Systemic and local inflammation in burn wound patients.

Upon tissue injury an inflammatory reaction is induced which is essential to
combat invading microorganisms, debride the wound from damaged tissue and
orchestra the healing process.
However in patients with burn wounds a massive inflammatory response is induced
that not only negatively affects the healing process of the burn wound, but
additionally exerts systemic effects resulting in secondary (organ) injury. The
biological factors involved in this secondary response to burn wounds are
largely unknown.
The inflammatory reaction comprises a wide range of reactions in which
different cell-types of the innate and adaptive immune system play an important
role.
The current study will investigate various immunological factors involved in
burns related effects.

Part 1. The acute phase response: Immunological factors and secondary organ
injury
An important factor in the inflammatory response is the acute phase response
(APR), in which complement is playing a central role. It is known that these
acute phase proteins are elevated up to months after the initial burn trauma.
We have shown that these proteins are also elevated locally in the wound up to
at least 4 weeks post-burn (pb). Recently, we described in a rat burn wound
model that application of C1-esterase inhibitor (C1-inh), a complement
inhibitor, improved healing of the burn wound and additionally reduced systemic
effects, more specific, inflammation of the heart. In humans the local and
systemic activation of the complement system is not resolved yet. Therefore we
will analyse different inflammatory factors related to complement activation
and the early inflammatory reaction in the blood (systemic) and in the burn
wound in patients. In addition we want to analyse the time frame of
infiltration of inflammatory cells (i.e. neutrophilic granulocytes, macrophages
and lymphocytes) in the burn wound.

Part 2. Inflammation and burn wound extension: Neutrophilic extracellular traps
in expansion of necrosis of the burn wound
Neutrophils are the first inflammatory cells to arrive at the wound site. These
cells are pivotal for debridement of the injured tissue and the clearance of
microorganisms. However they can also be detrimental to the healing process.
Secondary microvascular damage and thrombosis are important underlying factors
in the expansion of necrosis into vital tissues neighboring the initial burn
injury. Neutrophil extracellular trap formation (NETosis) has been shown to be
a major initiator of microvascular damage, coagulation and inflammation in
non-burn related major trauma, sepsis and autoimmune disease. However, its role
in burn injury, a pathology characterized by extensive (secondary) tissue
damage, prolonged inflammation and hypercoagulability is unknown. We recently
found evidence for the involvement of NETosis in the instigation of
microvascular thrombosis in the burn wound. In a rat burn wound model we showed
that thrombus formation in the blood vessels within the burn wound was
significantly higher than in the skin tissue taken from the non-burn hind leg
(internal control). Remarkable is the presence of thrombi in non-affected
healthy skin samples, which supports the idea of bigger generalised systemic
response. Furthermore, we found indications of causative factors resulting in
pb thrombi formation, like the formation of neutrophilic extracellular traps
(NETs), an increase of tissue factor (TF) (pro-coagulant) and a decrease of
DPP4/CD26 (anti-coagulant) expression in blood vessels, correlated with thrombi
formation within the burn wound.

Part 3. Inflammation, pain and psychological stress
Pain is a major complication following burns and it is still difficult to
treat. Burn pain results from tissue inflammation in which a range of
inflammatory mediators are released that in turn sensitise and stimulate pain
fibres throughout the wound healing process. Stress-induced hyperalgesia may
exacerbate the existing pain. We will explore associations between inflammation
of the wounds, pain and the psychological stress response

Objective:
Since the inflammatory reaction plays an important role in wound healing
problems and scar formation the main goal of this project is to unravel
different inflammatory mechanisms involved.
• Explore relationships between local and systemic inflammation and clinical
outcome with respect to complement activation. (part 1)
• Determine the relation between NETs in the blood and NETosis in burn wounds
and their prognostic potential with regards to clinical outcome. (part 2)
• Determine the timing of the different mechanisms involved in wound healing
and scar formation. (part 1 and 2)
• Determine the correlation between the above mentioned parameters with total
body surface area (TBSA %), co-morbidities and clinical course. (part 1 and 2)

Prospective observational cohort study in all burn patients admitted to the
ICU. In addition twenty healty volunteers will be included. Duration of the
study is 4 year. The study will be performed in the burn centre in Beverwijk
(Rode Kruis Ziekenhuis). For reference values 20 healthy volunteers will be
included.

Patients: The burden of the study is limited to blood withdrawals via
venipunctures and for patients with more severe burn wounds also eschar tissue
collection during planned operations. The venipuncture procedures are embedded
in the clinical routine. An extra blood tube for serum will be taken.
Venipunctures are common procedures at the burn centres and will be performed
by qualified people. The collection of (secondary necrosis) debridements will
take place during clinical routine.
Healthy volunteers: twice blood draw 1 tube 5 ml; minimal risks; blood draw can
be painful or cause a hematoma. The amount od blood drawn will not give
problems.