Local host response following an intramammary challenge with Staphylococcus fleurettii and different strains of Staphylococcus chromogenes in dairy heifers

Coagulase-negative staphylococci (CNS) are a common cause of subclinical mastitis in dairy cattle. The CNS inhabit various ecological habitats, ranging between the environment and the host. In order to obtain a better insight into the host response, an experimental infection was carried out in eight healthy heifers in mid-lactation with three different CNS strains: a Staphylococcus fleurettii strain originating from sawdust bedding, an intramammary Staphylococcus chromogenes strain originating from a persistent intramammary infection (S. chromogenes IM) and a S. chromogenes strain isolated from a heifer's teat apex (S. chromogenes TA). Each heifer was inoculated in the mammary gland with 1.0 x 10(6) colony forming units of each bacterial strain (one strain per udder quarter), whereas the remaining quarter was infused with phosphate-buffered saline. Overall, the CNS evoked a mild local host response. The somatic cell count increased in all S. fleurettii-inoculated quarters, although the strain was eliminated within 12 h. The two S. chromogenes strains were shed in larger numbers for a longer period. Bacterial and somatic cell counts, as well as neutrophil responses, were higher after inoculation with S. chromogenes IM than with S. chromogenes TA. In conclusion, these results suggest that S. chromogenes might be better adapted to the mammary gland than S. fleurettii. Furthermore, not all S. chromogenes strains induce the same local host response

Four-dimensional imaging and computer-assisted track analysis of nuclear migration in root hairs of Arabidopsis thaliana

Nuclear migration is a fundamental mechanism necessary for the proper growth and development of many eukaryotic organisms. In this study root hairs of Arabidopsis thaliana were used as a research model to gain insight into the dynamics of nuclear migration. Root hairs are long tubular outgrowths of epidermal cells and are responsible for the uptake of water and nutrients. During the development of root hairs, the nucleus migrates into the hair after the bulge is formed. The position of the nucleus relative to the tip plays an essential role in the growth process. However, what is happening to the nucleus in full-grown root hairs is still unclear. To study nuclear dynamics in living root hair cells, stably transformed plants with the fusion proteins Histone2B-YFP and NLS-GFP-GUS were used. Four-dimensional confocal laser scanning microscopy made it possible to monitor the exact position of the nucleus in different root hairs. To analyse the sequential positions of the nuclei in the root hairs, a new computer-assisted method was developed. After track analysis a number of parameters could be extracted from the movies, such as the average speed, the amplitude, direction factor and the range of movement in the root hairs. Our results show that nuclei do not reach a final position in full-grown root hairs and this sustained movement seems to be more similar in root hairs lying close to each other. Moreover, with this methodology it could be quantitatively demonstrated that the integrity of actin is necessary for nuclear movement.12 page(s

Antibody-induced endocytosis of viral glycoproteins, expressed on pseudorabies virus-infected monocytes protects these cells from complement-mediated lysis

Pseudorabies virus (PrV) can cause abortion in sows with an immune system activated by vaccination. Virus-carrying blood monocytes are essential for the spread of the virus from the respiratory tract to the pregnant uterus. Two major adaptive immune effector mechanisms should normally be capable of eliminating PrV-infected monocytes. First, newly synthesised viral proteins may be processed and coupled to the major histocompatibility complex class I (MHC I) which then is transported to the plasma membrane. This MHC I-antigen-complex can be recognised by cytotoxic T-lymphocytes (CTLs). Second, specific antibodies are capable of binding to newly synthesised viral envelope glycoproteins, which become expressed in the plasma membrane of the infected cell. Antibodies in association with complement or phagocytes may then result in the lysis of the infected cell. Addition of virus-specific antibodies to PrV-infected swine kidney cells in vitro is known to induce a redistribution of the plasma membrane-anchored viral glycoproteins. This redistribution finally leads to the release of the viral glycoproteins into the surrounding medium, leaving viable cells without visually detectable levels of viral glycoproteins on their plasma membrane. In the present study it was examined whether a similar phenomenon occurs in the natural carrier of the virus, the blood monocyte, in order to evaluate if this process may be significant to the immune evasion of the virus. Blood was collected from the vena jugularis from PrV-negative pigs and blood mononuclear cells were separated on Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden). Blood monocytes were purified by plastic adhesion, and were cultivated for 24 h. Afterwards, the cells were inoculated with PrV strain 89V87 or Kaplan and incubated at 37\,^\circC with 5% CO$_2$ for 13 h. After washing of the cells, FITC-labelled virus-specific antibodies were added (0.1 mg IgG/ml), and the cells were incubated at 37\,^\circC for different time periods (0, 5, 10, 30 and 60 min) before fixation with 0.4% formaldehyde and analysis by fluorescence microscopy and/or confocal laser scanning microscopy. Shortly after the addition of antibodies, viral plasma membrane glycoproteins become aggregated (patches). These patches are then internalised by the cell, leaving an infected cell with no visually detectable levels of viral glycoproteins on its plasma membrane. Antibody-induced endocytosis is a fast and efficient process. Endocytosis started at 10 min post-antibody addition, and was completed in 65% of the infected cells at 1 h post-antibody addition. Furthermore, only very few quantities of viral glycoproteins on the plasma membrane (reached after 7 h PI) and very low concentrations of antibodies (0.04 mg IgG/mL) were needed to induce endocytosis. Genistein, a specific inhibitor of tyrosine kinase activity, was found to be a very efficient inhibitor of viral glycoprotein internalisation (100% inhibition at 50 $\mu$g/mL). We also evaluated the effect of viral glycoprotein internalisation on complement-mediated lysis of the infected monocytes. Monocytes were infected for 10 h, and incubated with virus-specific antibodies for 2 h ($\pm 100\%$ of the infected cells displayed internalised viral glycoproteins at this time point). The control cells were incubated with antibodies in the presence of $50\,\mu$g/mL genistein, or were incubated without antibodies. Afterwards, the cells were washed and incubated with different concentrations of guinea pig complement (0-10 IU) for 1 h. Afterwards, 20 $\mu$g/mL of the DNA-staining fluorochrome, propidium iodide, was added for 5 min. Propidium iodide specifically stains dead cells which allows to determine the percentage of dead cells by flow cytometry. Compared relatively to the viability of the cells incubated without either antibodies or the complement, viability of the cells, incubated with the complement for 1 h decreased slightly to 79% $\pm$ 12% for cells incubated without antibodies (no activation of the complement), and to 84% $\pm$ 4% for cells incubated with antibodies (internalised viral glycoproteins and antibodies). The viability dropped to 24% $\pm$ 11% for cells incubated with antibodies and genistein (there was no internalisation of viral glycoproteins and antibodies), which was not caused by toxic effects of genistein. We can therefore state that antibody-induced endocytosis of viral glycoproteins protects PrV-infected cells from complement-mediated lysis. When performing double labelling experiments, we observed that the MHC I co-aggregates and undergoes co-endocytosis with the viral glycoproteins. This may indicate that the addition of virus-specific antibodies to PrV-infected monocytes can hide these cells from both humoral and cellular immune responses. To investigate this hypothesis, we are currently constructing an in vitro assay to evaluate the effect of MHC I co-endocytosis on the capacity of cytotoxic T-lymphocytes to eliminate PrV infected monocytes. Furthermore, we are examining whether the observed processes also occur in vivo. Preliminary experiments, consisting of the injection of colostrum-free piglets with biotinylated PrV-specific antibodies, followed by PrV-inoculation, already showed that endocytosis of antibodies occurs in vivo in infected cells, e.g. in alveolar macrophages