Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion

<p>Abstract</p> <p>Background</p> <p>RNA transfection into dendritic cells (DCs) is widely used to achieve antigen expression as well as to modify DC properties. CD40L is expressed by activated T cells and interacts with CD40 receptors expressed on the surface of the DCs leading to Th1 polarization. Previous studies demonstrated that ectopic CD40L expression via DNA transfection into DCs can activate the CD40 receptor signal transduction cascade. In contrast to previous reports, this study demonstrates that the same effect can be achieved when RNA encoding CD40L is electroporated into DCs as evidenced by secretion of IL-12. To achieve higher levels of IL-12 secretion, a systematic approach involving modification of coding and noncoding regions was implemented to optimize protein expression in the DCs for the purpose of increasing IL-12 secretion.</p> <p>Results</p> <p>Site-directed mutagenesis of each of the first five in-frame methionine codons in the CD40L coding sequence demonstrated that DCs expressing a truncated CD40L protein initiated from the second methionine codon secreted the highest levels of IL-12. In addition, a post-transcriptional method of capping was utilized for final modification of the CD40L RNA. This method enzymatically creates a type I cap structure identical to that found in most eukaryotic mRNAs, in contrast to the type 0 cap incorporated using the conventional co-transcriptional capping reaction.</p> <p>Conclusion</p> <p>The combination of knocking out the first initiation methionine and post-transcriptional capping of the CD40L RNA allowed for approximately a one log increase in IL-12 levels by the transfected DCs. We believe this is a first report describing improved protein expression of post-transcriptionally capped RNA in DCs. The post-transcriptional capping which allows generation of a type I cap may have broad utility for optimization of protein expression from RNA in DCs and other cell types.</p

The Immunosuppressive Properties of the HIV Vpr Protein Are Linked to a Single Highly Conserved Residue, R90

BACKGROUND: A hallmark of AIDS progression is a switch of cytokines from Th1 to Th2 in the plasma of patients. IL-12, a critical Th1 cytokine secreted by antigen presenting cells (APCs) is suppressed by Vpr, implicating it as an important virulence factor. We hypothesize that Vpr protein packaged in the virion may be required for disabling APCs of the first infected mucosal tissues. Consistent with this idea are reports that defects in the C-terminus of Vpr are associated with long-term non-progression. PRINCIPAL FINDINGS: Vpr RNA amplified from various sources was electroporated into monocyte-derived DC and IL-12 levels in supernatants were analyzed. The analysis of previously reported C-terminal Vpr mutations demonstrate that they do not alleviate the block of IL-12 secretion. However, a novel single conservative amino acid substitution, R90K, reverses the IL-12 suppression. Analysis of 1226 Vpr protein sequences demonstrated arginine (R) present at position 90 in 98.8%, with other substitutions at low frequency. Furthermore, none of sequences report lysine (K) in position 90. Vpr clones harboring the reported substitutions in position 90 were studied for their ability to suppress IL-12. Our data demonstrates that none of tested substitutions other than K relieve IL-12 suppression. This suggests a natural selection for sequences which suppress IL-12 secretion by DC and against mutations which relieve such suppression. Further analyses demonstrated that the R90K, as well as deletion of the C-terminus, directs the Vpr protein for rapid degradation. CONCLUSION: This study supports Vpr as an HIV virulence factor during HIV infection and for the first time provides a link between evolutionary conservation of Vpr and its ability to suppress IL-12 secretion by DC. DC activated in the presence of Vpr would be defective in the production of IL-12, thus contributing to the prevailing Th2 cytokine profile associated with progressive HIV disease. These findings should be considered in the design of future immunotherapies that incorporate Vpr as an antigen

The Freshman, vol. 5, no. 3 [issue misnumbered]

The Freshman was a weekly, student newsletter issued on Mondays throughout the academic year. The newsletter included calendar notices, coverage of campus social events, lectures, and athletic teams. The intent of the publication was to create unity, a sense of community, and class spirit among first year students. Included in this edition is an editorial authored on behalf of The Co-eds of \u2738 commenting on the divisive nature of Freshman Rules imposed by upper classmen and requesting first-year students be treated with respect

The Allen Telescope Array Pi GHz Sky Survey I. Survey Description and Static Catalog Results for the Bootes Field

