Research 6303 and EF3030 in multiple clonal cells

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Research paper report:

This paper is related
to a study focus on Pneumococcus
pneumoniae, a worldwide pathogen that infect humans in their first mounts
of life. The bacteria induce a pneumococcal pneumonia, when the infection
spread from the nasopharynx area to the deep respiratory tract and the alveoli.
The first encounter with the immune system will be with the alveolar
macrophages. As they encounter the bacteria first, they are highly important
for the activation of the innate immune response. This activation is linked to
the transcription factor nuclear factor ?B (NF- ?B). The production of NF- ?B
is essential for the expression of cytokines, which will induce many innate
immunity mediators. Another study shown that mice with defection of NF- ?B in
macrophages are more likely to develop severe respiratory infection. By this
observation, the researcher wants to attempt to link the macrophage NF- ?B
activation and the population of pneumococci. Furthermore, if the first link is
find, study if less triggered macrophage NF- ?B lead to a more severe infection
in the lugs.

The authors decided to
study the question because the possible outcomes could be the development of a
new drug that could help the immune system to respond to an infection from Pneumococcus pneumoniae, especially to
protect children and elderly people.

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experiment: The researcher notice that macrophages infected with
EF3030 strain were more likely to show a RelA nuclear translocation that if
they were infected with 6303 strains. To show that effect, the researcher
infected mice or in vitro raw alveolar macrophages with EF3030 or 6303. And to
show the RelA nuclear translocation, the cells were taken from the mice by bronchoalveolar,
washed 2 hours after intratracheal instillation of EF3030 or 6303 to C57BL/6
mice. In that way the NF- ?B RelA can be revealed by using
immunohistochemistry. Same things for the raw cells in vitro, and both samples
were observe using confocal microscopy. A second step of that experiment were focused
on the clonal cells line from RAW294.7 cells. The aim of that study was to
modulate the concentration of 6303 and EF3030 in multiple clonal cells lines
and link that concentration to the NF- ?B activation. The results support the
hypothesis where a low NF- ?B activation typify a severe pneumococcal

experiment: By screening pneumococcal isolates from true
positive and false positive patient, researcher found that the response from
macrophage NF- ?B activation levels were ascertain, which means that the NF- ?B
triggering is very different following the different isolates. The results are
shown by a histogram. To study this relation, the researcher recorded the NF- ?B
activation for different serotype. They observed that the serotype made a
difference between themselves, but there is a important variation among the
serotype. To conclude on that experiment, the researcher established that the
serotype-related capsular polysaccharide heterogeneity may be one of the
multiple variables that influence macrophage NF- ?B activation.  

experiment: Because macrophage NF- ?B is important for lung
defence against pneumococcus, the researcher asses the relation between the
percentage of macrophage NF- ?B triggering and the severity of pneumococcal
infection. The researcher takes out sample from different group of patients,
symptomatic and asymptomatic for pneumococcal. The area chosen were the
nasopharynx, the blood, the pleural fluid or the CSF (cerebrospinal fluid). The
data collected from the graph suggest that a low NF- ?B activation might be
critical for pneumococcal survival with special settings. To compare the
abilities and low NF- ?B-activating isolates of pneumococcus to survive inside
the lungs, the researcher used an animal model. By using mice, they compared
isolates which matched, but not for 2 different serotypes, 19A and 19F. To
study this difference, they focused on TNF-a and CXCL2, which are macrophage
derived and NF- ?B dependent during pneumococcal pneumonia. The results,
presented through histograms, support the idea that avoiding or subverting
macrophage NF- ?B activation during acute pneumonia enhances S. pneumoniae survival in the lung.

Fourth experiment:  To study the necrosis that occur
when the macrophages are infected, the researcher exited RAW264.7 cells with
high or low NF- ?B-activator S.
pneumoniae or vehicle control. The cells were put 2 hours in coculture, and
the cells with low NF- ?B activators shown an increasing of the plasma membrane
permeability, as shown by pictures of the cells with a membrane-impermeable fluorescent
dye. This increasing in permeability is conjunct to the cellular oncosis and
excess ROS production, which are indicative of the programmed necrosis (shown
as histograms). As necroptosis is linked to the necrosome, it’s include the
activity of the kinase RIPK1. And when the activity of RIPK1 were inhibited in
low concentration of NF- ?B, the data show that the percentage of cellular oncosis
ad of high oxidative activity, link to the ROS production, decrease
significantly. These result support the concept that the altered response of
the macrophage by low NF- ?B activators is related to the pneumococcal
induction of the programmed necrosis.

experiment: NF- ?B activity and necroptosis are correlated, and to
increase the necroptosis within the macrophages responding to pneumococci, researchers
inhibited NF- ?B. the BAY 11-7082 is an inhibitor of NF- ?B and has been used
in this study. The data, presented through a histogram, show that the inhibitor
increased necroptosis among RAW264.7 cells, while they were responding to a
high NF- ?B activator pneumococcus, and same thing for the number of cellular
oncosis. These results suggest that stimulating NF- ?B activity helps to avoid
the triggering of programmed necroptosis among macrophages.

Sixth experiment: The studies show that macrophages can have two faiths: NF- ?B-mediated
gene expression or necroptosis. The researchers designed a series of costimulation
and coinfection to see if it’s possible to reverse a necroptosis and a lung
infection by the presence of high NF- ?B activators. Through histograms, the
result show that presence of high NF- ?B-activating pneumococcus (EF3030) in an
infection made by a low NF- ?B-activating pneumococcus (6303) prevented the
effect of 6303. In vitro, these data show that the adding of high NF- ?B-activating
pneumococcus in an infection provoke by a low NF- ?B-activating pneumococcus
allow to stimulate a pathway that overcome the necroptosis pathways. The same
observation was possible in vivo, where the induction of EF3030 enhance the immune
response from a host that was experimenting a severe infection from 6303. These
results support the concept that the activation of the appropriate macrophages
including NF- ?B RelA enhance the elimination of a high necroptosis-inducing
isolate of S. pneumoniae while a lung

Those results, all
cumulate, show a new way to fight severe infection. Not by using drug and
vaccine, the use of pathogens can trigger and enhance the immune system when he
is disrupted by a severe pathogen which target him.

The researcher noted that,
while they were studying the different serotype of pneumococci, more isolates
were low activators than they expected.

For further studies, the
researcher suggests that new therapy, as immunostimulatory, would be possible to
treat pneumonia, for drug-resistant bacteria.

Categories: Development


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