Randomized, double-blind comparative trial of subunit and virosomal influenza vaccines for immunocompromised patients

BACKGROUND: To our knowledge, no study to date has compared the effects of a subunit influenza vaccine with those of a virosomal influenza vaccine on immunocompromised patients. METHODS: A prospective, double-blind, randomized study was conducted to compare the immunogenicity and reactogenicity of subunit and virosomal influenza vaccines for adult patients who had an immunosuppressive disease or who were immunocompromised as a result of treatment. RESULTS: There were 304 patients enrolled in our study: 131 with human immunodeficiency virus (HIV) infection, 47 with a chronic rheumatologic disease, 74 who underwent a renal transplant, 47 who received long-term hemodialysis, and 5 who had some other nephrologic disease. There were 151 patients who received the subunit vaccine and 153 patients who received the virosomal vaccine. A slightly higher percentage of patients from the subunit vaccine group were protected against all 3 influenza vaccine strains after being vaccinated, compared with patients from the virosomal vaccine group (41% vs. 30% of patients; P = .03). Among HIV-infected patients, the level of HIV RNA, but not the CD4 cell count, was an independent predictor of vaccine response. Among renal transplant patients, treatment with mycophenolate significantly reduced the immune response to vaccination. The 2 vaccines were comparable with regard to the frequency and severity of local and systemic reactions within 7 days after vaccination. Disease-specific scores for the activity of rheumatologic diseases did not indicate flare-ups 4-6 weeks after vaccination. CONCLUSIONS: For immunosuppressed patients, the subunit vaccine was slightly more immunogenic than the virosomal vaccine. The 2 vaccines were comparable with regard to reactogenicity. Vaccine response decreased with increasing degree of immune suppression. Among HIV-infected patients, the viral load, rather than the CD4 cell count, predicted the protective immune response to the vaccine. CLINICAL TRIALS REGISTRATION: NCT00783380 .

A 6'-Fluoro-Substituent in Bicyclo-DNA Increases Affinity to Complementary RNA Presumably by CF-HC Pseudohydrogen Bonds

The synthesis of a novel bicyclic thymidine analogue carrying a β-fluoro substituent at C6' (6'F-bcT) has been achieved. Key steps of the synthesis were an electrophilic fluorination/stereospecific hydrogenation sequence of a bicyclo sugar intermediate, followed by an N-iodo-succinimide-induced stereoselective nucleosidation. A corresponding phosphoramidite building block was then prepared and used for oligonucleotide synthesis. Tm measurements of oligonucleotides with single and double incorporations showed a remarkable stabilization of duplex formation particularly with RNA as complement without compromising pairing selectivity. Increases in Tm were in the range of +1-2 °C compared to thymidine and +1-3 °C compared to a standard bc-T residue. Structural investigations of the 6'F-bcT nucleoside by X-ray crystallography showed an in-line arrangement of the fluorine substituent with H6 of thymine, however, with a distance that is relatively long for a nonclassical CF-HC hydrogen bond. In contrast, structural investigations in solution by (1)H and (13)C NMR clearly showed scalar coupling of fluorine with H6 and C6 of the nucleobase, indicating the existence of at least weak electrostatic interactions. On the basis of these results, we put forward the hypothesis that these weak CF-HC6 electrostatic interactions increase duplex stability by orienting and partially freezing torsion angle χ of the 6'F-bcT nucleoside.

Characterization of the potential role for RNA-binding protein FUS/TLS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed RNA-binding protein of the hnRNP family, that has been discovered as fused to transcription factors, through chromosomal translocations, in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis (ALS) [1]. To date, FUS/TLS has been implicated in a variety of cellular processes such as gene expression control, transcriptional regulation, pre-mRNA splicing and miRNA processing [2]. In addition, some evidences link FUS/TLS to genome stability control and DNA damage response. In fact, mice lacking FUS/TLS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and in response to double-strand breaks, FUS/TLS gets phosphorylated by the protein kinase ATM [3,4,5]. Furthermore, the inducible depletion of FUS/TLS in a neuroblastoma cell line (SH-SY5Y FUS/TLS TET-off iKD) subjected to genotoxic stress (IR) resulted in an increased phosphorylation of γH2AX respect to control cells, suggesting an higher activation of the DNA damage response. The study aims to investigate the specific role of FUS/TLS in DNA damage response through the characterization of the proteomic profile of SH-SY5Y FUS/TLS iKD cells subjected to DNA damage stress, by mass spectrometry-based quantitative proteomics (e.g. SILAC). Preliminary results of mass spectrometric identification of FUS/TLS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS/TLS protein, highlighted the interactions with several proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS/TLS is involved in this pathway, even thou its exact role still need to be addressed.

