Non-conserved framework surface residues, which were not deemed too close to CDRs were replaced, but experimental analyses revealed that of these, 6 positions in Vl and 10 positions in Vh substantially affected antibody interaction with the antigen. For that reason a compromise between potential immunogenicity and retained binding specificity had to be made. Finally, 13 murine residues were replaced in the moScFv V5B2 to prepare a huScFv V5B2, which retained the significant ability to discriminate between CJD-affected and normal brain tissue. Nevertheless, the amino acid sequence of huScFv V5B2 shows high similarity with the human heavy chain subgroup III and light chain k subgroup I consensus sequences. In the process of humanization a computer model of antibody variable domains is often built to help design the humanized form. It is used for prediction of possible influence of mutations on CDR conformations, which usually results in loss of antibody binding BAY 73-4506 affinity or even specificity. Several reports showed that analysis of a computer model actually helped to avoid problems with the affinity reduction, which is particularly typical for CD-grafting. Moreover, it was demonstrated that during humanization antibody affinity could even be improved when the humanized form is carefully designed on the basis of a precise analysis of structural models. In our case, the structural model of the Fv V5B2 helped to determine framework surface residues of the V5B2, but it did not predict negative impact of several replacements we have introduced. It was however reasonable to expect that not all planned mutations could be introduced into variable domains without disturbing the structure of the antigen-binding site and influencing the binding. For that reason, several intermediate variants were prepared and tested for antigen-binding activity. A few resurfaced scFvs have been reported in the literature, generally containing from six to ten replacements. Any additional mutation usually resulted in reduced binding activity. It was also shown that even a single mutation in the antibody framework can improve or reduce the expression yield or binding affinity of a scFv tremendously. In our experiments, Western blot analyses indicated that humanized V5B2 scFv recognized the same epitope on PrP as the parent V5B2 mAb. When huScFv was assayed by IHC, it labelled less kuru plaque-like PrPSc deposits than V5B2 and failed to label the synaptic pattern of PrPSc deposition, which was clearly visualized by whole V5B2 mAb. This observation was attributed to expected reduced affinity of scFvs that hindered the detection of fine synaptic pattern and small plaques. Our IHC experiment clearly demonstrated that both murine and humanized form of scFv retained the ability to label PrPSc deposits specifically, although less potently, which is in agreement with results obtained by ELISA and immunoblotting. Even though several antibodies have been resurfaced in the last decade, their immunogenicity remains undetermined.

It was shown that it distinguishes between brain tissue samples from CJD – affected and nonCJD-affected patients reacting only with the native PrPSc deposits in immunohistochemical assays. Because of these properties, it has a great potential to be used in immunodiagnostic procedures or prion research. It was already used to induce anti-idiotypic response in mice and chicken in order to produce anti-idiotypic antibodies, which represented a new experimental approach in prion research. To better understand the narrow selectivity of V5B2, interaction between the antibody and P1 peptide was investigated, but only the most recent studies revealed that V5B2 selectively recognizes the newly discovered C-terminally truncated PrP, named PrP226*. To prepare a more suitable form of V5B2 for further immunotherapeutic development, recombinant single-chain antibody fragments have been derived from the mAb. However, murine antibody fragments are immunogenic, which is the major obstacle to their clinical application. For that reason, we decided to reduce its immunogenicity by humanization. The present data describe the humanization of the antibody single-chain fragment V5B2 and its characterization. To our knowledge, this is the first report of an anti-PrP mAb being humanized. We rationally designed four variants of humanized scFvs V5B2 by resurfacing of variable regions guided by computer modelling. By site-directed mutagenesis, human amino acid residues were stepwise introduced into murine variable regions. After being produced in E. coli using pMD204 expression vector, humanized antibody fragments were purified from the periplasm and their antigen-binding LEE011 molecular weight properties were analysed. The optimized construct was a scFv with 13 mutations introduced at key positions in the structure, which retained stability, binding specificity and affinity of the parent antibody. We believe that the recombinant humanized scFv with preserved functional properties of V5B2 could be used for designing new compounds with potentially diagnostic and therapeutic anti-prion properties. Single-chain Fvs are much smaller than whole antibodies, besides they do not require glycosylation and can be produced in a bacterial expression system. Such production is simpler, faster and significantly reduces production costs. Therefore, instead of full-length V5B2 antibody the scFv form was chosen to be humanized and produced. We have successfully humanized a single-chain antibody fragment of the murine monoclonal antibody V5B2, specific for PrPScfragment associated with prion-infected samples. We constructed over 30 scFvs on the DNA level and expressed, purified and examined a total of 14 humanized constructs: 4 for basic stages and 10 intermediates between different stages in search of destabilizing mutations. Construct with optimal binding properties and maximal possible amino acid replacements was denoted huScFv V5B2.

