Cross-presentation of a CMV pp65 epitope by human dendritic cells using bee venom PLA2 as a membrane-binding vector

We have used bee venom phospholipase A2 as a vector to load human dendritic cells ex vivo with a major histocompatibility complex (MHC) class I-restricted epitope fused to its C-terminus. The fusion protein bound to human monocyte-derived dendritic cells and was internalized into early endosomes. In vitro immunization experiments showed that these dendritic cells were able to generate specific CD8 T cell lines against the epitope carried by the fusion protein. Cross-presentation did not require proteasome, transporter associated with antigen processing, or endosome proteases, but required newly synthesized MHC molecules. Comparison of the antigen presentation pathway observed in this study to that followed by other toxins used as vectors is discussed.     

The plant multigenic family of thiol peroxidases

Thiol peroxidases are ubiquitous recently characterized heme-free peroxidases, which catalyze the reduction of peroxynitrites and of various peroxides by catalytic cysteine residues and thiol-containing proteins as reductants. In plants, five different classes can be distinguished, according to the number and the position of conserved catalytic cysteines. Four classes are defined as peroxiredoxins and were already identified by phylogenetic sequence analysis, 1-Cys, 2-Cys, type II, and type Q peroxiredoxins, and the fifth is represented by glutathione peroxidases, which were recently shown to possess a thioredoxin-dependent activity in plants. Since the discovery of peroxiredoxins in plants in 1996, a lot of work has been devoted to the biochemical and functional characterization of the different peroxiredoxin isoforms, but in contrast, few structural data are available. The analysis of the Arabidopsis thaliana genome indicates that at least 17 isoforms of thioredoxin-dependent peroxidases are expressed in various plant compartments. The role of these proteins is discussed in terms of electron donor and substrate specificities and in light of their expression and localization. These enzymes are expressed in many plant tissues and are involved notably in the protection of the photosynthetic apparatus, in the response to various biotic or abiotic stresses by fighting reactive oxygen or nitrogen species and lipid peroxidation.     

Central,but not peripheral application of motilin increases c-Fos expression in hypothalamic nuclei in the rat brain

Previous immunocytochemical studies have shown the presence of motilin-immunoreactive neurons in specific brain areas of rats and autoradiographic studies in rabbits demonstrated motilin-binding sites in the central nervous system as well. Therefore, the aim of this study was to determine the anatomical localisation and neurochemical features of neurons activated by central administration of motilin (Mo) in rats. One week after cannulation, an intracerebroventricular injection of Mo (ICV, 3 g/6 l 0.9% saline) was given. For comparative purposes, a group of animals received an intravenous injection of motilin (IV, 9 g/300 l 0.9% saline) or an equal volume of saline. Neuronal excitation was assessed by c-Fos immunocytochemistry and combined with immunostaining for neurotransmitter markers. In contrast to the IV motilin-treated animals, the ICV motilin-treated animals displayed a significant increase in c-Fos expression in the supraoptic nuclei (SO) and paraventricular nuclei of the hypothalamus (PVH). At the level of the dorsomedial, ventromedial and lateral hypothalamic nuclei, ICV administration of motilin did not induce changes in c-Fos expression. In addition, the cerebellum did not show c-Fos expression after ICV motilin administration either. These findings might suggest distinct pathways and actions of centrally released and systemic motilin, but, particularly in rodents, do not rule out the possibility that the effects seen in the SO and PVH after ICV application are aspecific in nature. At present, we cannot exclude the fact that the results observed with motilin in rodents are due to cross-interaction with other related (e.g. ghrelin) or not yet identified receptors.     

In vitro and ex vivo permissivity of hepatocytes for Leishmania donovani

Using models of ex vivo infection of murine, rat, and human primary hepatocytes by Leishmania donovani, we showed that hepatocytes are permissive for Leishmania at a low level. We then modeled the in vitro infection of a human hepatoma-derived cell line to examine the parasite's capability to proliferate and to cause direct damage to hepatocytes. Results showed that L. donovani can infect hepatocytes, but do not massively proliferate. This slight infection under our experimental conditions resulted in limited damage to hepatocytes. These results bring into question a possible role for hepatocytes as a parasite reservoir during latent infection.     

