Terje Traavik

Lactoferrin and cyclic lactoferricin inhibit the entry of human cytomegalovirus into human fibroblasts

Lactoferrin is mainly produced by polymorphonuclear leukocytes and has been demonstrated in mammalian milk and external secretions. Lactoferrin is an iron-binding, multifunctional protein and may play an important role in immune regulation and in defense mechanisms against bacteria, fungi and viruses. Lactoferricin is a potent antimicrobial peptide generated from the N-terminal part of lactoferrin by pepsin cleavage. We demonstrate that lactoferrins from different species and its N-terminal peptide lactoferricin (particularly the cyclic form) inhibit expression of early and late antigens, as well as production of infectious viral progeny during human cytomegalovirus (HCMV) infection in vitro. Iron-saturated lactoferrin did not affect HCMV antigen expression. Heparin had the same effects as iron-depleted lactoferrin. Yet, mixtures of lactoferrin and heparin did not inhibit HCMV multiplication i.e. lactoferrin and heparin seemed to mutually block each others antiviral activities. HCMV-infected cells exposed to lactoferrin and cyclic lactoferricin contained less intracellular virus than unexposed cells. The antiviral activity of cyclic lactoferricin was more than seven-fold weaker than that of the maternal molecule. Lactoferrin and cyclic lactoferricin prevented HCMV entrance into the host cell.

Reduced Fitness of Daphnia magna Fed a Bt-Transgenic Maize Variety

Genetically modified (GM) maize expressing the Bt-toxin Cry1Ab (Bt-maize) was tested for effects on survival, growth, and reproduction of the water flea Daphnia magna, a crustacean arthropod commonly used as a model organism in ecotoxicological studies. In three repeated experiments, D. magna were fed 100% ground maize in suspension, using either GM or isogenic unmodified (UM) maize. D. magna fed GM-maize showed a significantly reduced fitness performance: The mortality was higher, a lower proportion of females reached sexual maturation, and the overall egg production was lower compared to D. magna fed UM isogenic maize. We conclude that the tested variety of Bt-maize and its UM counterpart do not have the same quality as food sources for this widely used model organism. The combination of a reduced fitness performance combined with earlier onset of reproduction of D. magna fed Bt-maize indicates a toxic effect rather than a lower nutritional value of the GM-maize.

Noncoding control region of naturally occurring BK virus variants: sequence comparison and functional analysis.

The human polyomavirus BK (BKV) has a proven oncogenic potential, but its contribution to tumorigenesis under natural conditions remains undetermined. As for other primate polyomaviruses, the approximately 5.2 kbp double-stranded circular genome of BKV has three functional regions: the coding regions for the two early (T, t antigens) and four late (agno, capsid proteins; VP1-3) genes separated by a noncoding control region (NCCR). The NCCR contains the origin of replication as well as a promoter/enhancer with a mosaic ofcis-acting elements involved in the regulation of both early and late transcription. Since the original isolation of BKV in 1971, a number of other strains have been identified. Most strains reveal a strong sequence conservation in the protein coding regions of the genome, while the NCCR exhibits considerable variation between different BKV isolates. This variation is due to deletions, duplications, and rearrangements of a basic set of sequence blocks. Comparative studies have proven that the anatomy of the NCCR may determine the transcriptional activities governed by the promoter/enhancer, the host cell tropism and permissivity, as well as the oncogenic potential of a given BKV strain. In most cases, however, the NCCR sequence of new isolates was determined after the virus had been passaged several times in more or less arbitrarily chosen cell cultures, a process known to predispose for NCCR rearrangements. Following the development of the polymerase chain reaction (PCR), it has become feasible to obtain naturally occurring BKV NCCRs, and their sequences, in samples taken directly from infected human individuals. Hence, the biological significance of BKV NCCR variation may be studied without prior propagation of the virus in cell culture. Such variation has general interest, because the BKV NCCRs represent typical mammalian promoter/enhancers, with a large number of binding motifs for cellular transacting factors, which can be conveniently handled for experimental purposes. This communication reviews the naturally occurring BKV NCCR variants, isolated and sequenced directly from human samples, that have been reported so far. The sequences of the different NCCRs are compared and analyzed for the presence of proven and putative cellular transcription factor binding sites. Differences in biological properties between BKV variants are discussed in light of their aberrant NCCR anatomies and the potentially modifying influence of transacting factors.

NIH 3T3 cells stably transfected with the gene encoding phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus acquire a transformed phenotype.

In order to determine whether chronic elevation of intracellular diacylglycerol levels generated by hydrolysis of phosphatidylcholine (PC) by PC-hydrolyzing phospholipase C (PC-PLC) is oncogenic, we generated stable transfectants of NIH 3T3 cells expressing the gene encoding PC-PLC from Bacillus cereus. We found that constitutive expression of this gene (plc) led to transformation of NIH 3T3 cells as evidenced by anchorage-independent growth in soft agar, formation of transformed foci in tissue culture, and loss of contact inhibition. The plc transfectants displayed increased intracellular levels of diacylglycerol and phosphocholine. Expression of B. cereus PC-PLC was confirmed by immunoperoxidase and immunofluorescence staining with an affinity-purified anti-PC-PLC antibody. The NIH 3T3 clones expressing plc induced DNA synthesis, progressed through the cell cycle in the absence of added mitogens, and showed significant growth in low-concentration serum. Transfection with an antisense plc expression vector led to a loss of PC-PLC expression accompanied by a complete reversion of the transformed phenotype, suggesting that plc expression was required for maintenance of the transformed state. Taken together, our results show that chronic stimulation of PC hydrolysis by an unregulated PC-PLC enzyme is oncogenic to NIH 3T3 cells.

