Phage display-selected single chain antibodies confer high levels of resistance against Tomato spotted wilt virus

Rational design of antibodies targeting essential viral proteins can complement the palette of antiviral resistance strategies. Here, stable and high expression of single-chain monoclonal antibodies targeting the nucleoprotein of the economically important plant virus Tomato spotted wilt virus, a protein that is involved in multiple steps in the viral infection cycle, is reported. High cytoplasmic expression levels of three selected phage display-derived anti-viral single-chain antibodies were established. Of these antibodies, two led to high levels of resistance against this plant virus. Protoplast experiments provided evidence that the two resistance-conferring antibodies may have a different mode of action and could be combined for higher durability of resistance in the fiel

Epitope identification and in silico prediction of the specificity of antibodies binding to the coat proteins of Potato Virus Y strains

A phage library containing 2.7 × 10(9) randomly expressed peptides was used to determine the epitopes of three monoclonal antibodies that bind to the coat protein of Potato Virus Y. Construction of the consensus sequences for the peptides obtained after three selection rounds indicated that each antibody recognized a different epitope located within the first 50 N-terminal amino acids of the coat protein. The location of the epitopes was confirmed by heterologous expression of the N-terminal part of the coat protein in Escherichia coli, and, subsequently, by performing an immunological test with the three antibodies. The accuracy of the phage library was demonstrated by predicting in silico the cross-reactivity of the three antibodies with other potyvirus family members. ELISA and in silico predictions revealed the same results in almost every case. The potential of peptide phage libraries to optimize the use of antibodies in plant virology is discusse

Functional characterization of Schistosoma mansoni fucosyltransferases in Nicotiana benthamiana plants

Helminth parasites secrete a wide variety of immunomodulatory proteins and lipids to dampen host immune responses. Many of these immunomodulatory compounds are modified with complex sugar structures (or glycans), which play an important role at the host–parasite interface. As an example, the human blood fluke Schistosoma mansoni produces highly fucosylated glycan structures on glycoproteins and glycolipids. Up to 20 different S. mansoni fucosyltransferase (SmFucT) genes can be found in genome databases, but thus far only one enzyme has been functionally characterized. To unravel the synthesis of highly fucosylated N-glycans by S. mansoni, we examined the ability of ten selected SmFucTs to modify N-glycans upon transient expression in Nicotiana benthamiana plants. All enzymes were localized in the plant Golgi apparatus, which allowed us to identify the SmFucTs involved in core fucosylation and the synthesis of complex antennary glycan motifs. This knowledge provides a starting point for investigations into the role of specific fucosylated glycan motifs of schistosomes in parasite-host interactions. The functionally characterized SmFucTs can also be applied to synthesize complex N-glycan structures on recombinant proteins to study their contribution to immunomodulation. Furthermore, this plant expression system will fuel the development of helminth glycoproteins for pharmaceutical applications or novel anti-helminth vaccines

Comparison of chop chemotherapy with autologous bone marrow transplantation for slowly responding patients with aggressive non-Hodgkin's lymphoma

High-dose chemoradiotherapy combined with autologous bone marrow transplantation can cure patients with disseminated, aggressive non-Hodgkin's lymphoma in whom first-line chemotherapy has failed. In contrast, cure is rare with second-line chemotherapy. It has been suggested that patients with slow responses to the initial phase of first-line chemotherapy are at high risk for relapse. Therefore, such patients are potential candidates for early bone marrow transplantation

Intestinal Damage Determines the Inflammatory Response and Early Complications in Patients Receiving Conditioning for a Stem Cell Transplantation