Regarding the H1N1pdm virus, it has been shown that prolonged virus shedding in cancer patients may occur, although such a phenomenon has only been documented through cases involving a single studied patient. Here, we prospectively and systematically collected information from a cohort of hospitalized cancer patients with severe H1N1pdm infections. These patients presented high mortality, prolonged viral shedding and H1N1pdm evolution without the emergence of oseltamivir resistance. This is the first study to address viral shedding and resistance in cancer patients with H1N1pdm infections; thus, it may provide insight into the role of cancer patients as potential human reservoirs for this pandemic virus. Unlike previous reports, our population was composed of hospitalized, severely immunocompromised cancer patients. Most of them were young, had hematologic malignancies and received chemotherapy and systemic steroids in the weeks that preceded the H1N1pdm infection. The patients were treated with oseltamivir in the early course of the infection. A total of 13 patients required intensive care and presented severe respiratory distress. In these patients, the mortality rates were higher than those observed for general ICU patients suffering from H1N1pdm infections as well as for non-critically ill cancer patients. Interestingly, during the influenza season, 14 patients with febrile neutropenia were identified as H1N1pdm cases, a condition that is not usually investigated in this scenario. However, febrile neutropenic cancer patients have an increased risk of developing respiratory distress and multi-organ failure. Therefore, screening for respiratory viruses and prompt initiation of oseltamivir treatment should be considered in these patients. Febrile neutropenia indicates a poor prognostic with respect to a patient’s outcome, but neutropenia duration in our cohort of patients was less than seven days. Thus, prolonged viral shedding might not have a correlation with neutropenia. We observed the persistence of H1N1pdm in 5 out of 10 patients studied for this purpose. In these individuals, viral shedding continued for at least 11 days, despite the use of oseltamivir. The median duration of viral shedding in our population was 23 days, and two pediatric patients with acute lymphoblastic leukemia showed even longer virus secretions, although it is TH-302 difficult to determine whether viral persistence was due to cancer per se or to acute lung injury and mechanical ventilation. Influenza shedding is not considered to last long, and it disappears seven days after the onset. Studies aimed at monitoring 2009 H1N1pdm virus shedding using randomized trials with appropriate controls, such as outpatients and hospitalized or immunocompromised.

Of interest, it has been shown that the expression of a nucleolin-related protein was moderately up-regulated following phagocytosis of Mycobaterium paratuberculosis, suggesting that nucleolin could be involved in phagocytosis. One remarkable characteristic of nucleolin is that it shuttles constantly between the nucleus and the cytoplasm and additionally serves in some cell types as a cell surface receptor. Nucleolin has been also described to be present in the LY2157299 TGF-beta inhibitor endoplasmic reticulum. Thus, nucleolin could play a role in the dynamic network of membrane compartments. In conclusion, we had previously demonstrated that nucleolin, when localized on the cell surface of human monocytes, factor Tu of LVS. We herein confirm the importance of nucleolin expression for LVS binding and its specificity as nucleolin is not involved in binding of another intracellular pathogen as L. monocytogenes or an inert particle. We also demonstrate that association of nucleolin with F. tularensis during infection continues intracellularly after endocytosis of the bacteria. The co-localization of nucleolin, with bacteria and LAMP-1, in the phagosomes suggests that nucleolin shuttles with F. tularensis during the early stages of infection. The dissociation of nucleolin from LVS seems to parallel bacterial release from the phagosomes, as iglC bacteria and inert particles remain associated with nucleolin. Recent studies have identified some mammalian host factors required for modulation of phagosome biogenesis and intracellular proliferation of F. tularensis within the cytosol. It remains to be seen whether nucleolin plays an active role at the latter stages of F. tularensis infection. Emerging data on the clinical course of severe H1N1pdm infection have allowed the identification of high-risk groups, which include pregnant women and patients with morbid obesity. However, an analysis of the impact of this novel virus in a highly susceptible population, such as cancer patients, through clinical and virological perspectives, needs to be highlighted. The atypical clinical presentation of influenza infections in cancer patients, which delays clinical suspicion, antiviral treatment and adequate prevention of viral transmission, is a major challenge for clinical management in this population. Cancer patients are more likely to suffer from severe seasonal influenza infections and prolonged viral shedding, as has been reported for an H3N2 seasonal virus. Prolonged shedding and the development of oseltamivir resistance in cancer patients infected with the H1N1pdm virus have not been thoroughly evaluated. Data on these aspects could have major implications for the clinical management and infection control practices for H1N1pdm-infected cancer patients.

We described also a procedure for selecting a partition among a set of candidate ones and a measure of cluster reliability. Note that our definition of the likelihood of a partition is not an absolute measure of the “goodness” of the partition, but expresses how much a partition is similar to all the generated partitions and is of use for assessing cluster reliability. In principle, the TBC might be applied also to next-generation sequencing data alignments, and the PLX-4720 sequence length should not be a serious problem if Roche 454 Life Science technology is used to amplify specific regions. With shotgun sequencing the data need to be analysed via sliding windows, and then a problem of variant reconstruction arises. We do not know how the TBC would behave in presence of sequencing errors that should be -at least- corrected before running the clustering. However, another approach that uses the Chinese restaurant process as a prior to infer clusters via Gibbs’ sampling has been recently proposed, and performs both clustering and error correction at the same time. TBC was also evaluated in the problem of transmission event identification. Using a data set of patients followed up at the Catholic University of Sacred Heart in Rome, Italy, with known transmission history, TBC was able to identify transmission events in 25% of cases, whilst CTree assessed on 16.7%. The transmission event dataset was also evaluated using a previously published method, specifically tuned for HIV transmission cluster identification, and that method identified 25% of transmission events. With a human-visual evaluation of subtrees and node reliability of a maximum-likelihood phylogenetic tree, we were able to infer correctly 50% of transmission events. Thus, even a detailed phylogenetic analysis was not able to resolve all transmission events. In fact, for HIV it has been shown previously that many factors can limit the concordance of phylogenetic reconstruction and the reported epidemiological evidence. The transmission event data set of CUSH was composed by sequence samples of patients taken at different times and disease stage: some patients were sequenced multiple times either before treatment initiation or at treatment failures, whilst others had only one sequence sample taken. We recognise that a larger and less sparse data set would be desirable in order to assess better the TBC performance on this particular problem. A way to optimise the percentile threshold -without knowing a priori the sequence grouping is to run the TBC using different thresholds and then calculate for each partition a cluster validity measure, such as the Goodman-Kruskal index, the Dunn’s index, or the average silhouette value.