Cellular and humoral immunogenicity of hamster polyomavirus-derived virus-like particles harboring a mucin 1 cytotoxic T-cell epitope.

In this study, we examined hamster polyomavirus (HaPyV) major capsid protein VP1-derived virus-like particles (VLPs) as a carrier for a human tumor-associated cytotoxic T lymphocyte (CTL) epitope. The VP1 tolerated the insertion of an HLA-*A2-restricted CTL epitope from human mucin 1 (MUC1) into two sites independently and simultaneously, without interfering with assembly of chimeric VLPs. Chimeric VLPs did not differ in the entry pathway or maturation potential of human dendritic cells (hDCs) compared to unmodified VLPs. Recently we demonstrated that immunization of BALB/c mice with chimeric VLPs harboring two MUC1 insertions resulted in the generation of MUC1-specific monoclonal antibodies. Here we demonstrate that the monoclonal antibodies generated react specifically with human tumor cells. Co-cultivation of chimeric VLP-primed hDCs with autologous peripheral blood leukocytes resulted in the activation of MUC1 epitope-specific CD8(+) T cells. This was evidenced by IFN-gamma secretion of an expanded MUC1-specific CD8(+) T-cell pool. The induction of epitope-specific T cells in a human in vitro model and of murine MUC1-reactive antibodies in vivo indicate the potential of chimeric HaPyV VP1-derived VLPs as a delivery vehicle for immunotherapeutic targets.

Hamster polyomavirus-derived virus-like particles are able to transfer in vitro encapsidated plasmid DNA to mammalian cells.

The authentic major capsid protein 1 (VP1) of hamster polyomavirus (HaPyV) consists of 384 amino acid (aa) residues (42 kDa). Expression from an additional in-frame initiation codon located upstream from the authentic VP1 open reading frame (at position -4) might result in the synthesis of a 388 aa-long, amino-terminally extended VP1 (aa -4 to aa 384; VP1(ext)). In a plasmid-mediated Drosophila Schneider (S2) cell expression system, both VP1 derivatives as well as a VP1(ext) variant with an amino acid exchange of the authentic Met1Gly (VP1(ext-M1)) were expressed to a similar high level. Although all three proteins were detected in nuclear as well as cytoplasmic fractions, formation of virus-like particles (VLPs) was observed exclusively in the nucleus as confirmed by negative staining electron microscopy. The use of a tryptophan promoter-driven Escherichia coli expression system resulted in the efficient synthesis of VP1 and VP1(ext) and formation of VLPs. In addition, establishment of an in vitro disassembly/reassembly system allowed the encapsidation of plasmid DNA into VLPs. Encapsidated DNA was found to be protected against the action of DNase I. Mammalian COS-7 and CHO cells were transfected with HaPyV-VP1-VLPs carrying a plasmid encoding enhanced green fluorescent protein (eGFP). In both cell lines eGFP expression was detected indicating successful transfer of the plasmid into the cells, though at a still low level. Cesium chloride gradient centrifugation allowed the separation of VLPs with encapsidated DNA from "empty" VLPs, which might be useful for further optimization of transfection. Therefore, heterologously expressed HaPyV-VP1 may represent a promising alternative carrier for foreign DNA in gene transfer applications.

Cytopathogenicity of Balamuthia mandrillaris, an opportunistic causative agent of granulomatous amebic encephalitis.

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of lethal granulomatous amebic encephalitis in humans and other mammals. Balamuthia mandrillaris is highly cytopathic but, in contrast to the related Acanthamoeba, does not feed on bacteria and seems to feed only on eukaryotic cells instead. Most likely, the cytopathogenicity of B. mandrillaris is inseparable from its infectivity and pathogenicity. To better understand the mechanisms of B. mandrillaris cytopathogenicity, an assay for measuring amebic cytolytic activity was adapted that is based on the release of a reporter enzyme by damaged target cells. The ameba is shown to lyse murine mastocytoma cells very efficiently in a time- and dose-related manner. Furthermore, experiments involving semipermeable membranes and phagocytosis inhibitors indicate that the cytolytic activity of B. mandrillaris is essentially cell contact-dependent. Standard and fluorescence light microscopy, as well as scanning and transmission electron microscopy support and extend these findings at the ultrastructural level.

