Introduction: In recent scientific literature, 2 proteins, macrophage migration inhibitory
factor (MIF) and high-mobility group-1 protein (HMG-1), have emerged as important mediators
of inflammation and sepsis.
Hypothesis: 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. The presence of MIF and HMG-1 in the serum and MIF in the myocardium
of children undergoing bypass will correlate with clinical outcome.
Methods: We will study a group of infants and children undergoing operative repair of
congenital heart disease during which there is an expectation of cardiac tissue removal.
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, MIF,
and HMG-1 prior to surgery. Cardiac tissue, removed as a planned part of the procedure, will
be obtained from the cardiothoracic surgeons and assayed for MIF and for apoptosis, a
potential mechanism of myocardial dysfunction mediated by MIF and/or HMG-1. The patient will
be admitted to the cardiac intensive care unit post operatively for routine care. Blood will
be obtained at 1, 8, 24, 28, and 72 hours post operatively for the cytokine assays detailed
above. The blood will be drawn from indwelling arterial or venous catheters routinely placed
at the time of surgery. The amount of blood drawn (-4cc per sample) is unlikely to cause any
hemodynamic compromise or result in additional blood product replacement.
Sample size and Analysis Plan: 30 subjects will be enrolled to determine the presence or
absence of MIF/HMG-1 in the serum and cardiac tissue pre and post cardiopulmonary bypass.
Descriptive statistics of patient demographics and clinical outcome variables will be
correlated to serum and myocardial concentrations of the various cytokines.
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 1999).
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 chart.
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
1. Any child under the age of 5 undergoing operative repair of congenital heart disease
on CPB where there is an expectation of cardiac tissue removal.
2. Written informed consent obtained from a parent/legal guardian.
1. Unable to obtain informed consent
2. Evidence of ongoing infection
3. Known immunodeficiency