Macrophage Inhibitory Factor (MIF) and High-Mobility Group-1 Protein (HMG-1) in Children Undergoing Cardiopulmonary Bypass
PURPOSE: To document the presence or absence of the proinflammatory cytokines macrophage
migration inhibitory factor (MIF) and high-mobility group-1 protein (HMG-1) in the serum and
myocardium of children undergoing cardiopulmonary bypass (CPB) and to correlate the presence
or absence of these cytokines with clinical outcome after CPB
BACKGROUND: MIF, a 12.5 kD protein discovered in the 1960s as a substance produced by
sensitized T lymphocytes involved in delayed type hypersensitivity, has emerged as a key
cytokine in the innate immune response to infectious and inflammatory stimuli. Sources of
MIF include monocytes/macrophages, the anterior pituitary, liver, kidney, spleen, and brain.
MIF is released in response to various stimuli including lipopolysaccharide (LPS), toxic
shock syndrome toxin-1, tumor necrosis factor (TNF), and interferon (INF). Once released,
MIF promotes the secretion of other proinflammatory mediators by macrophages and T-cells
thus intensifying the body's immune response. In addition, MIF has the unique ability to
override the anti-inflammatory and immunosuppressive effects of glucocorticoids (Calandra et
al., Nature Medicine, Feb 2000). MIF in combination with LPS potentiates lethality in
murine endotoxemia models, and administration of anti-MIF antibodies increases survival in
murine sepsis models. (Calandra et al., Nature Medicine, Feb 2000). Additionally, MIF
knockout mouse models are resistant to lethal doses of LPS (Bozza et al., J Exp Med, Jan
HMG-1, a 30kD protein discovered in 1973, is a nonhistone chromatin-associated protein that
serves as a DNA binding protein involved in nucleosome stabilization, facilitation of gene
transcription, and as a modulator of steroid home receptor activity. More recently HMG-1
has been implicated as a monocyte/macrophage derived cytokine that serves as a late mediator
of endotoxin lethality (Wang et al., Science 1999). Murine and human macrophages/monocytes
release large amounts of HMG-1 18 hours after exposure to bacterial endotoxin. Serum HMG-1
levels rise 16-36 hours after LPS administration in murine lethal endotoxemia models.
Administration of anti-HMG antibodies attenuates lethality in these models even when
administered 2 hours after LPS exposure. Purified rHMG-1 is lethal to LPS-responsive and
LPS-resistant mice. HMG-1 levels are increased in patients with sepsis and are higher in
non-survivors than survivors. (Wang et al., Science 1999). HMG levels are also increased in
patients with hemorrhagic shock (Ombrellino et al., Lancet 1999). HMG-1 induces TNF release
by cultured human peripheral blood monocytes (Andersson et al., J Exp Med, 2000).
Cardiopulmonary bypass (CPB) triggers an inflammatory state associated with endotoxemia and
cytokine elevation (Lequier et al., Chest, Jun 2000). Cytokines are known to have profound
effects on cardiac function in sepsis. Cardiac depression occurs commonly after CPB and may
be related to cytokine elevation. The role of MIF and HMG-1 in the CPB-induced
inflammatory state are unknown. We have demonstrated the presence of MIF in murine heart
tissue in response to LPS and are currently performing time course studies and functional
assays examining murine cardiac myocyte response to MIF stimulus. In order to examine the
clinical relevance of our laboratory data and to further characterize the inflammatory state
occurring after CPB, we would like to obtain human data correlating MIF levels in human
serum and myocardium with clinical outcome following CPB. Additionally, since the
myocardial depression seen after CPB occurs 12-24 hours after CPB suggesting that a late
mediator of inflammation may be important in post-CPB myocardial depression, we would like
to assess for HMG-1 in the serum of patients undergoing CPB and correlate the presence or
absence of HMG-1 with clinical outcome.
CONCISE SUMMARY OF PROJECT: We propose to study a population of infants and children
undergoing operative repair of congenital heart disease during which there is an expectation
that cardiac tissue will be removed. Patients will have an assessment of cardiac function
by echocardiography as well as blood assays for tumor necrosis factor (TNF), interleukin-6,
interleukin-8, interleukin-10, complement 3a, MIF, and HMG-1 prior to surgery. Cardiac
tissue, removed as a planned part of the surgical procedure will be assayed for MIF as well
as for evidence of apoptosis, a potential mechanism of cell death and myocardial dysfunction
in response to MIF or HMG-1. The patients will be monitored in the PICU for a number of
clinical outcome indicators including vital signs, fluid status, inotropic requirement,
acid-base status, etc. Blood will be obtained prior to surgery and at 1, 8, 24, 48, and 72
hours post-operatively for cytokine assays to characterize the inflammatory response of each
patient. We will then determine if MIF and/or HMG-1 levels correlate with clinical outcome.
SOURCES OF RESEARCH MATERIAL: Blood will be obtained pre- and postoperatively as described.
Heart tissue will be obtained at the time of surgery as a routine part of the procedure.
The biochemical assays performed on the samples are for research purposes only. Clinical
data will be obtained from routinely recorded data from each patient's electronic PICU
RECRIUTMENT OF SUBJECTS: Subjects will be recruited preoperatively in the cardiology clinic
or PICU. Written informed consent will be obtained from a parent/legal guardian by one of
the study investigators.
POTENTIAL RISKS: Study participation will not pose any additional discomfort or stress to
the patient/family. Cardiac tissue removal will be a planned portion of the cardiac
surgical repair regardless of study participation. Blood samples will be obtained from an
indwelling vascular catheter routinely placed prior to surgery and will be collected at
times when routine pre and post-operative tests are normally obtained. The amount of blood
drawn (~4cc/sample) should not cause any hemodynamic compromise or result in additional
blood product replacement. All patients will receive the same quality care and monitoring
in the PICU regardless of study participation. Patients will not be responsible for any
costs generated solely from research.
SPECIAL PRECAUTIONS: All blood samples will be obtained in a sterile fashion. Cardiac
tissue will be obtained in the operating room as a routine part of the surgical procedure.
BIOSTATISTICS: A random sample of 30 subjects will be selected to determine whether or not
MIF and HMG-1 are present in the cardiac tissue as well as serum obtained prior to and
following CPB as outlined above. Descriptive statistics of patient demographics and plasma
and tissue concentrations of indicated cytokines will be correlated to clinical outcome
Observational Model: Cohort, Time Perspective: Prospective
MIF and HMG-1 will be present in the serum of children who have undergone cardiopulmonary bypass. MIF will be present in the myocardium of children who have undergone cardiopulmonary bypass.
Leslie Garner, MD
United States: Institutional Review Board
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