The effect of endotoxin tolerance on ischemia-reperfusion injury in humans in vivo

Animal experiments demonstrate that induction of endotoxin tolerance results in
protection from subsequent ischemia-reperfusion injury. For example, animals
treated with endotoxin are protected from cardial or renal ischemic damage.
Endotoxin-induced protection is more potent than any pharmacological form of
protection studied up to now.

To demonstrate attenuated ischemia-reperfusion damage in subjects that have
become tolerant due to repeated endotoxin administrations.

In 10 subjects endotoxin tolerance will be induced by repeated infusions of
increasing dosages of endotoxin (0.2-2.0 ng/kg/day) in 5 days.
Prior to and directly following the induction of endotoxine tolerance (day 6),
ischemia-reperfusion damage is examined in the thenar muscle using annexine A5
scintigraphy.
In a second group of 10 subjects will serve as time-controls, in which
ischemia-reperfusion damage is examined in the thenar muscle using annexine A5
scintigraphy without the pretreatment with endotoxin.

Endotoxin tolerance is induced by repeated infusions of endotoxine (E. coli
O:113).
Ischemia-reperfusion damage is investigated using annexine A5 scintigraphy.
No additional drugs/agents are investigated in this project.

- Concerning the use of endotoxin.
The described protocol has been conducted many times before by our group. The
subjects experience flue-like symptoms, but endotoxin administration is
considered safe and it has never resulted in persistent damage.
Endotoxin administration is an accepted model of systemic inflammation, that is
used in several European countries and the United States. Thousands of subjects
have received endotoxin and apart from vagal collapse, no serious adverse
events or prolonged hospitalisation have been reported. The dosage of endotoxin
used is 2-4 ng/kg in the literature. In our project a maximal dose of 2 ng/kg
is administered. Endotoxin is 'dead' material, there is no risk of infection.
The inflammatory response to endotoxin is predictable and reproducible. After
the administration of 2 ng/kg, the volunteers experience flue-like symptoms
like fever (max. 38.5 oC), chills, headache, muscleache, backache. These
symptoms are qualified as 'mild' by the subjects. All subjects are closely
monitored: symptoms, general appearance, heart rate, blood pressure. During the
first 4 hours after the administration of the endotoxin, a medical docter is
constantly present. It is important to notice that the described symptoms occur
after the administration of 2 ng/kg endotoxin. During previously conducted
endotoxin tolerance experiments, it was found that (due to the induction of
tolerance) the subjects experienced no, or very mild symptoms.

- Concerning the repeated administrations of endotoxin.
In our previous study (CMO nr 2005-087), in which the subjects received 2 ng/kg
endotoxin each day for 5 days, the flue-like symptoms occured the first 2 days
and then faded. Subjects that are exposed to the increasing dosages of
endotoxin (0.2-2.0 ng/kg/day), as in the present project, experience no or very
mild symptoms.

- Concerning the 10 minutes of ischemic exercise and the administration of
labeled annexine during the ischemia-reperfusion experiments.
In approximately 140 subjects this protocol was conducted without any side
effects. In theory, an allergic reaction to annexine is possible. This never
occured up to now, but it is mentioned in the information for the participants.
Also, the first 30 minutes and 1 hr and 4 hrs after the administration of
annexine, a medical doctor is with the subjects.

In view of the potential clinical implications we feel it justified to conduct
the present project to confirm or reject the hypothesis of the study.