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1. Hypothesis: The key hypothesis in
this paper is recombinant human vimentin can abate inflammation by targeting neutrophil
attachment to blood vessel endothelial cells and platelets by thwarting the
selectin-receptor interaction.

 

2. Rationale: There are two key pieces
of evidence for the hypothesis: the discovery of circulating vimentin via
secretion and vimentin binding to N-acetylglucosamine. A precedent study with Vimentin
resolved its binding capability with N-acetylglucosamine, a subunit of PSGL-1
on leukocytes. Hence there is a probability that it can target neutrophils with
this property. Vimentin was
previously assumed to be a cellular structural component until recently when a
soluble form was found in certain conditions, which is postulated to be
involved in regulatory function.

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3. Methods and Research
Questions:

a) in vitro
parallel-plate flow-adhesion studies

Flow
adhesion studies simulate fluid dynamics to enable a controlled cell to cell
interaction analysis. A cell type or molecule (example: platelets and fibrin) was
affixed to a plate and another cell type (isolated neutrophil) or media (whole
blood) is delivered thru fluidic action. Binding using rolling analysis is then
visualized thru the use of microscope, which can then be quantified for
statistical analysis. This was used to determine rhVim effect with different
experimental conditions a) leukocyte-platelet interaction on whole blood  b) leukocyte-platelet interaction with
isolated neutrophils c) human umbilical vein endothelial cells (HUVEC)
P-selectin upregulation using 1l-4 and IL-1ß or inhibition with blocking antibody.

b) rhVim binding assay

This
assay is a modified ELISA (Enzyme Linked Immunosorbent Assay) which is
dependent on antibody recognition and binding, typically with a detectable
enzymatic signal in this case HRP conjugated anti-polyhistidine Ab. A chimeric
protein (P-selectin/Fc) or PSGL-1/Fc was used as antibody. This was used to
quantify the rhVim binding to receptors.

c) Surface plasmon
resonance

This
is a light based method of detecting electron vibration in a metal surface
where there is an embedded ligand to determine binding activity. This was used
to determine the rhVim binding to receptors (P-selectin and L-selectin). The
difference with ELISA described above, is that this is a label free method and
serves as another layer of proof of binding.

d) Biolayer
interferometry(BLI)

Detects
changes in binding affinity using alterations in light signal in an optical
fiber tip, where a layer of molecule is immobilized. rhVim was layered in the
optical fiber tip and antibody chimera for the different receptors where made
allowed to interact (P-selectin/Fc, E-selectin/Fc, PSGL-1/Fc). This is also
label free method of determining molecular interaction but is able to
accommodate more samples per run compared to surface plasmon resonance.

e) Endotoxin induced
muri ALI

This
lipopolysaccharide triggered injury models bacterial mediated lung injury.

Leukocyte infiltration reduction capability of rhVim on an in vivo model was
tested to determine the actual therapeutic value rhVim. Histopathology was done
to measure visually the impact of rhVim.

 

4. Structural components
that can be secreted might have regulatory function. This is a demonstration
that P-selectin is a possible therapeutic target. This study has a more
mechanistic approach in proving using several detection methods, the binding of
the drug with the targets. This has extended potential methods in determining drugs
that can bind with P-selectin.

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