Resuscitation for repair of endothelial permeability in endotoxemia

Acute critical illness can impair endothelial barrier function. In acute
critical illness, such as sepsis, activation of the endothelium occurs,
resulting in shedding of constituents of the glycocalyx (a layer of proteins
lining the luminal vessel wall), resulting in endothelial permeability, which
clinically manifests as accumulation of protein-rich fluid in the extravascular
space, leading to edema. Together with inflammatory and coagulation responses,
these changes impair microcirculatory perfusion and tissue oxygenation,
resulting in multiple organ dysfunction. Also, non-infectious causes of acute
critical illness can result in strong pro-inflammatory host-responses with
ensuing endothelial hyperpermeability, such as pancreatitis or major surgery.

Acute critical illness often requires volume resuscitation with fluids.
However, the downside of resuscitation is the occurrence of edema. Compared to
a liberal fluid balance, a restrictive fluid balance reduces the occurrence of
organ failure. Most likely, fluid resuscitation results in an increased
gradient of leakage over the hyper-permeable endothelium. This poses a dilemma
to the treatment of sepsis, as fluid therapy is both a cornerstone of therapy
as well as a foe in the occurrence of organ failure, calling for strategies
which improve endothelial barrier function.

Different fluid strategies may have differential effects on endothelial
permeability. Fluids with a low protein-content, such as crystalloids, seem to
have negative effects on the endothelial glycocalyx, aggravating endothelial
permeability. In sepsis patients, the amount of crystalloid fluid is
associated with increased glycocalyx breakdown, which may be driven by
inflammatory mediators, as shown in vitro. These effects may be related to the
low protein content. Albumin, but not crystalloids, was found to protect
against endothelial dysfunction and improved survival by inhibiting
inflammatory and oxidative stress in a rodent endotoxemia model as well as in a
hemorrhagic shock model. In clinical practice, albumin iscurrently used as an
additive solution to supplement crystalloid fluid therapy.

The effect of albumin on the glycocalix appears less than with the use of
plasma. Indeed, plasma was found to restore glycocalyx thickness and
endothelial barrier integrity, resulting in less organ failure in models of
hemorrhagic shock and sepsis. In critically ill patients, we have shown that
plasma resulted in a reduction in markers of endothelial activation and a
reduction in pro-inflammatory cytokines. Other clinical data on the effect of
plasma in sepsis are limited. In retrospective studies, plasma exchange was
performed as a rescue therapy with the aim to remove toxic compounds. An
improved survival was found compared to historical survival data.

Solvent-detergent (SD) plasma is a pooled and washed multiple donor (varying
from 10 - 1000 male and female donors) product. In a prospective cohort in
critically ill children, SD plasma, but not fresh frozen plasma, was
independently associated with reduced mortality. In a randomized pilot trial in
patients undergoing surgery for aortic dissection, SD plasma reduced glycocalyx
shedding and reduced endothelial tight junction injury compared to FFP. This
difference is possibly due to the washing process in the manufacturing of SD
plasma, which might dilute harmful substances in plasma products such as
anti-human leukocyte antigens. Alternatively, the centrifugation steps may
remove cellular debris such as extracellular vesicles, as these substances are
linked to complications such as transfusion-related acute lung injury (TRALI).
In line with this, no TRALI has been reported with the use of SD plasma, and
the rate of allergic or anaphylactic reactions with SD plasma is between 76%
and 94% lower than that of FFP.

Specific mechanisms mediating protective effects of plasma may be related to a
release of sphingosine-1-phosphate (S1P), limiting metalloproteases from
cleaving the endothelial glycocalyx. Alternatively, plasma may be protective by
replenishing ADAMTS13, as we have shown in observational studies, thereby
balancing the excess release of von Willebrand Factor, limiting von Willebrand
Factor multimerization, microthrombi formation and subsequent edema and organ
failure. It will be important to determine which proteins contribute to
protective effects, in order to enable the manufacturing of these specific
proteins for targeted therapy.


In this study in a human endotoxemia model, we hypothesize that fluid therapy
with protein rich fluids is superior to crystalloids, which is the standard
used resuscitation fluid in acute critical illness, in terms of preservation of
the endothelial barrier integrity.

The main objective of the proposed study is to investigate the effect of plasma
products and albumin on markers of endothelial function in a human endotoxemia
model.

Open label, randomised interventional trial in healthy male volunteers

Group 1 (n=6): 2 ng/mL LPS + 10 ml/kg crystalloids (Plasma-Lyte, control group)
Group 2 (n=6): 2 ng/mL LPS + 10 ml/kg SD plasma (Omniplasma®)

Benefits: none

Investigational products:
1. All volunteers will receive LPS injection. Discomfort from LPS can consist
of nausea, chills, fever, and hypotension, which will subside in 3-4 hours.
Subject monitoring will take place at our ICU until after symptoms have
subsided. A dose of 2 ng/kg LPS in healthy volunteers has proven to be safe in
previous studies at this and other institutions. The LPS is distributed by the
National Institute of Health (NIH), USA. The analysis and concentration of the
endotoxin is determined by a gel-clot method and its standard of reference in
the USA.
2. Omniplasma: Due to the manufacturing process which also dilutes allergens,
allergic reactions are seldom, and usually mild (skin erythema, urticaria) (<
1:100 recipients, BIJSLUITER). More severe reactions are hypotension, bronchial
hyperreactivity, tachycardia and dyspnea (< 1:10.000 recipients). These side
effects are caused by the citrate in Omniplasma causing hypocalcemia. This is a
particular problem in recipients with liver dysfunction. This will not be
present in the healthy volunteers. High dose or high speed of infusion rate can
cause circulatory overload. This is not expected in the volunteers who will
have received LPS and hence are volume depleted. Circulatory overload is a
particular risk in recipients with heart failure (23). This will not be present
in the healthy volunteers. The infusion speed will be set at a rate that is
generally well tolerated in recipients without heart failure.
3. Plasma-Lyte: This is a so-called *balanced* crystalloid solution, as it has
an electrolyte composition that resembles that of plasma. However, this
solution is protein-free. Crystalloids are the current standard resuscitation
fluid. There are no known allergies to crystalloids.

The experiment:
1. During the experiment an artery line will be inserted in one of the arteries
of the arms of the test person. The placing of the artery line is performed by
an experienced doctor. There is a small chance this procedure results in
bruises or blood cloth formation in the blood vessel. During the last years no
major complications occurred with this procedure in our center.

Risks assessment:
1. Infusion of E. coli LPS with a dose of 2 ng/kg has previously been proven to
be safe in healthy adult volunteers in our institution.
2. Omniplasma is considered to have low risk of allergic reactions.
Transfusions will be prepared and transfused using the standard clinical
protocols by Sanquin and our hospital.
3. The combination of LPS with infusion is expected only to cause mild
temporary side effects because subjects tolerate this LPS dose well in previous
experiments, and the risk of an adverse events with the intervention fluids is
very low.