Endotoxin-induced Inflammatory and Behavioral Responses and Predictors of Individual Differences
Condition: Sickness Behavior
MeSH terms: Illness Behavior
Study type: Interventional

Summary: 
      The objective of the present study is to specifically assess the effect of lipopolysaccharide
      (LPS) administration on the development of behavioral symptoms and the underlying
      contribution of inflammatory processes. In particular, the investigators will assess the
      development of subjective and objective behavioral symptoms. In addition, the investigators
      will determine whether some psychological trait or state can predict and/or modulate the
      LPS-induced inflammatory and behavioral responses.

      Twenty-five healthy subjects will be included. A placebo-controlled, double-blinded and
      cross-over design will be used. Subjects will receive an intravenous injection of endotoxin
      at 2 nanogram/kilogram (ng/kg) of body weight and an intravenous injection of sodium chloride
      as placebo of endotoxin injection at two different occasions.

      Prior to inclusion and randomization, subjects will come at the hospital and will receive a
      medical examination. Psychological variables that could affect the behavioral (or immune)
      response to LPS will be assessed at that time, using several self-assessment questionnaires.

      On the trial days, injection of endotoxin or sodium chloride will be performed and blood
      samples will be taken just before the endotoxin or sodium chloride injection and 1, 1.5, 2,
      3, 4, 5, 6 and 7.5 hours after the injection. Blood samples will be used to measure several
      inflammatory and immune markers. Urine samples will be taken before the endotoxin or sodium
      chloride injection and as late as possible after the injection. Subjects will wear T-shirt
      all day. Urine and T-shirt samples will be used for behavioral assessment and analysis of
      body odor compound.

      Self-assessment questionnaires assessing behavioral and psychological variables will be
      completed by participants just before the endotoxin or sodium chloride injection, three hours
      and 7.5 hours after the injection. A short questionnaire assessing sickness behavior
      (SicknessQ) will be repeatedly completed by participants from just before to 7.5 hours after
      the endotoxin or sodium chloride injection.

      Several behavioral tests will be used, including a motivation task, a test assessing
      behavioral response to negative and sickness stimuli. Analysis of gait and motion, as well as
      of social interactions, will be performed. Photographs will be taken for the further rating
      of the faces.
    

Detailed description: 
      Study design

      Twenty-five subjects will be included in the present study, which will follow a prospective,
      placebo-controlled and cross-over design. Participants will be blinded, as well as research
      personnel in charge of the participants and the laboratory.

      Subjects will be randomly assigned in the cross-over design with two trials, with about four
      weeks wash-out between LPS stimulations:

        1. Trial 1: endotoxin. Subjects will receive an intravenous injection of endotoxin at 2
           ng/kg of body weight.

        2. Trial 2: placebo. Subjects will receive an intravenous injection of sodium chloride as
           placebo of endotoxin injection.

      Ten other healthy volunteers will be recruited in a pilot study prior the beginning of the
      main study, in order to test the behavioral tasks that will be used. These subjects will be
      recruited among the university staff or among university students. They will perform four
      specific behavioral tasks and will complete the questionnaires assessing psychological and
      behavioral variables in order to associate outcomes from the behavioral tasks to
      psychological and subjective behavioral assessments. No biological samples (blood, urine or
      tee-shirt samples) will be taken in subjects recruited in the pilot study.

      Treatment

      The LPS is provided by LGC Promochem AB, Albanoliden 5, 4tr, 506 30 Borås, Sweden. The USP
      (United States Pharmacopeial Convention) Endotoxin (Cat. No. 1235503) does come as a
      lyophilized (freeze-dried) powder and each vial contains 10,000 USP Endotoxin Units. Each
      vial contains very approximately 1 microgram of Endotoxin and is mixed with 5 milliliter (mL)
      of sterile water.

      Study procedure

      The study will be conducted at Department of Clinical Sciences, Danderyd Hospital.

      Prior to inclusion and randomization, subjects will come at the hospital in order to be
      subjected to a medical examination, including an electrocardiogram and blood laboratory
      analyses. Socio-demographic data and medical history will be assessed at this occasion, as
      well as body variables (weight, height, body composition) and physical activity using the
      Swedish version of the IPAQ (International Physical Activity Questionnaire). History of major
      depression will be assessed using the Mini-International Neuropsychiatric Interview
      (M.I.N.I.) for major depression. Psychological variables that could affect the behavioral (or
      immune) response to LPS, and which are not believed to change between the screening day and
      the trial days, will be also assessed at that time, using several web-based self-assessment
      questionnaires. These measures will include the subjects' perception of the body and health
      risk (e.g., interoceptive awareness, perceived vulnerability to disease, health anxiety and
      disgust reactivity, somatosensory amplification, fear of anxiety-related sensations) and
      psychological well-being (e.g., perceived stress, optimism, social support, cognitive
      well-being). The overall time for the medical examination will be of 30-45 minutes. The time
      to complete questionnaires is estimated to approximately 45 minutes but can be done from
      home. The participants will also receive a kit with a T-shirt (with sewn-in cotton pads) and
      a scent-free soap. Participants will be asked to wear the T-shirt in the morning (after
      washing armpits) on each trial day, and take it off after their arrival. This will allow
      collecting pre-treatment (baseline) samples of body odor.