The influence of the Internet on immunology education

The potential of the Internet as a medium through which to teach basic and applied immunology lies in the ability to illustrate complex concepts in new ways for audiences that are diverse and often geographically dispersed. This article explores two collaborative Internet-based learning projects (also known as e-learning projects) that are under development: Immunology Online, which will present an Internet-based curriculum in basic and clinical immunology to Swiss undergraduate and graduate students across five campuses; and the OCTAVE project, which will offer online training to an international cadre of new investigators, the members of which are carrying out clinical trials of vaccines against HIV infection.     

Reprogrammed genetic decoding in cellular gene expression

Reprogrammed genetic decoding signals in mRNAs productively overwrite the normal decoding rules of translation. These "recoding" signals are associated with sites of programmed ribosomal frameshifting, hopping, termination codon suppression, and the incorporation of the unusual amino acids selenocysteine and pyrrolysine. This review summarizes current knowledge of the structure and function of recoding signals in cellular genes, the biological importance of recoding in gene regulation, and ways to identify new recoded genes.     

KMD

The Keck Microarray Database (KMD) is a port of the ArrayExpress database from Oracle to the MySQL environment. The requirements for a locally available, open-source microarray database solution based on ArrayExpress are analysed in this article. The differences between the Oracle and MySQL environments are identified and the method to port to MySQL is described, providing a unified relational database management system (RDBMS) platform for both MIAMExpress and ArrayExpress. AVAILABILITY: The software and documentation are available from the Keck Graduate Institute of Applied Life Sciences website at http://public.kgi.edu/~jmainguy/applied-bioinformatics.htm.     

Automated, on-line two-dimensional nano liquid chromatography tandem mass spectrometry for rapid analysis of complex protein digests

Evaluation of cellular processes and their changes at the level of protein expression and post-translational modifications may allow identification of novel proteins and the mechanisms involved in pathogenic processes. However, the number of proteins and, after tryptic digestion, of peptides from a single cell can be tremendously high. Separation and analysis of such complex peptide mixtures can be performed using multidimensional separation techniques such as two-dimensional gel electrophoresis or two-dimensional-high-performance liquid chromatography (2-D-HPLC). The aim of this work was to establish a fully automated on-line 2-D-HPLC separation method with column switching for the separation of complex tryptic digests. A model mixture of five proteins as well as a nuclear matrix protein sample were digested with trypsin and separated using a strong cation exchange (SCX) column in the first dimension and nano reversed phase in the second dimension. Separated peptides were detected using an ion trap mass spectrometer. The advantages of this new fully automated method are rapid sample loading, the possibility of injecting large volumes and no introduction of salt into the mass spectrometer. Furthermore, column switching allows the independent control and optimization of the two dimensions independently.     

Gamma interferon-dependent protection of the mouse upper respiratory tract following parenteral immunization with a respiratory syncytial virus G protein fragment

The protective mechanisms induced in the mouse upper respiratory tract (URT) after intraperitoneal immunization with G2Na, a recombinant respiratory syncytial virus (RSV) G protein fragment (amino acid residues 130 to 230), were investigated. This protection was recently shown to be mediated by CD4(+) T cells and to be critically dependent on the cysteines and amino acids 193 and 194 (H. Plotnicky-Gilquin, A. Robert, L. Chevalet, J.-F. Haeuw, A. Beck, J.-Y. Bonnefoy, C. Brandt, C.-A. Siegrist, T. N. Nguyen, and U. F. Power, J. Virol. 74:3455-3463, 2000). On G2Na, we identified a domain (amino acid residues 182 to 198) responsible for the T-helper-cell activity. This region coincided with a peptide designed AICK (residues 184 to 198) which includes the previously identified murine and human T-helper-cell epitope on the native G protein (P. W. Tebbey, M. Hagen, and G. E. Hancock, J. Exp. Med. 188:1967-1972, 1998). Immunization with AICK, in alum or complete Freund's adjuvant, significantly reduced nasal RSV titers in normal BALB/c mice. However, although lung protection was induced, in contrast to the case with live RSV, neither AICK nor G2Na was able to prevent nasal infection in gamma interferon (IFN-gamma)-knockout mice. Anti-IFN-gamma neutralizing antibodies partially inhibited URT protection after administration to G2Na-immunized BALB/c mice. Furthermore, while purified CD4(+) T cells from BALB/c mice immunized with G2Na or AICK significantly reduced lung and nasal infection of naive recipient mice after adoptive transfer, the cells from IFN-gamma-knockout mice had no effect. Together, these results demonstrated for the first time that the T-helper-cell epitope of RSV G protein induces URT protection in mice after parenteral immunization through a Th1-type, IFN-gamma-dependent mechanism.