Demographic responses of Daphnia magna fed transgenic Bt-maize

The food/feed quality of a variety of genetically modified (GM) maize expressing Cry1Ab Bt-toxin was tested over the life-cycle of Daphnia magna, an arthropod commonly used as model organism in ecotoxicological studies. Demographic responses were compared between animals fed GM or unmodified (UM) near isogenic maize, with and without the addition of predator smell. Age-specific data on survival and birth rates were integrated and analysed using life tables and Leslie matrices. Survival, fecundity and population growth rate (PGR) data generally disfavoured transgenic Bt-maize as feed for D. magna compared to animals fed the unmodified (UM) near isogenic line of maize. Decomposition of age-specific effects revealed that the most important contributions to a reduced PGR in the GM-fed group came from both fecundity and survival differences early in life. We conclude that juvenile and young adult stages are the most sensitive experimental units and should be prioritized in future research. These stages are often omitted in toxicological/ecotoxicological studies and in feeding trials.

The precautionary principle: Scientific uncertainty and omitted research in the context of GMO use and release

Commercialization of genetically modified organisms (GMOs) have sparked profound controversies concerning adequate approaches to risk regulation. Scientific uncertainty and ambiguity, omitted research areas, and lack of basic knowledge crucial to risk assessmentshave become apparent. The objective of this article is to discuss the policy and practical implementation of the Precautionary Principle. A major conclusion is that the void in scientific understanding concerning risks posed by secondary effects and the complexity ofcause-effect relations warrant further research. Initiatives to approach the acceptance or rejection of a number of risk-associated hypotheses is badly needed. Further, since scientific advice plays a key role in GMOregulations, scientists have a responsibility to address and communicate uncertainty to policy makers and the public. Hence, the acceptance of uncertainty is not only a scientific issue, but is related to public policy and involves an ethical dimension.

Age-dependent prevalence of BK virus IgG and IgM antibodies measured by enzyme-linked immunosorbent assays (ELISA).

Enzyme immunoassays (ELISA) have been developed for the detection of BK virus IgG- and IgM-antibodies. Specific IgG is detected by an antigen-coated solid phase test; IgM by an antibody capture method. These methods have been used to study the age-distribution of BK virus antibodies in Tromsø county in Northern Norway. The serum panels tested were: 60 sera from paediatric patients aged 0-1 year; 220 sera from healthy persons aged 1-82 years; 74 sera from healthy blood donors; 107 sera from healthy pregnant women. The age-distribution of BKV-IgG antibodies showed that primary infections took place predominantly between the ages of 1 and 6 years, and that there were no sex differences, either in the age-specific prevalence or in the level of BKV-IgG. We found no significant differences in the prevalence of BKV-IgM antibodies in healthy children and adults and pregnant women. BKV-IgM was detected in 26 of the 461 sera tested (5.6%).

SEROSURVEY FOR ORTHOPOXVIRUSES IN RODENTS AND SHREWS FROM NORWAY

Two hundred and twenty one blood samples representing eight different rodent species and the common shrew (Sorex araneus), collected in Norway between 1993 and 1995, were examined for anti-orthopoxvirus antibodies by a competition enzyme linked imunnosorbent assay (ELISA) and, when possible, an indirect immunofluorescence assay. The serological results indicated that the bank vole (Clethrionomys glareolns), woodmouse (Apodemus sylvaticus) and Norway lemming (Lemmus lemmus) may be reservoir species for orthopoxviruses in Norway, with antibody prevalences of 17 (12/69), 30 (24/81) and 56% (19/34), respectively. Orthopoxvirus infection in lemmings has not been reported previously. On some other small rodent species such as field voles (Microtus agrestis), common rats (Rattus norvegicus), and common shrews, seropositive individuals were detected. However, the total number of tested animals was low, and the role of these species in the epidemiology of orthopoxvirus infections remains unclear. Attempts to isolate orthopoxviruses from these small mammals failed, although orthopoxvirus specific DNA sequences were detected previously in the same animals by the polymerase chain reaction (PCR). The serological results were compared with and discussed in the context of the occurrence of orthopoxvirus-specific DNA sequences, and it is concluded that orthopoxviruses are widely distributed among wildlife in Norway.

Prevalence of anti BK virus antibody in Portugal and Norway.

Specific IgG antibodies against BK virus measured by ELISA were used as a marker of previous infection. Results with sera from healthy people of different counties in Portugal were compared with previous findings in Norwegian sera. No significant difference between the prevalence and level of BKV IgG could be found between Portugal and Norway, and when comparing the different counties of Portugal. Thus, the way of transmission seemed to follow the same routes both in rural and urban counties in Portugal, and in Norway.

Human endothelial cells allow passage of an archetypal BK virus (BKV) strain--a tool for cultivation and functional studies of natural BKV strains.

Summary.The ubiquitous human polyomavirus BK (BKV) causes the serious condition BKV-nephropathy in an increasing number of renal-transplant patients. The lack of authentic cell cultures for multiplication of naturally occurring strains has hampered cultivation and functional studies of BKV.Here we demonstrate that the most common urine shed BKV strain, the archetype, multiplies in the human endothelial cell line HUV-EC-C. Additional variants with deletions in the non-coding control region (NCCR) appear upon prolonged propagation. Although the titer produced was low, at the present HUV-EC-C is the only cell line shown to allow propagation of archetypal BKV. HUV-EC-C may therefore be a useful tool for BKV cultivation as well as functional studies.