Characterization of the potential role for RNA-binding protein FUS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed protein of the hnRNP family, that has been discovered as fused to transcription factors in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis [Vance C. et al., 2009]. FUS is a 53 kDa nuclear protein that contains structural domains, such as a RNA Recognition Motif (RRM) and a zinc finger motif, that give to FUS the ability to bind to both RNA and DNA sequences. It has been implicated in a variety of cellular processes, such as pre-mRNA splicing, miRNA processing, gene expression control and transcriptional regulation [Fiesel FC. and Kahle PJ., 2011]. Moreover, some evidences link FUS to genome stability control and DNA damage response: mice lacking FUS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and, in response to double-strand breaks, FUS is phosphorylated by the protein kinase ATM [Kuroda M. et al., 2000; Hicks GG. et al., 2000; Gardiner M. et al., 2008]. Furthermore, preliminary results of mass spectrometric identification of FUS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS protein, highlighted the interactions with proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS is involved in this pathway, even though its role still needs to be clarified. This study aims to investigate the biological roles of FUS in human cells and in particular the putative role in DNA damage response through the characterization of the proteomic profile of the neuroblastoma cell line SH-SY5Y upon FUS inducible depletion, by a quantitative proteomic approach. The SH-SY5Y cell line that will be used in this study expresses, in presence of tetracycline, a shRNA that targets FUS mRNA, leading to FUS protein depletion (SH-SY5Y FUS iKD cells). To quantify changes in proteins expression levels a SILAC strategy (Stable Isotope Labeling by Amino acids in Cell culture) will be conducted on SH-SY5Y FUS iKD cells and a control SH-SY5Y cell line (that expresses a mock shRNA) and the relative changes in proteins levels will be evaluated after five and seven days upon FUS depletion, by nanoliquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) and bioinformatics analysis. Preliminary experiments demonstrated that the SH-SY5Y FUS iKD cells, when subjected to genotoxic stress (high dose of IR), upon inducible depletion of FUS, showed a increased phosphorylation of gH2AX with respect to control cells, suggesting an higher activation of the DNA damage response.

The synthesis and application of a diazirine-modified uridine analogue for investigating RNA-protein interactions

The roles played by many ncRNAs remain largely unknown. Similarly, relatively little is known about the RNA binding proteins involved in processing ncRNA. Identification of new RNA/RNA binding protein (RBP) interactions may pave the way to gain a better understanding of the complex events occurring within cells during gene expression and ncRNA biogenesis. The development of chemical tools for the isolation of RBPs is of paramount importance. In this context, we report on the synthesis of the uridine phosphoramidite U Dz that bears a diazirine moiety on the nucleobase. RNA probes containing U Dz units were irradiated in the presence of single-stranded DNA binding protein (SSB), which is also known to bind ssRNAs, and shown to efficiently (15% yield) and selectively cross-link to the protein. The corresponding diazirine-modified uridine triphosphate U DzTP was synthesized and its capacity to act as a substrate for the T7 RNA polymerase was tested in transcription assays. U DzTP was accepted with a maximum yield of 38% for a 26mer RNA containing a single incorporation and 28% yield for triple consecutive incorporations. Thus, this uridine analogue represents a convenient biochemical tool for the identification of RNA binding proteins and unraveling the role and function played by ncRNAs.

Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations.

We have used three beta-thalassemic mutations, IVS2-654, -705 and -745, that create aberrant 5' splice sites (5' ss) and activate a common cryptic 3' ss further upstream in intron 2 of the human beta-globin gene to optimize a generally applicable exon-skipping strategy using antisense derivatives of U7 small nuclear RNA (snRNA). Introducing a modified U7 snRNA gene carrying an antisense sequence against the cryptic 3' ss into cultured cells expressing the mutant beta-globin genes, restored correct beta-globin mRNA splicing for all three mutations, but the efficiency was much weaker for IVS2-654 than for the other mutations. The length of antisense sequence influenced the efficiency with an optimum of approximately 24 nucleotides. Combining two antisense sequences directed against different target sites in intron 2, either on separate antisense RNAs or, even better, on a single U7 snRNA, significantly enhanced the efficiency of splicing correction. One double-target U7 RNA was expressed on stable transformation resulting in permanent and efficient suppression of the IVS2-654 mutation and production of beta-globin. These results suggest that forcing the aberrant exon into a looped secondary structure may strongly promote its exclusion from the mRNA and that this approach may be used generally to induce exon skipping.