Phage Display Technology for Human Monoclonal Antibodies.

During the last 20 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection and screening procedures to identify lead candidates. One of the most successful methods is based on the use of filamentous phages. Functional Antibodies (Abs) fragments can be displayed on the surface of phages by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridoma technology can be isolated by a series of cycles of selection on the antigen of interest. In this system, antibody genes can be recovered simultaneously with selection and can be easily further engineered, for example by increasing their affinity to levels unobtainable in the immune system, or by modulating their specificity and their effector functions (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction and selection of binder with desired specificity.

Impact of IFN lambda 3/4 single nucleotide polymorphisms on the cytomegalovirus reactivation in autologous stem cell transplant patients.

Cytomegalovirus (CMV) infection represents one of the main cause mortality after Stem Cell Transplantation. Recently, a protective effect of the T allele of rs12979860 IL28B Single Nucleotide Polymorphisms (SNPs) against CMV infection in the allogenic stem cell transplantation was suggested. We investigate whether the rs12979860 IL28B SNP and the relative rs368234815 (IFNλ4) genotype may affect the incidence of active CMV infection in Autologous stem cell transplantation (Auto-SCT) setting. The study included 99 patients who underwent to Auto-SCT. IL28 and IFNΔ4 SNPs were correlated with CMV reactivation along with other clinical and treatment parameters. CMV reactivation by CMV DNAemia was evaluated once a week until day 100 from Auto-SCT. CMV reactivation was documented in 50% (TT-ΔG/ΔG), 35% (CC-TT/TT) and 29.2% (CT-TT/ΔG) of the patients respectively. No differences in CMV copies number were recorded at reactivation between different IL28/IFNλ4 genotypes. The analysis of patients older than 60 years showed a significantly higher incidence of active CMV infection in the TT-ΔG/ΔG (83%) population with respect to CC-TT/TT (21%) and CT-TT/ΔG (40%) patients. Our data suggest a negative role of TT-ΔG/ΔG genotype in the CMV reactivation in Auto-SCT. The exposure to rituximab and the pre-infusion presence of anti CMV IgG also significantly influenced CMV reactivation.

Regulation of telomere metabolism by the RNA processing protein Xrn1.

Telomeric DNA consists of repetitive G-rich sequences that terminate with a 3΄-ended single stranded overhang (G-tail), which is important for telomere extension by telomerase. Several proteins, including the CST complex, are necessary to maintain telomere structure and length in both yeast and mammals. Emerging evidence indicates that RNA processing factors play critical, yet poorly understood, roles in telomere metabolism. Here, we show that the lack of the RNA processing proteins Xrn1 or Rrp6 partially bypasses the requirement for the CST component Cdc13 in telomere protection by attenuating the activation of the DNA damage checkpoint. Xrn1 is necessary for checkpoint activation upon telomere uncapping because it promotes the generation of single-stranded DNA. Moreover, Xrn1 maintains telomere length by promoting the association of Cdc13 to telomeres independently of ssDNA generation and exerts this function by downregulating the transcript encoding the telomerase inhibitor Rif1. These findings reveal novel roles for RNA processing proteins in the regulation of telomere metabolism with implications for genome stability in eukaryotes.