A serological approach to the identification of potato cyst nematodes

The potato cyst nematode species G.rostochiensis and G.pallida are a threat to the cultivation of potatoes. Their presence in the soil embodies a potential financial loss to the farmer either because of harvest reduction, or because of rejection of seed potatoes, and other crops with adhering soil, for certification. Together with crop rotation and the use of nematicides, resistant potato varieties are essential for the control of these parasites. A reliable, simple and quick screening test to characterize and monitor field infestations of potato cyst nematodes according to species should improve possibilities for control, by means of cultivating resistant potato varieties. Both parasites are hard to distinguish, and considerable attention has been paid to the identification of these nematode species by methods other than those based on morphological characteristics. Such methods have mostly been based on biochemical entities, but have not proven suitable for routine applications. In this thesis, the outlines are presented of an immunoassay, which is based on species-differentiating monoclonal antibodies, and which can be used for routine purposes.To raise species-differentiating (monoclonal) antibodies, species-specific antigens must be available. In chapter II the purification of two major groups of heat-stable proteins from homogenates of eggs of G.rostochiensis and G.pallida is described. SDS-polyacrylamide gel electrophoresis revealed two bands specific for G.rostochiensis with apparent molecular weights of 20.7 kD and 18.0 kD, and three bands specific for G.pallida with apparent molecular weights of 21.0 kD, 20.5 kD and 17.0 kD. Two-dimensional gel electrophoresis revealed that actually four thermostable proteins are present in either nematode species, of which two (present in the 17.0 kD and 18.0 kD band respectively) have the same apparent molecular weight but differ in iso-electric point. Conventional antisera made against proteins of either one of the Globodera species were shown to exhibit a strong cross-reactivity with these species-specific proteins.Monoclonal antibodies (McAbs) hold the promise of better differentiating reagents since they recognize a single epitope with a possible reduction in the extent of cross-reactivity. The development of such antibodies using thermostable proteins of either nematode species as an antigen source is described in chapter IV. The antibodies produced by three of the hybridomas (WGP 1, WGP 2 and WGP 3) bind preferentially to thermostable protein antigens of G.pallida while two other hybridomas (WGR 11 and WGR 12) produce antibodies which prefer binding to proteins of G.rostochiensis . Most of the hybridomas which were isolated, however, produce antibodies which bind to thermostable proteins from both potato cyst nematode species. To quantitate the differences in affinity, binding constants were determined (chapter IV) according to the method as described in chapter III. In immunoblotting experiments, it was demonstrated that all G.pallida specific, and some aspecific, McAbs bind to the same two proteins in a mixture of four thermostable proteins from either G.pallida alone, or G.rostochiensis and G.pallida . Besides their very similar physico-chemical characteristics this can be interpreted as another indication that these proteins are homologous. In addition, the reactivity with thermostable proteins from other commonly occurring cyst nematodes was also investigated. Three categories of McAbs could be distinguished: i) WGP 2 and WGP 3, which only bind to thermostable proteins from G.pallida , ii) WGP 1, which binds to proteins of both G.pallida and G.rostochiensis , and iii) all other McAbs, which bind to thermostable proteins of potato cyst, and other cyst nematodes. In chapter V, the topological relationship between the antigenic sites on the G.rostochiensis and G.pallida antigens as defined by six of these McAbs was determined with a competition ELISA.From the McAbs so far characterized, a deliberate choice was made with respect to the development of a routine test for the identification of the potato cyst nematodes G.rostochiensis and G.pallida . In chapter VI, the reactivity of these McAbs in a direct ELISA was predicted and verified with the use of the formerly established binding constants.Thus, the McAbs obtained and characterized in this study establish a basis for the serological identification of the potato cyst nematode species G.rostochiensis and G.pallida by an immunoassay. In chapter VII, the implications of such an assay for the reduction of nematicide applications, and the utilization of resistant potato varieties is discussed

Design of a confocal microfluidic particle sorter using fluorescent photon burst detection

