed the long-term effects of highly active antiretroviral therapy (HAART) on the stability of the CD8(+) T cell receptor (TCR) repertoire in a cohort of 15 treated and 10 untreated individuals diagnosed with human immunodeficiency virus (HIV) infection. The CD4(+) TCR repertoire was studied in a second cohort receiving interleukin-2 infusions in addition to HAART. Analysis was based on kinetic (quantitative) reverse-transcription PCR (kRT-PCR) of the TCR variable B gene (TCRbetaV). Expression of each of the 24 Vbeta families was assessed at baseline immediately after infection and following initiation of HAART at 2, 4, 12, 24, and up to 192 weeks in 24-week intervals. Statistically significant family-specific expression changes were observed between treated and untreated individuals for 10 TCRbetaV families. Overall, when compared to untreated patients, a more stable expression of TCR genes was observed for HAART-treated individuals. Interestingly, this difference did not correlate with either CD4 or CD8 counts, which follow the expected curves for treated and untreated patients. When we applied our quantitative analysis to IL-2-treated patients we observed a rapid polyclonal activation of the repertoire. These results suggest that homeostasis in the T cell receptor repertoire is more robust in those patients who stay on HAART for a long time and confirm the polyclonal stimulating capacity of IL-2.
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ABSTRACT: Monodispersed zinc oxide (ZnO) particles with various morphologies were successfully prepared via a simple solution route at low temperature. SEM, XRD, TEM, HRTEM, SAED, FTIR and UV–Vis DRS were used to characterize the morphology and microstructure of the products. Photocatalysis of the as-prepared ZnO powders was evaluated by degradation of methyl orange in an aqueous solution exposed to ultraviolet light. Results suggested a close relationship between the photocatalytic activity and the particle morphology and size.Graphical AbstractMonodispersed ZnO particles with various morphologies were prepared via a simple solution route. Photocatalysis of the as-prepared ZnO powders was evaluated by degradation of methyl orange in an aqueous solution exposed to ultraviolet light. Results suggested a close relationship between the photocatalytic activity and the particle morphology and size.Research Highlights►ZnO particles with various morphologies were prepared via a simple solution route. ►The morphology of ZnO particles depends on the crystal nucleation and growth rates. ►Photocatalytic activity of ZnO particles is related to their morphology and size.Powder Technology 01/2011; 207:140-144. · 2.50 Impact Factor
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ABSTRACT: T cell receptor (TCR) diversity is critical for adaptive immunity. Existing methods for measuring such diversity are qualitative, expensive, and/or of uncertain accuracy. Here, we describe a method and associated reagents for estimating the absolute number of unique TCR Vβ rearrangements present in a given number of cells or volume of blood. Compared to next generation sequencing, this method is rapid, reproducible, and affordable. Diversity of a sample is calculated based on three independent measurements of one Vβ-Jβ family of TCR rearrangements at a time. The percentage of receptors using the given Vβ gene is determined by flow cytometric analysis of T cells stained with anti-Vβ family antibodies. The percentage of receptors using the Vβ gene in combination with the chosen Jβ gene is determined by quantitative PCR. Finally, the absolute clonal diversity of the Vβ-Jβ family is determined with the AmpliCot method of DNA hybridization kinetics, by interpolation relative to PCR standards of known sequence diversity. These three component measurements are reproducible and linear. Using titrations of known numbers of input cells, we show that the TCR diversity estimates obtained by this approach approximate expected values within a two-fold error, have a coefficient of variation of 20%, and yield similar results when different Vβ-Jβ pairs are chosen. The ability to obtain accurate measurements of the total number of different TCR gene rearrangements in a cell sample should be useful for basic studies of the adaptive immune system as well as in clinical studies of conditions such as HIV disease, transplantation, aging, and congenital immunodeficiencies.Journal of immunological methods 03/2011; 368(1-2):45-53. · 2.35 Impact Factor
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ABSTRACT: HIV-1-infected individuals progressively loss CD4(+) T cells leading to immunosuppression and raising the risk of opportunistic infections. CD8(+) T-cells play an important role in the immune response against virus infections through their TCR. To evaluate the CD8-TCR repertoire and immunologic markers in HIV-1-infected patients. Ten chronic HIV-1-infected individuals on prolonged effective antiretroviral treatment (ART) were analyzed at baseline (before treatment interruption), after at least one year of treatment interruption (TI) and after at least one year from ART resume (TR). Twenty-four TCR-Vβ gene families were analyzed by a modified CDR3 spectratyping method in isolated CD8(+) T-cells. Immune activation, exhaustion and subpopulation markers were analyzed by flow cytometry. Expansion of Vβ10, Vβ14 and Vβ15 families, associated with low cell activation and stable exhaustion markers, were found at TI. Moreover, an increment of effector memory cells was found. Besides, depletion of Vβ20, Vβ28, and Vβ29 families, associated with an increase in cell activation and exhaustion markers, at TI were also found. These alterations seemed to be more pronounced in patients who had longer time from diagnosis. ART seemed to restore altered CD8(+) T-cell repertoire and most of the immunologic markers. During TI (that was more pronounced in patients with longer HIV-1 infection) it was observed the expansion of Vβ families correlated with decreased cell activation, while Vβ families correlated with cell activation and exhaustion were depleted. These specific repertoire alterations reverted after ART resume.Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 10/2013; · 3.12 Impact Factor