Miriam Ossevoort

Dendritic cells as carriers for a cytotoxic T-lymphocyte epitope-based peptide vaccine in protection against a human papillomavirus type 16-induced tumor

Previously we have demonstrated that two doses of a cytotoxic T lymphocyte (CTL) epitope-based peptide vaccine of human papillomavirus type 16 (HPV 16) E7 aa 49-57 elicit protection against outgrowth of HPV 16-transformed tumor cells (C3 cells) in B6 mice. Incomplete Freunds adjuvant (IFA), as a carrier, was used to induce this response. To avoid side effects caused by the use of external adjuvants, we have now investigated the effectiveness of highly purified spleen dendritic cells (DC) that efficiently induce primary peptide-specific CTL responses in vitro, as physiological carriers for the HPV 16 E7(49-57) peptide-based vaccine. This is the first report demonstrating that mice immunized once i.v. with syngeneic spleen DCs pulsed with the HPV 16 E7(49-57) peptide in vitro were protected against the outgrowth of C3 tumor cells. In comparison, a single injection of the HPV 16 E7(49-57) peptide in IFA s.c. also resulted in effective induction of tumor-specific immunity in vivo. In both immunization protocols, protective tumor-specific immunity was mediated by CTL that recognized HPV 16 E7(49-57) peptide-pulsed target cells, as well as C3 cells in vitro. Peptide affinity of the CTL induced by both protocols was similar. Thus under the conditions tested, a single injection of spleen DCs pulsed with a CTL epitope-based peptide in vitro elicited tumor-antigen-specific CTL in vivo, which protected mice against a subsequent tumor inoculation. This result indicates that spleen DCs pulsed with a CTL epitope can effectively serve as a tumor-specific vaccine.

The influence of student characteristics on the use of adaptive e-learning material

Adaptive e-learning materials can help teachers to educate heterogeneous student groups. This study provides empirical data about the way academic students differ in their learning when using adaptive e-learning materials. Ninety-four students participated in the study. We determined characteristics in a heterogeneous student group by collecting demographic data and measuring motivation and prior knowledge. We also measured the learning paths students followed and learning strategies they used when working with adaptive e-learning material in a molecular biology course. We then combined these data to study if and how student characteristics relate to the learning paths and strategies they used. We observed that students did follow different learning paths. Gender did not have an effect, but (mainly Dutch) BSc students differed from (international) MSc students in the intrinsic motivation they had and the learning paths and strategies they followed when using the adaptive e-learning material. Highlights? Adaptive e-learning materials can help teachers to educate heterogeneous student groups. ? Students differ in the learning paths they follow and strategies they use when working with adaptive e-learning materials. ? University graduate and undergraduate students participated in the study. ? Results from traces, test scores, intrinsic motivation inventory responses and student self-reports were analysed.

A rapid isolation procedure for dendritic cells from mouse spleen by centrifugal elutriation

The standard isolation procedure for antigen presenting dendritic cells (DC) takes 2 days and includes selective adherence to tissue culture plates which may lead to the activation of these cells. This report describes the isolation of DC by centrifugal elutriation (CE). Murine spleen cells were separated on the basis of size and density into 7 CE fractions. This method took 90 min. Cells from each CE fraction were characterized by fluorescence activated cell sorter (FACS) analysis and their antigen presenting cell (APC) activity was determined by a secondary Sendai virus specific T cell proliferation assay. CE fraction 5 contained most of the DC with a concentration of 6-10%, representing an approximately 15-fold enrichment compared to unseparated spleen cells (< 1% DC). This CE fraction also exhibited the highest APC activity, which was almost completely abolished after depletion of DC by treatment with monoclonal antibody 33D1 (DC-marker) and complement. Further enrichment of CE fraction 5 by discontinuous density gradient centrifugation resulted in a cell population containing 35-55% 33D1-positive cells with similar characteristics as DC isolated by the standard procedure, such as the capacity to induce a primary viral peptide specific CTL response. Two-color FACS analysis showed an increase in MHC expression on 33D1-positive cells of CE fraction 5 after 18 h culture involving cell adhesion to a similar level as the MHC expression on DC isolated by the standard procedure. During this same period their morphology changed from a round to a dendritic appearance. In conclusion, our results indicate that CE is well suited for isolating DC more rapidly and without activation of these cells by adherence, a process which readily occurs in the standard isolation procedure.

A Teaching Strategy with a Focus on Argumentation to Improve Undergraduate Students' Ability to Read Research Articles.

This article describes a teaching strategy for first-year undergraduate life sciences students at a research university, in which they learn to read authentic research articles by focusing on rhetorical moves that play an important role in the authors argument. We used cognitive apprenticeship as the pedagogical approach.