      Participants will then be subjected to the two trials performed in a randomized order, with
      at least six weeks of wash-out: endotoxin versus placebo.

      The participants will be asked to restrain from strenuous exercise and alcohol taking 48
      hours before the trial day, to avoid strong spices 24 hours before the trial day, and to take
      a light breakfast the morning of the trial day. Participants will arrive on the morning (at
      7.30AM). They will turn in their morning T-shirt (see above) and change to a new T-shirt
      (with sewn-in cotton pads in the armpit) to wear during all day. The T-shirts will be used
      for behavioral assessment and analysis of body odor compound. Shortly after their arrival,
      two intravenous catheters will be placed: one for blood sampling at one arm and one at one
      hand for the injection of endotoxin or sodium chloride that will be removed after the
      injection. An anesthetic cream will be provided to spread on their arm in order to minimize
      the pain of venipuncture. Subjects will then rest for at least 30 minutes.

      Injection of endotoxin or sodium chloride will be performed when the rest is completed
      (around 8.30AM). Blood samples (10 mL in EDTA (Ethylenediaminetetraacetic acid) tubes and 10
      mL in sodium citrate tubes) will be taken just before the endotoxin or sodium chloride
      injection and 1, 1.5, 2, 3, 4, 5, 6 and 7.5 hours after the injection. In addition, 2.5 mL in
      PAXGene™ tubes will be taken before and 2, 4 and 7.5 hours after the injection. Two
      additional samples in EDTA tubes (8 mL of blood) will be taken before the injection and 3 and
      7.5 hours after the injection. The total quantity of blood taken will be of about 200 mL,
      corresponding to a third of the amount given at a blood donation. Blood samples will be used
      to measure concentrations of pro- and anti-inflammatory cytokines and other inflammatory
      markers (e.g., C-Reactive protein - CRP) using Enzyme-Linked Immunosorbent Assay (ELISA),
      immune cell subpopulations and concentrations of microparticles using flow cytometry, genetic
      markers using Cap analysis gene expression (CAGE) analyses and white blood cell count and
      differential.

      Urine samples will be taken before the endotoxin or sodium chloride injection (i.e., before
      the catheters are placed) and as late as possible after the injection (classically around
      four hours after the injection).

      Participants will be monitored with continuous electrocardiogram. Oxygen saturation (SO2),
      blood pressure and body temperature will be monitored every 30 minutes. Nitrogen dioxide
      (NO2) will be monitored continuously, methemoglobin checked at the same time than each blood
      sample.

      Self-assessment questionnaires assessing behavioral and psychological variables will be
      completed by participants just before the endotoxin or sodium chloride injection (during the
      rest period), three hours and 7.5 hours after the injection. A short questionnaire assessing
      sickness behavior (SicknessQ) will be repeatedly completed by participants from just before
      to 7.5 hours after the endotoxin or sodium chloride injection. Subjects will be filmed when
      in bed and will wear a sociometric badge, in order to analyze participants' behavioral
      response to a standardized situation of social interaction.

      Photographs will be taken before (i.e., before the catheters are placed), two hours after and
      7.5 hours after the injection, for the further rating of the faces (e.g., health,
      attractiveness, tiredness). In parallel, skin color will be measured at the same time than
      blood samples (except T1.5) in order to assess a potential mediating effect of faces' health
      ratings. Analysis of subjects' gait and motion will be performed two and half hours after the
      injection. Short computerized cognitive tests will be performed after these tests, just
      before three hours after the injection. Subjects will perform a motivation task after
      completing the questionnaires three hours after the injection. In addition, two tests
      assessing behavior in response to positive/negative and healthy/sickness stimuli (an
      approach-avoidance behavior test and a regulation of emotion test) will be executed three
      hours and half after the injection.

      Participants will be allowed to take a light meal (snack and juice drink) around lunch time
      (12.30) and will hand in their T-shirt just before five and half hours after the injection.

      The last blood samples will be taken 7.5 hours after the injection and participants will be
      discharged.

      Monitoring of the experiment will be done by a medical doctor, supervised by a specialist in
      anesthesia and intensive care.
    

Intervention name(s): Endotoxin; Placebo
Intervention type(s): Biological; Biological
Other intervention name(s): LPS; lipopolysaccharide; NaCl 0.9%; saline
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: Triple (Participant, Investigator, Outcomes Assessor)
Primary purpose: Basic Science
Keywords: Sickness behavior; Cytokines; Psychoneuroimmunology