Human immunodeficiency virus type I-specific CD8+ T cell subset abnormalities in chronic infection persist through effective antiretroviral therapy

Background: Effective highly active antiretroviral therapy (HAART) reduces human immunodeficiency virus (HIV) replication, restores CD4 +T lymphocyte counts and greatly reduces the incidence of opportunistic infections. While this demonstrates improved generalized immune function, rapid rebound to pre-treatment viral replication levels following treatment interruption indicates little improvement in immune control of HIV replication. The extent to which HAART can normalize HIV-specific CD8 +T cell function over time in individuals with chronic infection remains an important unresolved issue. In this study, we evaluated the magnitude, general specificity and character of HIV specific CD8 +T cell responses at four time points across 2-9 years in 2 groups of chronically infected individuals separated on the basis of either effective antiretroviral suppression or ongoing replication of HIV.Methods: Peripheral blood mononuclear cells (PBMC) were stimulated with overlapping 15mer peptides spanning HIV Gag, Pol, Env and Nef proteins. Cells producing interferon-γ (IFN-γ) or interleukin-2 (IL-2) were enumerated by ELISPOT and phenotyped by flow cytometry.Results and Conclusions: The magnitude of the HIV-specific CD8 +T cell response ranged from < .01 to approximately 1.0% of PBMC and was significantly greater in the group with detectable viral replication. Stronger responses reflected higher numbers of CD8 +CD45RA -effector memory cells producing IFN-γ, but not IL-2. Magnitude, general specificity and character of the HIV-specific CD8 +T cell response changed little over the study period. While antiretroviral suppression of HIV in chronic infection reduces HIV-specific CD8 +T cell response magnitude in the short term, it had no significant effect on response character over periods up to 9 years

Chemical imaging of lignocellulosic biomass by CARS microscopy

Chemical and structural composition of wood biomass is studied by label-free and chemically specific Coherent Anti-Stokes Raman Scattering (CARS) microscopy. A concept developed for assignment and semi-quantitative imaging of sample components; cellulose, hemicellulose, and lignin; by multiplex CARS microspectroscopy and subsequent data analysis is presented. Specific imaging without fluorescence backround is achieved an order of magnitude faster compared with conventional Raman microscopy. Laser polarization control yield information on molecular arrangement in wood fibers. Narrowband CARS excitation of single vibrations allows for three-dimensional volume imaging. Thus, CARS microscopy has potential as an important instrument for characterization of lignocellulosic materials

Positioning of APOBEC3G/F Mutational Hotspots in the Human Immunodeficiency Virus Genome Favors Reduced Recognition by CD8+ T Cells

Due to constitutive expression in cells targeted by human immunodeficiency virus (HIV), and immediate mode of viral restriction upon HIV entry into the host cell, APOBEC3G (A3G) and APOBEC3F (A3F) have been considered primarily as agents of innate immunity. Recent bioinformatic and mouse model studies hint at the possibility that mutation of the HIV genome by these enzymes may also affect adaptive immunity but whether this occurs in HIV-infected individuals has not been examined. We evaluated whether APOBEC-mediated mutations within common HIV CD8+ T cell epitopes can potentially enhance or diminish activation of HIV-specific CD8+ T cells from infected individuals. We compared ex vivo activation of CD8+ T lymphocytes from HIV-infected individuals by wild type HIV peptide epitopes and synthetic variants bearing simulated A3G/F-induced mutations by measuring interferon-c (IFN-c) production. We found that A3G/F-induced mutations consistently diminished HIV-specific CD8+ T cell responses against the common epitopes we tested. If this reflects a significant trend in vivo, then adaptation by HIV to enrich sequences that are favored for mutation by A3G/F (A3G/F hotspots) in portions of its genome that encode immunogenic CD8+ T cell epitopes would favor CTL escape. Indeed, we found the most frequently mutated A3G motif (CCC) is enriched up to 6-fold within viral genomic sequences encoding immunodominant CD8+ T cell epitopes in Gag, Pol and Nef. Within each gene, A3G/F hotspots are more abundant in sequences encoding epitopes that are commonly recognized due to their HLA restriction. Thus, in our system, mutations of the HIV genome, mimicking A3G/F activity, appeared to abrogate or severely reduce CTL recognition. We suggest that the physiological significance of this potential effect in facilitating CTL escape is echoed in the adaptation of the HIV genome to enrich A3G/F hotspots in sequences encoding CTL epitopes that are more immunogenic at the population level

Visualization of structural changes in cellulosic substrates during enzymatic hydrolysis using multimodal nonlinear microscopy

Enzymatic hydrolysis of cellulose provides a renewable source of monosaccharides for production of variety of biochemicals and biopolymers. Unfortunately, the enzymatic hydrolysis of cellulose is often incomplete, and the reasons are not fully understood. We have monitored enzymatic hydrolysis in terms of molecular density, ordering and autofluorescence of cellulose structures in real time using simultaneous CARS, SHG and MPEF microscopy with the aim of contributing to the understanding and optimization of the enzymatic hydrolysis of cellulose. Three cellulose-rich substrates with different supramolecular structures, pulp fibre, acid-treated pulp fibre and Avicel, were studied at microscopic level. The microscopy studies revealed that before enzymatic hydrolysis Avicel had the greatest carbon-hydrogen density, while pulp fibre and acid-treated fibre had similar density. Monitoring of the substrates during enzymatic hydrolysis revealed the double exponential SHG decay for pulp fibre and acid-treated fibre indicating two phases of the process. Acid-treated fibre was hydrolysed most rapidly and the hydrolysis of pulp fibre was spatially non-uniform leading to fractioning of the particles, while the hydrolysis of Avicel was more than an order of magnitude slower than that of both fibres