Contains fulltext : 87954.pdf (publisher's version ) (Open Access)BACKGROUND: Stem cell transplantation (SCT) is still complicated by the occurrence of fever and inflammatory complications attributed to neutropenia and subsequent infectious complications. The role of mucosal barrier injury (MBI) of the intestinal tract therein has received little attention. METHODS: We performed a retrospective analysis in 163 SCT recipients of which data had been collected prospectively on intestinal damage (citrulline), inflammation (C-reactive protein), and neutrophil count. Six different conditioning regimens were studied; 5 myeloablative (MA) and 1 non-myeloablative (NMA). Linear mixed model multivariate and AUC analyses were used to define the role of intestinal damage in post-SCT inflammation. We also studied the relationship between the degree of intestinal damage and the occurrence of early post-SCT complications. RESULTS: In the 5 MA regimen there was a striking pattern of inflammatory response that coincided with the occurrence of severe intestinal damage. This contrasted with a modest inflammatory response seen in the NMA regimen in which intestinal damage was limited. With linear mixed model analysis the degree of intestinal damage was shown the most important determinant of the inflammatory response, and both neutropenia and bacteremia had only a minor impact. AUC analysis revealed a strong correlation between citrulline and CRP (Pearson correlation r = 0.96). Intestinal damage was associated with the occurrence of bacteremia and acute lung injury, and influenced the kinetics of acute graft-versus-host disease. CONCLUSION: The degree of intestinal damage after myeloablative conditioning appeared to be the most important determined the inflammatory response following SCT, and was associated with inflammatory complications. Studies should explore ways to ameliorate cytotoxic therapy-induced intestinal damage in order to reduce complications associated with myeloablative conditioning therapy

No improvement of survival with reduced- versus high-intensity conditioning for allogeneic stem cell transplants in Ewing tumor patients

Background: Outcomes of Ewing tumor (ET) patients treated with allogeneic stem cell transplantation (allo-SCT) were compared regarding the use of reduced-intensity conditioning (RIC) and high-intensity conditioning (HIC) regimens as well as human leukocyte antigen (HLA)-matched and HLA-mismatched grafts. Patients and methods: We retrospectively analyzed data of 87 ET patients from the European Group for Blood and Marrow Transplantation, Pediatric Registry for Stem Cell Transplantations, Asia Pacific Blood and Marrow Transplantation and MetaEICESS registries treated with allo-SCT. Fifty patients received RIC (group A) and 37 patients received HIC (group B). Twenty-four patients received HLA-mismatched grafts and 63 received HLA-matched grafts. Results: Median overall survival was 7.9 months [±1.24, 95% confidence interval (CI) 5.44-10.31] for group A and 4.4 months (±1.06, 95% CI 2.29-6.43) for group B patients (P = 1.3). Death of complications (DOC) occurred in 4 of 50 (0.08) and death of disease (DOD) in 33 of 50 (0.66) group A and in 16 of 37 (0.43) and 17 of 37 (0.46) group B patients, respectively. DOC incidence was decreased (P < 0.01) and DOD/relapse increased (P < 0.01) in group A compared with group B. HLA mismatch was not generally associated with graft-versus-Ewing tumor effect (GvETE). Conclusions: There was no improvement of survival with RIC compared with HIC due to increased DOD/relapse incidence after RIC despite less DOC incidence. This implicates general absence of a clinically relevant GvETE with current protocol

Prognostic impact of progression to induction chemotherapy and prior paclitaxel therapy in patients with germ cell tumors receiving salvage high-dose chemotherapy in the last 10 years: A study of the European Society for Blood and Marrow Transplantation Solid Tumors Working Party