The Pi GHz Sky Survey (PiGSS) is a key project of the Allen Telescope Array. PiGSS is a 3.1 GHz survey of radio continuum emission in the extragalactic sky with an emphasis on synoptic observations that measure the static and time-variable properties of the sky. During the 2.5-year campaign, PiGSS will twice observe ~250,000 radio sources in the 10,000 deg^2 region of the sky with b > 30 deg to an rms sensitivity of ~1 mJy. Additionally, sub-regions of the sky will be observed multiple times to characterize variability on time scales of days to years. We present here observations of a 10 deg^2 region in the Bootes constellation overlapping the NOAO Deep Wide Field Survey field. The PiGSS image was constructed from 75 daily observations distributed over a 4-month period and has an rms flux density between 200 and 250 microJy. This represents a deeper image by a factor of 4 to 8 than we will achieve over the entire 10,000 deg^2. We provide flux densities, source sizes, and spectral indices for the 425 sources detected in the image. We identify ~100$ new flat spectrum radio sources; we project that when completed PiGSS will identify 10^4 flat spectrum sources. We identify one source that is a possible transient radio source. This survey provides new limits on faint radio transients and variables with characteristic durations of months.Comment: Accepted for publication in ApJ; revision submitted with extraneous figure remove

Multiplex RT-PCR Amplification of HIV Genes to Create a Completely Autologous DC-Based Immunotherapy for the Treatment of HIV Infection

BACKGROUND: Effective therapy for HIV-infected individuals remains an unmet medical need. Promising clinical trials with dendritic cell (DC)-based immunotherapy consisting of autologous DC loaded with autologous virus have been reported, however, these approaches depend on large numbers of HIV virions to generate sufficient doses for even limited treatment regimens. METHODOLOGY/PRINCIPAL FINDINGS: The present study describes a novel approach for RT-PCR amplification of HIV antigens. Previously, RT-PCR amplification of autologous viral sequences has been confounded by the high mutation rate of the virus which results in unreliable primer-template binding. To resolve this problem we developed a multiplex RT-PCR strategy that allows reliable strain-independent amplification of highly polymorphic target antigens from any patient and requires neither viral sequence data nor custom-designed PCR primers for each individual. We demonstrate the application of our RT-PCR process to amplify translationally-competent RNA encoding regions of Gag, Vpr, Rev and Nef. The products amplified using this method represent a complex mixture of autologous antigens encoded by viral quasispecies. We further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro. CONCLUSION/SIGNIFICANCE: This study describes a strategy to overcome patient to patient viral diversity enabling strain-independent RT-PCR amplification of RNAs encoding sequence divergent quasispecies of Gag, Vpr, Rev and Nef from small volumes of infectious plasma. The approach allows creation of a completely autologous therapy that does not require advance knowledge of the HIV genomic sequences, does not have yield limitations and has no intact virus in the final product. The simultaneous use of autologous viral antigens and DCs may provoke broad patient-specific immune responses that could potentially induce effective control of viral loads in the absence of conventional antiretroviral drug therapy

The functional ALDH2 polymorphism is associated with breast cancer risk: A pooled analysis from the Breast Cancer Association Consortium.

BACKGROUND: Epidemiological studies consistently indicate that alcohol consumption is an independent risk factor for female breast cancer (BC). Although the aldehyde dehydrogenase 2 (ALDH2) polymorphism (rs671: Glu>Lys) has a strong effect on acetaldehyde metabolism, the association of rs671 with BC risk and its interaction with alcohol intake have not been fully elucidated. We conducted a pooled analysis of 14 case-control studies, with individual data on Asian ancestry women participating in the Breast Cancer Association Consortium. METHODS: We included 12,595 invasive BC cases and 12,884 controls for the analysis of rs671 and BC risk, and 2,849 invasive BC cases and 3,680 controls for the analysis of the gene-environment interaction between rs671 and alcohol intake for BC risk. The pooled odds ratios (OR) with 95% confidence intervals (CI) associated with rs671 and its interaction with alcohol intake for BC risk were estimated using logistic regression models. RESULTS: The Lys/Lys genotype of rs671 was associated with increased BC risk (OR = 1.16, 95% CI 1.03-1.30, p = 0.014). According to tumor characteristics, the Lys/Lys genotype was associated with estrogen receptor (ER)-positive BC (OR = 1.19, 95% CI 1.05-1.36, p = 0.008), progesterone receptor (PR)-positive BC (OR = 1.19, 95% CI 1.03-1.36, p = 0.015), and human epidermal growth factor receptor 2 (HER2)-negative BC (OR = 1.25, 95% CI 1.05-1.48, p = 0.012). No evidence of a gene-environment interaction was observed between rs671 and alcohol intake (p = 0.537). CONCLUSION: This study suggests that the Lys/Lys genotype confers susceptibility to BC risk among women of Asian ancestry, particularly for ER-positive, PR-positive, and HER2-negative tumor types.Includes EU, FP7 and CRU