An instrumental system is described for detecting and sorting single fluorescent particles such as microspheres, bacteria, viruses, or even smaller macromolecules in a flowing liquid. The system consists of microfluidic chips (biochips), computer controlled high voltage power supplies, and a fluorescence microscope with confocal optics. The confocal observation volume and detection electro-optics allow measurements of single flowing fluorescent particles. The output of the avalanche photodiode (single photon detector) is coupled to a real-time photon-burst detection device, which output can address the control of high voltage power supplies for sorting purposes. Liquid propulsion systems like electro-osmotic flow and plain electric fields to direct the particles through the observation volume have been tested and evaluated. The detection and real-time sorting of fluorescent microspheres are demonstrated. Applications of these biochips for screening of bacteriophages-type biolibraries are briefly discussed. (C) 2004 American Institute of Physics

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

3D Domain Swapping Causes Extensive Multimerisation of Human Interleukin-10 When Expressed In Planta

Heterologous expression platforms of biopharmaceutical proteins have been significantly improved over the last decade. Further improvement can be established by examining the intrinsic properties of proteins. Interleukin-10 (IL-10) is an anti-inflammatory cytokine with a short half-life that plays an important role in re-establishing immune homeostasis. This homodimeric protein of 36 kDa has significant therapeutic potential to treat inflammatory and autoimmune diseases. In this study we show that the major production bottleneck of human IL-10 is not protein instability as previously suggested, but extensive multimerisation due to its intrinsic 3D domain swapping characteristic. Extensive multimerisation of human IL-10 could be visualised as granules in planta. On the other hand, mouse IL-10 hardly multimerised, which could be largely attributed to its glycosylation. By introducing a short glycine-serine-linker between the fourth and fifth alpha helix of human IL-10 a stable monomeric form of IL-10 (hIL-10mono) was created that no longer multimerised and increased yield up to 20-fold. However, hIL-10mono no longer had the ability to reduce pro-inflammatory cytokine secretion from lipopolysaccharide-stimulated macrophages. Forcing dimerisation restored biological activity. This was achieved by fusing human IL-10mono to the C-terminal end of constant domains 2 and 3 of human immunoglobulin A (Fca), a natural dimer. Stable dimeric forms of IL-10, like Fca-IL-10, may not only be a better format for improved production, but also a more suitable format for medical applications

Unmasking motherhood : a poststructuralist study of motherhood and its meanings for women

The intention of the following paper is to explore from a poststructuralist perspective the reasons why women choose to have children and the consequences resulting from this choice. Interviews were conducted with women who were mothers, and a selection of contemporary feminist literature, some related to motherhood and some to poststructuralist feminist theory, reviewed, in order to develop an understanding of the subject positions available to women in the discourses of motherhood. Two paradoxical and contradictory discourses of motherhood emerge, which are described in the concluding chapter of the paper

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

Beta-hexosaminidases along the secretory pathway of nicotiana benthamiana have distinct specificities toward engineered helminth N-glycans on recombinant glycoproteins

Secretions of parasitic worms (helminths) contain a wide collection of immunomodulatory glycoproteins with the potential to treat inflammatory disorders, like autoimmune diseases. Yet, the identification of single molecules that can be developed into novel biopharmaceuticals is hampered by the limited availability of native parasite-derived proteins. Recently, pioneering work has shown that helminth glycoproteins can be produced transiently in Nicotiana benthamiana plants while simultaneously mimicking their native helminth N-glycan composition by co-expression of desired glycosyltransferases. However, efficient "helminthization" of N-glycans in plants by glyco-engineering seems to be hampered by the undesired truncation of complex N-glycans by beta-N-acetyl-hexosaminidases, in particular when aiming for the synthesis of N-glycans with antennary GalNAc beta 1-4GlcNAc (LacdiNAc or LDN). In this study, we cloned novel beta-hexosaminidase open reading frames from N. benthamiana and characterized the biochemical activity of these enzymes. We identified HEXO2 and HEXO3 as enzymes responsible for the cleavage of antennary GalNAc residues of N-glycans on the model helminth glycoprotein kappa-5. Furthermore, we reveal that each member of the HEXO family has a distinct specificity for N-glycan substrates, where HEXO2 has strict beta-galactosaminidase activity, whereas HEXO3 cleaves both GlcNAc and GalNAc. The identification of HEXO2 and HEXO3 as major targets for LDN cleavage will enable a targeted genome editing approach to reduce undesired processing of these N-glycans. Effective knockout of these enzymes could allow the production of therapeutically relevant glycoproteins with tailor-made helminth N-glycans in plants.Host-parasite interactio

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

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<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