Little is known about the prognostic impact of prior paclitaxel therapy and response to induction chemotherapy defined as the regimen preceding high-dose chemotherapy (HDCT) for the salvage therapy of advanced germ cell tumors. Twenty European Society for Blood and Marrow Transplantation centers contributed data on patients treated between 2002 and 2012. Paclitaxel used in either prior lines of therapy or in induction-mobilization regimens was considered. Multivariable Cox analyses of prespecified factors were undertaken on PFS and overall survival (OS). As of October 2013, data for 324 patients had been contributed to this study. One hundred and ninety-two patients (59.3%) had received paclitaxel. Sixty-one patients (19%) had a progression to induction chemotherapy, 234 (72%) a response (29 (9%) missing or granulocyte colony-stimulating factor without chemotherapy). Both progression to induction chemotherapy and prior paclitaxel were significantly associated with shorter OS univariably (P&lt;0.001 and P=0.032). On multivariable analysis from the model with fully available data (N=216) progression to induction was significantly prognostic for PFS and OS (P=0.003), but prior paclitaxel was not (P=0.674 and P=0.739). These results were confirmed after multiple imputation of missing data. Progression to induction chemotherapy could be demonstrated as an independent prognostic factor, in contrast to prior paclitaxel

Carfilzomib and dexamethasone versus bortezomib and dexamethasone for patients with relapsed or refractory multiple myeloma (ENDEAVOR): And randomised, phase 3, open-label, multicentre study

Background: Bortezomib with dexamethasone is a standard treatment option for relapsed or refractory multiple myeloma. Carfilzomib with dexamethasone has shown promising activity in patients in this disease setting. The aim of this study was to compare the combination of carfilzomib and dexamethasone with bortezomib and dexamethasone in patients with relapsed or refractory multiple myeloma. Methods: In this randomised, phase 3, open-label, multicentre study, patients with relapsed or refractory multiple myeloma who had one to three previous treatments were randomly assigned (1:1) using a blocked randomisation scheme (block size of four) to receive carfilzomib with dexamethasone (carfilzomib group) or bortezomib with dexamethasone (bortezomib group). Randomisation was stratified by previous proteasome inhibitor therapy, previous lines of treatment, International Staging System stage, and planned route of bortezomib administration if randomly assigned to bortezomib with dexamethasone. Patients received treatment until progression with carfilzomib (20 mg/m2 on days 1 and 2 of cycle 1; 56 mg/m2 thereafter; 30 min intravenous infusion) and dexamethasone (20 mg oral or intravenous infusion) or bortezomib (1·3 mg/m2; intravenous bolus or subcutaneous injection) and dexamethasone (20 mg oral or intravenous infusion). The primary endpoint was progression-free survival in the intention-to-treat population. All participants who received at least one dose of study drug were included in the safety analyses. The study is ongoing but not enrolling participants; results for the interim analysis of the primary endpoint are presented. The trial is registered at ClinicalTrials.gov, number NCT01568866. Findings: Between June 20, 2012, and June 30, 2014, 929 patients were randomly assigned (464 to the carfilzomib group; 465 to the bortezomib group). Median follow-up was 11·9 months (IQR 9·3-16·1) in the carfilzomib group and 11·1 months (8·2-14·3) in the bortezomib group. Median progression-free survival was 18·7 months (95% CI 15·6-not estimable) in the carfilzomib group versus 9·4 months (8·4-10·4) in the bortezomib group at a preplanned interim analysis (hazard ratio [HR] 0·53 [95% CI 0·44-0·65]; p<0·0001). On-study death due to adverse events occurred in 18 (4%) of 464 patients in the carfilzomib group and in 16 (3%) of 465 patients in the bortezomib group. Serious adverse events were reported in 224 (48%) of 463 patients in the carfilzomib group and in 162 (36%) of 456 patients in the bortezomib group. The most frequent grade 3 or higher adverse events were anaemia (67 [14%] of 463 patients in the carfilzomib group vs 45 [10%] of 456 patients in the bortezomib group), hypertension (41 [9%] vs 12 [3%]), thrombocytopenia (39 [8%] vs 43 [9%]), and pneumonia (32 [7%] vs 36 [8%]). Interpretation: For patients with relapsed or refractory multiple myeloma, carfilzomib with dexamethasone could be considered in cases in which bortezomib with dexamethasone is a potential treatment option. Funding: Onyx Pharmaceuticals, Inc., an Amgen subsidiary

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery