Rational design of a multiepitope vaccine encoding T-lymphocyte epitopes for treatment of chronic hepatitis B virus infections

Protein sequences from multiple hepatitis B virus (HBV) isolates were analyzed for the presence of amino acid motifs characteristic of cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes with the goal of identifying conserved epitopes suitable for use in a therapeutic vaccine. Specifically, sequences bearing HLA-A1, -A2, -A3, -A24, -B7, and -DR supertype binding motifs were identified, synthesized as peptides, and tested for binding to soluble HLA. The immunogenicity of peptides that bound with moderate to high affinity subsequently was assessed using HLA transgenic mice (CTL) and HLA cross-reacting H-2^bxd (BALB/c × C57BL/6J) mice (HTL). Through this process, 30 CTL and 16 HTL epitopes were selected as a set that would be the most useful for vaccine design, based on epitope conservation among HBV sequences and HLA-based predicted population coverage in diverse ethnic groups. A plasmid DNA-based vaccine encoding the epitopes as a single gene product, with each epitope separated by spacer residues to enhance appropriate epitope processing, was designed. Immunogenicity testing in mice demonstrated the induction of multiple CTL and HTL responses. Furthermore, as a complementary approach, mass spectrometry allowed the identification of correctly processed and major histocompatibility complex-presented epitopes from human cells transfected with the DNA plasmid. A heterologous prime-boost immunization with the plasmid DNA and a recombinant MVA gave further enhancement of the immune responses. Thus, a multiepitope therapeutic vaccine candidate capable of stimulating those cellular immune responses thought to be essential for controlling and clearing HBV infection was successfully designed and evaluated in vitro and in HLA transgenic mice.     

Predicting changes in knee adduction moment due to load-altering interventions from pressure distribution at the foot in healthy subjects

The purpose of this pilot study of healthy subjects was to determine if changes in foot pressure patterns associated with a lateral wedge can predict the changes in the knee adduction moment. We tested two hypotheses: (1) increases or decreases in the knee adduction moment and ankle eversion moment due to load-altering footwear interventions can be predicted from foot pressure distribution and (2) changes in magnitude of the knee adduction moment and ankle eversion moment due to lateral wedges can be predicted from pressure distribution at the foot during walking. Fifteen healthy adults performed walking trials in three shoes: 0 degrees , 4 degrees , and 8 degrees laterally wedged. Maximum heel pressure ratio, first peak knee adduction moment, and peak ankle eversion moment were assessed using a pressure mat, motion capture system, and force plate. Increases or decreases in the knee adduction moment and ankle eversion moment were predicted well from foot pressure distribution. However, the magnitude of the pressure change did not predict the magnitude of the peak knee adduction moment change or peak ankle eversion moment change. Factors such as limb alignment or trunk motion may affect the knee adduction moment and override a direct relationship between the pressure distribution at the shoe-ground interface and the load distribution at the knee. However, changes (increases or decreases) in the peak knee adduction moment due to load-altering footwear interventions predicted from pressure distribution during walking can be important when evaluating these types of interventions from a clinical perspective.     

The Human Cytomegalovirus US28 Protein Is Located in Endocytic Vesicles and Undergoes Constitutive Endocytosis and Recycling

Genes encoding chemokine receptor-like proteins have been found in herpes and poxviruses and implicated in viral pathogenesis. Here we describe the cellular distribution and trafficking of a human cytomegalovirus (HCMV) chemokine receptor encoded by the US28 gene, after transient and stable expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX(3)C-chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role in the sequestration of host chemokines, thereby modulating antiviral immune responses. In addition, the distribution of US28 mainly on endosomal membranes may allow it to be incorporated into the viral envelope during HCMV assembly.     

Vergleich der Schadstoffbelastung in Eiern des Sperbers (Accipiter nisus) aus Brandenburg und Nordrhein-Westfalen, Deutschland

Zusammenfassung Eier von Sperbern aus Brandenburg und aus Nordhrein-Westfalen wurden auf ihren Gehalt an PCB, DDT, HCH, HCB und die Schwermetalle Blei und Cadmium untersucht. Bei den PCB und beim DDT und seinen Abbauprodukten traten signifikante Unterschiede in der Belastung der beiden Populationen auf, während die anderen untersuchten Stoffe in den Eiern beider Untersuchungsgebiete nur in geringen Mengen nachzuweisen waren. Dabei waren in den Proben aus der ehemaligen DDR die DDT-Konzentrationen deutlich höher als in den Eiern aus dem westfälischen Münsterland. Bei den PCB-Konzentrationen ergab sich ein genau umgekehrtes Ergebnis, im Münsterland waren diese erheblich höher. Die Ergebnisse stehen im klaren Zusammenhang mit der unterschiedlichen Anwendung und dem zeitlich unterschiedlich erfolgtem Verbot der untersuchten Schadstoffe in den beiden ehemaligen deutschen Staaten. Trotz der Belastungsunterschiede bezüglich der PCB ist das PCB-Muster in den Eiern aus den Untersuchungsgebieten aufgrund des artspezifischen Metabolismus nahezu identisch.

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Comparison between the burden of xenobiotics in eggs of the Sparrowhawk (Accipiter nisus) from Brandenburg and North-Rhine-Westphalia, Germany

Summary Eggs from Sparrowhawks from Brandenburg (former GDR) and North-Rhine-Westphalia (former FRG) were examined for their burden of PCB, DDT, HCH, HCB and the heavy metals lead and cadmium. Significant differences emerged in the cases of PCB and DDT and its metabolites between the populations of the two regions. The other examined xenobiotics were detectable only in low concentrations. The DDT concentrations were distinctly higher in the samples from the former GDR, while PCB concentrations were higher in the eggs from North-Rhine-Westphalia. The results reflect clearly the different use and prohibition of the examined xenobiotics in the two former german states. In spite of the differences in the PCB burden, the PCB pattern in the eggs of the different regions is nearly identical. Eggshell indices correlate negatively with DDT burden in both sample areas.

     

Genetic engineering of shikonin biosynthesis hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene

The biosynthetic pathway to 4-hydroxybenzoate (4HB), a precursor of the naphthoquinone pigment shikonin, was modified in Lithospermum erythrorhizon hairy root cultures by introduction of the bacterial gene ubiC. This gene of Escherichia coli encodes chorismate pyruvate-lyase (CPL), an enzyme that converts chorismate into 4HB and is not normally present in plants. The ubiC gene was fused to the sequence for a chloroplast transit peptide and placed under control of a constitutive plant promoter. This construct was introduced into L. erythrorhizon by Agrobacterium rhizogenes-mediated transformation.The resulting hairy root cultures showed high CPL activity. 4HB produced by the CPL reaction was utilized for shikonin biosynthesis, as shown by in vivo inhibition of the native pathway to 4HB with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase. A feeding experiment with [1,7-13C2]shikimate showed that in the absence of AIP the artificially introduced CPL reaction contributed ca. 20% of the overall 4HB biosynthesis in the transgenic cultures. ubiC transformation did not lead to a statistically significant increase of shikonin formation, but to a 5-fold increase of the accumulation of menisdaurin, a nitrile glucoside which is presumably related to aromatic amino acid metabolism.     

Topography and distribution of nerve fibers in the posterior longitudinal ligament of the rat: an immunocytochemical and electron-microscopical study

The distribution and immunocytochemical characterization of nerve fibers and their terminals in the posterior longitudinal ligament of the rat lumbar vertebral column was studied in whole-mount preparations and serial semithin and ultrathin sections. Differences in the localization, distribution pattern and density of peptidergic and catecholaminergic nerve fibers were found in the vertebral and intervertebral regions of the posterior longitudinal ligament. For immunocytochemistry, free floating specimens were incubated with primary antibodies against protein gene product 9.5, substance P, calcitonin gene-related peptide, dopamine-beta-hydroxylase, vasoactive intestinal polypeptide and neuropeptide Y together with the avidin-biotin-peroxidase method. In whole-mount preparations, the neural marker protein gene product 9.5 is immunostained in all unmyelinated nerve fibers in the posterior longitudinal ligament, thus giving a panoramic view of the nerve fiber plexus. The most striking nerve fiber plexus is localized in the intervertebral region. In this region, the posterior longitudinal ligament is rich in capillaries that form a dense plexus within its ventral part and extend to the outer layer of the annulus fibrosus. The peptidergic and catecholaminergic innervation of the posterior longitudinal ligament is discussed in the context of pain syndromes related to the vertebral column and degenerative lumbar spine diseases.