Characterization of Bacillus anthracis-like bacteria isolated from wild great apes from Cote d'Ivoire and Cameroon.

We present the microbiological and molecular characterization of bacteria isolated from four chimpanzees and one gorilla thought to have died of an anthrax-like disease in Côte d'Ivoire and Cameroon. These isolates differed significantly from classic Bacillus anthracis by the following criteria: motility, resistance to the gamma phage, and, for isolates from Cameroon, resistance to penicillin G. A capsule was expressed not only after induction by CO(2) and bicarbonate but also under normal growth conditions. Subcultivation resulted in beta-hemolytic activity and gamma phage susceptibility in some subclones, suggesting differences in gene regulation compared to classic B. anthracis. The isolates from Côte d'Ivoire and Cameroon showed slight differences in their biochemical characteristics and MICs of different antibiotics but were identical in all molecular features and sequences analyzed. PCR and Southern blot analyses confirmed the presence of both the toxin and the capsule plasmid, with sizes corresponding to the B. anthracis virulence plasmids pXO1 and pXO2. Protective antigen was expressed and secreted into the culture supernatant. The isolates possessed variants of the Ba813 marker and the SG-749 fragment differing from that of classic B. anthracis strains. Multilocus sequence typing revealed a close relationship of our atypical isolates with both classic B. anthracis strains and two uncommonly virulent Bacillus cereus and Bacillus thuringiensis isolates. We propose that the newly discovered atypical B. anthracis strains share a common ancestor with classic B. anthracis or that they emerged recently by transfer of the B. anthracis plasmids to a strain of the B. cereus group.

Expression of the human endogenous retrovirus-K transmembrane envelope, Rec and Np9 proteins in melanomas and melanoma cell lines.

The human endogenous retrovirus-K encodes two potential tumor proteins, Rec and Np9. Rec is related to the Rev protein of HIV-1 and has been shown to be associated with tumor development in nude mice. Having shown the expression of human endogenous retrovirus-K in human melanomas and melanoma cell lines, tools were developed to allow the expression of the transmembrane envelope, Rec and Np9 mRNA and proteins to be studied in more detail. The expression of spliced env, rec and np9 was investigated by reverse transcriptase-polymerase chain reaction using a set of primers developed to discriminate between full-length and spliced mRNA. Env-specific, Rec-specific and Np9-specific antisera were produced, characterized and used to study protein expression in melanomas and melanoma cell lines by immunohistochemistry, immunofluorescence and Western blot analyses. Existence of human endogenous retrovirus-K Rec and Np9-specific antibodies in the sera of melanoma patients were analyzed by Western blot of immunofluorescence studies. The expression of both spliced env and rec mRNA was detected in 39% of the melanomas and in 40% of the melanoma cell lines and np9 mRNA was detected in 29 and 21%, respectively. In normal neonatal melanocytes, spliced rec mRNA was detected in the absence of spliced env mRNA. Using antisera specific for Rec and Np9, Rec protein was found in 14% of the melanomas but Np9 in none. In addition, cell surface expression of the putatively immunosuppressive transmembrane envelope protein and release of virus particles were shown. Antibodies specific for neither Rec nor Np9 were detected. The transmembrane envelope protein, Rec and Np9 proteins are expressed in melanoma cells with a pattern similar to that seen in teratocarcinoma cell lines. Additional experiments are needed to determine their involvement, if any, in cell proliferation and tumor progression.

Balamuthia mandrillaris, free-living ameba and opportunistic agent of encephalitis, is a potential host for Legionella pneumophila bacteria.

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment.

Detection of human polyomaviruses in urine from bone marrow transplant patients: comparison of electron microscopy with PCR.

