70 research outputs found
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
for 13Â h. After washing of the cells, FITC-labelled
virus-specific antibodies were added (0.1Â mg IgG/ml), and the cells were
incubated at 37\,^\circC for different time
periods (0, 5, 10, 30 and 60Â 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 10Â min
post-antibody addition, and was
completed in 65% of the infected cells at 1Â h post-antibody addition. Furthermore,
only very few quantities of viral glycoproteins
on the plasma membrane (reached after 7Â h PI)
and very low concentrations of antibodies (0.04Â 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
50Â g/mL). We also evaluated the effect of viral glycoprotein internalisation on
complement-mediated lysis of the infected monocytes. Monocytes were infected for
10Â h, and incubated with virus-specific antibodies for
2Â h ( of the infected cells
displayed internalised viral glycoproteins at this time point). The control cells
were incubated with antibodies in the presence of
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 1Â h.
Afterwards, 20Â g/mL of the
DNA-staining fluorochrome, propidium iodide, was added for 5Â 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 1Â h
decreased slightly to 79% 12% for cells incubated without antibodies (no activation
of the complement), and to 84% 4% for cells
incubated with antibodies (internalised viral
glycoproteins and antibodies). The viability dropped to 24% 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
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