First Experiences with Reading Primary Literature by Undergraduate Life Science Students

Learning to read and understand research articles (primary literature) is an important step in the enculturation of higher education students into the scientific community. We presume, based on ideas from the field of genre analysis, that it is important for the development of reading skills to become conscious of the rhetorical structures in research articles. So, we determined how well science students are able to identify 2 important elements of this rhetorical structure: conclusions and grounds. First-year undergraduate life science students who followed a course called ‘Biomedical Research’ made assignments in which they had to identify these 2 elements. We analysed the answers of 20 students in detail and compared their answers with 2 expert readers. Furthermore, we conducted task-based interviews with 4 students to gain more insight into their reading strategies and to determine how they identify conclusions and grounds. Our results show that students and experts defined conclusions and grounds in different ways. Students and experts agreed on the most important conclusion of the articles. However, students identified a wide range of sentences which were not seen as conclusions by the experts. The grounds students mentioned mostly matched their conclusions. Students sometimes failed to mention important grounds for a particular conclusion. In conclusion, our study shows the differences between student and expert readers of primary literature. Based on our results, we formulated criteria for the design of a teaching strategy that aims to improve students skills for reading primary literature.

A web-based adaptive tutor to teach PCR primer design

When students have varying prior knowledge, personalized instruction is desirable. One way to personalize instruction is by using adaptive e‐learning to offer training of varying complexity. In this study, we developed a web‐based adaptive tutor to teach PCR primer design: the PCR Tutor. We used part of the Taxonomy of Educational Objectives (the three cognitive processes: remember, understand, and apply) to design exercises of varying complexity. Using this method, we demonstrated that we were able to systematically categorize exercises. There was also a good learning effect and a positive student perception when using the PCR Tutor.

Peptide specificity of alloreactive CD4 positive T lymphocytes directed against a major histocompatibility complex class I disparity

The mouse strains C57BL/6 (B6, H2b) and Kbm1 mutant bm1 have a defined difference of three amino acids at position 152, 155, and 156 in the MHC class I K molecule. This causes a change in the side and the bottom of the antigen presenting groove of the K molecule resulting in strong allogeneic responses in vitro and in vivo. Here we report on the peptide specificity of CD4+ T cells of B6 origin directed against the Kbm1 mutant and speculate on the peptide specificity of CD8+ bm1-specific T lymphocytes of B6 origin. Bm1-specific CD4+ T helper cells recognized a peptide derived from the Kbm1 molecule encompassing the three mutations, presented by MHC class II molecules on syngeneic cells. The ability of this peptide to bind to MHC class II resulted from amino acid mutations at positions 155 and 156. Furthermore, the recognition of the natural peptide derived from the Kbm1 molecule presented by MHC class II I-Ab molecules on cells of bml origin could be blocked by addition of an MHC class II I-Ab binding competitor peptide. Thus, due to the mutations in an MHC class I molecule, indirect presentation via MHC class II molecules and MHC class II-restricted recognition of a peptide derived from such a MHC class I molecule is demonstrable.

Functional and Ultrastructural Aspects of Antigen Processing by Dendritic Cells

Major histocompatibility (MHC) class II molecules present peptides derived from exogenous antigens to CD4+ T lymphocytes (reviewed 1,2,3). The MHC class II molecule is a heterodimer of two transmembrane subunits, an α chain (33 kDa) and β chain (29 kDa), both encoded in the MHC region4. The ends of the peptide-binding groove of MHC class II molecules are open so peptides can extend out. As a result, MHC class II-associated peptides have a length varying between 12–24 amino acids residues5. MHC class II molecules are primarily expressed on antigen presenting cells (APC), such as B cells, macrophages and dendritic cells (DC).

Scientific Argumentation Model (SAM): A Heuristic for Reading Research Articles by Science Students

Research articles are the typical means scientists use for publishing their scientific results. Therefore, it is important that science students acquire genre knowledge about research articles. This will not only help them with reading science texts but will also provide them with knowledge about the way scientists obtain scientific findings. However, studies have shown that students have difficulties with reading original scientific texts. To support students in acquiring this skill, we have developed a model, the Scientific Argumentation Model (SAM), which can be used as a heuristic in secondary or higher education. This model is based on ideas from argumentation theory and genre analysis and consists of descriptions of seven rhetorical moves that play an important role in a research article’s argumentation: motive, objective, support, counterargument, refutation, main conclusion, and implication. The relations between these moves are depicted in an argumentation scheme. In this study, SAM was validated by investigating its use on research articles from astronomy and biomedical science. The average frequencies of motives, main conclusions, implications, and support chains seem somewhat higher in astronomy papers than in biomedical papers. This might be explained by the different natures of these two disciplines.

The MHC Expression of Dendritic Cells from Mouse Spleen Isolated by Centrifugal Elutriation is Upregulated During Short Term Culture

Dendritic cells (DC) are extremely potent antigen presenting cells (APC), which can even induce an anti-viral specific primary T cell response in vitro (Macatonia et al, 1989). Both the quantitatively and qualitatively (fewer sialic acids) superior MHC expression and the large surface area of DC with long dendritic projections possibly allow a better interaction of the MHC-peptide complex on the DC with the T cell receptor (Boog et al., 1989). Generally, fewer DC than other types of APC are required to induce a specific T cell response (Kast et al., 1988).