BACKGROUND: We studied electron microscopy (EM) as an appropriate test system for the detection of polyomavirus in urine samples from bone marrow transplant patients. METHODS: We evaluated direct EM, ultracentrifugation (UC) before EM, and solid-phase immuno-EM (SPIEM). The diagnostic accuracy of EM was studied by comparison with a real-time PCR assay on 531 clinical samples. RESULTS: The detection rate of EM was increased by UC and SPIEM. On 531 clinical urine samples, the diagnostic sensitivity of EM was 47% (70 of 149) with a specificity of 100%. We observed a linear relationship between viral genome concentration and the proportion of urine samples positive by EM, with a 50% probability for a positive EM result for urine samples with a polyomavirus concentration of 10(6) genome-equivalents (GE)/mL; the probability of a positive EM result was 0% for urine samples with <10(3) GE/mL and 100% for urine samples containing 10(9) GE/mL. CONCLUSIONS: UC/EM is rapid and highly specific for polyomavirus in urine. Unlike real-time PCR, EM has low sensitivity and cannot quantify the viral load.

Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR.

Bacillus anthracis spores have been shown to be an efficient biological weapon and their recent use in bioterrorist attacks has demonstrated the need for rapid and specific diagnostics. A TaqMan real-time PCR for identification of B. anthracis was developed, based on the two plasmids, pX01 and pX02, both of which are necessary for pathogenicity, as well as on the chromosomally encoded rpoB gene. Bacteria picked from colonies or pelleted from liquid cultures were directly inoculated into the PCR mix, thus avoiding time-consuming DNA preparation and minimizing handling risks. B. anthracis spores were cultivated for a few hours in enrichment broth before PCR analysis, or used directly for real-time PCR, thus allowing to confirm or exclude potential attacks approximately 2-3 h after the material has arrived in the laboratory.

Production and characterization of Escherichia coli enterohemolysin and its effects on the structure of erythrocyte membranes.

Hemolysins are cell-damaging protein toxins produced by pathogenic bacteria, which are usually released into the extracellular medium. Escherichia coli enterohemolysin is an intracellular toxin produced during the log phase of growth, with a maximal intracellular accumulation in the late log phase. In the present study, we have employed electron microscopy and SDS-PAGE to assess the effects of enterohemolysin on erythocyte membranes from different species. The erythrocyte cell damage began immediately after exposure to enterohemolysin with chemically detectable changes in cell membrane permeability, and the formation of surface lesions which increased rapidly in size. This process resulted in complete cell destruction. Ring-shaped structures with a diameter of 10nm were observed by electron microscopy after treatment of horse erythrocyte membranes with enterohemolysin. The ring structures were found clustered and irregularly distributed on the surface of the membranes. Following incubation of the toxin with horse erythrocyte ghosts and detergent-solubilization, the enterohemolysin was isolated from the cytoplasm in its membrane-bound form by sucrose density gradient. SDS-PAGE and silver staining of deoxycholate-solubilized target membranes revealed heterogeneous forms of the toxin. By using SDS-PAGE and gel filtration, the molecular weight of the toxin was estimated to be 35 kDa. With respect to species specificity, horse erythrocytes showed the highest sensitivity to the enterohemolysin, followed by human and guinea pig erythrocytes. The hemolytic sensitivity correlated with the toxin binding capacity of erythrocyte membranes of different animal species. The degree of hemolysis was unaffected by temperature in the range of 4 degrees C-37 degrees C and was optimal at pH 9.0. In contrast to pore-forming cytolysins, the hemolytic activity of enterohemolysin was enhanced continuously in the presence of increasing concentrations of dextran 4 and dextran 8 within the range of 5 to 30 mM. Trypsin sensitivity of membrane-bound enterohemolysin indicates that the cell surface is the most likely target site for this toxin. Additionally, the fact that proteinase and phosphatase inhibitors failed to inhibit lysis suggests that enterohemolysin alters and disrupts cell membranes by a detergent-like mechanism.