Unexpected Diversity of Cellular Immune Responses against Nef and Vif in HIV-1-Infected Patients Who Spontaneously Control Viral Replication

Background

HIV-1-infected individuals who spontaneously control viral replication represent an example of successful containment of the AIDS virus. Understanding the anti-viral immune responses in these individuals may help in vaccine design. However, immune responses against HIV-1 are normally analyzed using HIV-1 consensus B 15-mers that overlap by 11 amino acids. Unfortunately, this method may underestimate the real breadth of the cellular immune responses against the autologous sequence of the infecting virus.

Methodology and Principal Findings

Here we compared cellular immune responses against nef and vif-encoded consensus B 15-mer peptides to responses against HLA class I-predicted minimal optimal epitopes from consensus B and autologous sequences in six patients who have controlled HIV-1 replication. Interestingly, our analysis revealed that three of our patients had broader cellular immune responses against HLA class I-predicted minimal optimal epitopes from either autologous viruses or from the HIV-1 consensus B sequence, when compared to responses against the 15-mer HIV-1 type B consensus peptides.

Conclusion and Significance

This suggests that the cellular immune responses against HIV-1 in controller patients may be broader than we had previously anticipated.     

Iron-sulfur cluster reconstitution of spinach chloroplast Rieske protein requires a partially prefolded apoprotein

The Rieske 2Fe-2S protein is a central component of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for expression in Escherichia coli of full-length and truncated Spinacia oleracea Rieske (PetC) proteins fused to the MalE, maltose binding protein. The expressed Rieske fusion proteins were found predominantly in soluble form in the E. coli cytoplasm. These proteins could be readily purified for further experimentation. In vitro reconstitution of the characteristic, "Rieske-type" 2Fe-2S cluster into these fused proteins was accomplished by a chemical method employing reduced iron and sulfide. Cluster incorporation was monitored by electron paramagnetic resonance and optical circular dichroism (CD) spectroscopy. CD spectral analysis in the ultraviolet region suggests that the spinach Rieske apoprotein must be in a partially folded conformation to incorporate an appropriate iron-sulfur cluster. These data further suggest that upon cluster integration, further folding occurs, allowing the Rieske protein to attain a final, native structure. The data presented here are the first to demonstrate successful chemical reconstitution of the 2Fe-2S cluster into a Rieske apoprotein from higher plant chloroplasts.     

Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB 42

Although bacterial polyketides are of considerable biomedical interest, the molecular biology of polyketide biosynthesis in Bacillus spp., one of the richest bacterial sources of bioactive natural products, remains largely unexplored. Here we assign for the first time complete polyketide synthase (PKS) gene clusters to Bacillus antibiotics. Three giant modular PKS systems of the trans-acyltransferase type were identified in Bacillus amyloliquefaciens FZB 42. One of them, pks1, is an ortholog of the pksX operon with a previously unknown function in the sequenced model strain Bacillus subtilis 168, while the pks2 and pks3 clusters are novel gene clusters. Cassette mutagenesis combined with advanced mass spectrometric techniques such as matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization mass spectrometry revealed that the pks1 (bae) and pks3 (dif) gene clusters encode the biosynthesis of the polyene antibiotics bacillaene and difficidin or oxydifficidin, respectively. In addition, B. subtilis OKB105 (pheA sfp(0)), a transformant of the B. subtilis 168 derivative JH642, was shown to produce bacillaene, demonstrating that the pksX gene cluster directs the synthesis of that polyketide. The GenBank accession numbers for gene clusters pks1(bae), pks2, and pks3(dif) are AJ 634060.2, AJ 6340601.2, and AJ 6340602.2, respectively.     

The neutrophil recombinatorial TCR-like immune receptor is expressed across the entire human life span but repertoire diversity declines in old age

Recent evidence has revealed the existence of T cell receptor (TCR) αβ-based recombinatorial immune receptors in phagocytes. Here, we performed a systematic survey of the variable β-chain repertoires of the neutrophil TCR-like αβ immunoreceptor (referred to as TCRL(n)αβ) in defined cohorts of young and old individuals. Peripheral blood CD15(+) neutrophils from young adults (age 30 ± 7 years, n=12) expressed an average number of 13 ± 6 distinct TCRL(n) Vβ-chains from the total pool of 25 human Vβ-chains. Neutrophils from aged subjects (age 76 ± 6 years, n=12) also consitutively express the TCRL(n), however, only a small number of Vβ-chains is used (4 ± 2). Consistent with this, the average number of expressed CDR3 Vβ length variants was fourfold higher in young individuals than in aged subjects (33 ± 24 vs. 8 ± 3). Young adults showed broad usage of all TCRL(n) Vβ-chains. In contrast, >70years individuals displayed a striking repertoire polarization towards the TCRL(n) Vβ1 and Vβ5b chains and a high degree of Vβ5b clonotype sharing. Our study reveals broad TCRL(n) repertoire diversity in young adults and demonstrates that the neutrophil variable immune receptor is expressed throughout the entire human life span. The marked decline in TCRL(n) repertoire diversity in old age identifies a novel mechanism of immunosenescence in neutrophils.     

CD57 Expression and Cytokine Production by T Cells in Lesional and Unaffected Skin from Patients with Psoriasis

Background

The immunopathogenic mechanisms leading to psoriasis remain unresolved. CD57 is a marker of replicative inability and immunosenescence on CD8+ T cells and the proportion of CD57 expressing CD8+ T cells is increased in a number of inflammatory conditions.

Methodology

We examined the expression of CD57 on T cells in the skin of patients affected with psoriasis, comparing lesional and unaffected skin. We also assessed functionality of the T cells by evaluating the secretion of several inflammatory cytokines (IL-17A, IFN-gamma, IL-2, IL-33, TNF-alpha, IL-21, IL-22, and IL-27), from cell-sorted purified CD4+ and CD8+ T cells isolated from lesional and unaffected skin biopsies of psoriasis patients.

Principal Findings

We observed that the frequency of CD57+CD4+ and CD57+CD8+ T cells was significantly higher in unaffected skin of psoriasis patients compared to lesional skin. Sorted CD4+ T cells from psoriatic lesional skin produced higher levels of IL-17A, IL-22, and IFN-gamma compared to unaffected skin, while sorted CD8+ T cells from lesional skin produced higher levels of IL-17, IL-22, IFN-gamma, TNF-alpha, and IL-2 compared to unaffected skin.

Conclusions/Significance

These findings suggest that T cells in unaffected skin from psoriasis patients exhibit a phenotype compatible with replicative inability. As they have a lower replicative capacity, CD57+ T cells are less frequent in lesional tissue due to the high cellular turnover.     

Kinetic microplate assay for determining immobilized antimicrobial peptide activity

Antimicrobial peptide immobilization onto surfaces is of great interest, although characterization of activity can be problematic. The kinetic microplate method described here determines the minimum bactericidal concentration (MBC) of immobilized antimicrobial peptides through a combination and modification of traditional solution assays, overcoming the difficulties of working with a solid substrate. The technique enables rapid, accurate evaluation of immobilized peptide lytic behavior, elucidating both dose- and time-dependent activity at multiple concentrations. Furthermore, the method yields information regarding sublethal concentrations not realized in the traditional assays.