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PROTOCOL #:
COMIRB Protocol
(Use Protocol Manager on the COMIRB Website)
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD
CAMPUS BOX F-490 TELEPHONE: 303-724-1055 Fax: 303-724-0990
Project Title:
Burn Trauma and Infection: Mechanisms by which inhalation injury exacerbates
burn injury-induced pulmonary pathology
Principal Investigator: Ellen Burnham
I. Hypotheses and Specific Aims:
Hypothesis of the Study
The working hypothesis is that in burn patients, inhalation injury and/or infection cause a marked
increase in both the pulmonary and systemic inflammatory response relative to burn injury alone.
Technical Objectives
To address this hypothesis, experiments separated into 2 aims are designed to:
1) characterize effects of inhalation injury on the pulmonary and systemic inflammatory response in
burn patients; and
2) correlate levels of inflammatory mediators in the lungs and blood of burn patients with and
without inhalation injury with outcomes, including pneumonia.
Aim 1: To characterize effects of inhalation injury on the pulmonary and systemic inflammatory
response in burn patients.
Aim 2: To correlate the levels of inflammatory mediators in the lungs and blood of burn patients
with and without inhalation injury with outcomes, including pneumonia
II. Background and Significance:
Trauma, including burn injury, is the leading cause of death among those under the age of 50 in the
U.S. While improvements in resuscitation strategies have markedly improved early mortality,
deaths due to sepsis and multiple organ failure (MOF) remain unchanged. Two million people per
year suffer burn injury in the U.S., and 40,000 of these subjects require hospitalization. Patients
who sustain burn injury with inhalation injury are more likely to suffer long term pulmonary
complications (in part, as a result of mechanical ventilation), require more surgical procedures and
more rigorous antibiotic therapy, and stay in the hospital longer than those who sustain burn injury
without inhalation injury.
Inhalation injury occurs in about 30% of all major burns and accounts for a significant number of
deaths. Results of proposed studies will provide a better understanding of pulmonary infections
associated with burn injury and thereby potentially lead to more effective treatment modalities that
will decrease the associated morbidity and mortality
This proposal is designed to examine the production of acute mediators of inflammation. The
resuscitation and stabilization phase will be the focus of this proposal. Pulmonary fluid, exhaled
breath condensate peripheral blood will be collected at the time of admission bronchoscopy. The
first samples obtained upon admission will provide important information regarding the initial
inflammatory response that could lead to the successful development of diagnostic and therapeutic
strategies. Additional BAL samples and exhaled breath will be obtained if bronchoscopy is done
for usual care. The sampled cells will be quantified by microscopy and cultured for reactive
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properties. The fluid will be tested for TNF-α, IL-1β, IL-6, IL-8, MIP-1α, MCP-1, PDGF , IL-10 and
TGF-1.
In addition, the measurement of neutrophil elastase, alpha(1)-antitrypsin (A1AT) levels and
assessment of ubiquitin and proteosomes in the BAL fluid will be performed.
Laboratory measurements will be examined for correlation with clinical indicators
of injury severity, acute lung injury scores, multiple organ failure index, and
peripheral cellular response and reactivity.
.
III. Preliminary Studies/Progress Report:
Previous studies (8) reveal that the lungs of burn patients with more severe grades of inhalation
injury have higher levels of pro-inflammatory mediators in bronchoalveolar lavage (BAL) fluid and
greater production of those mediators by BAL cells stimulated in vitro when compared with burned
subjects with little or no inhalation injury. In addition, evidence suggests that lungs are particularly
susceptible to systemic inflammatory signals as well as those generated in the lung itself. This
may be in part because the lung has a large capillary bed and is the first organ exposed to
activated leukocytes returning from the systemic circulation. Systemic and local production of proinflammatory mediators, like TNF-, IL-1 and IL-6, may facilitate the accumulation of leukocytes in
the lung through up-regulation of adhesion molecules and stimulation of chemokine production.
Chemokines, including IL-8 and monocyte chemoattractant protein-1 (MCP-1), in turn, recruit
neutrophils and monocytes, respectively, from the blood to the pulmonary interstitium and alveolar
space. Because of the additional airway damage caused by inhaled smoke, it is expected that the
production of mediators in the lungs of patients with higher grades of inhalation injury in addition to
burn injury will be greater, thus resulting in excessive tissue damage.
IV. Research Methods
A. Outcome Measure(s):
The sampled cells will be quantified by microscopy and cultured for reactive properties. The fluid
(both BAL and exhaled breath) will be tested for TNF-α, IL-1β, IL-6, IL-8, MIP-1α, MCP-1, PDGF ,
IL-10 and TGF-1.
In addition, the measurement of neutrophil elastase, alpha(1)-antitrypsin (A1AT) levels and
assessment of ubiquitin and proteosomes in the BAL fluid will be performed.
Laboratory measurements will be examined for correlation with clinical indicators
of injury severity, acute lung injury scores, multiple organ failure index, and
peripheral cellular response and reactivity.
Initial assessments will focus on the levels of pro-inflammatory and fibrogenic cytokines in the
lungs and blood obtained from burn patients with and without inhalation injury. We will also
quantitate the pulmonary leukocyte number and phenotype in both the pulmonary compartment
and the circulation, and the leukocytes will be cultured and stimulated in vitro to determine their
cytokine production capacity. In addition, studies will examine the levels of ubiquitin and
proteosomes as well as the degree of neutrophil activation in lung and blood. Evidence reveals
that extracellular ubiquitin is an endogenous immune modulator with anti-inflammatory actions.
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Increased levels of ubiquitin are found in fluids obtained from trauma and sepsis patients (40), and
burn patients who develop sepsis/MOF often have a relative ubiquitin deficiency (41). Similarly,
recent studies show that proteosomes are also released into the alveolar space after injury and
suggest that proteosomes are involved in the clearance of lung edema after lung injury (42).
Proteases are normally regulated tightly by their naturally occurring endogenous inhibitors.
However, in some pathological conditions protease levels are increased, which in turn can cause
excessive tissue damage by hydrolyzing extracellular matrix components such as elastin,
fibronectin, and collagen. A major hallmark of lung injury is the recruitment of neutrophils and
release of proteases such as elastase. In addition, it is possible that an increase in neutrophil
elastase release could result from a decrease in proteases inhibitors, such as A1AT. Although
multiple mechanisms may exist for an increase in neutrophil elastase activity following burn injury,
results from this study will help delineate whether there are further increases in elastase levels after
smoke inhalation and burn injury, and if that increase is related to a reduction in anti-protease
activity.
B. Study Design and Research Methods
Bronchoscopy is performed in burn patients admitted to the ICU at UCH when smoke inhalation
injury is suspected by history (i.e. delayed extrication from burning area, loss of consciousness,
stridor/shortness of breath as symptoms/signs), physical examination (i.e. soot in nares, or in
tracheal secretions), or by laboratory findings (i.e. elevated carboxyhemoglobin level on an arterial
blood gas). Additionally, the presence and severity of inhalation injury is also documented as a
way to guide subsequent treatment decisions (i.e. additional fluid resuscitation, need for inhaled
medications, need for pulmonary toilet). Grading injury may also serve in a prognostic role to
determine future outcomes [ref: Mosier MJ 2011]. Serial bronchoscopies may be performed for
changes in patient condition (such as mucous plugging), or to re-address findings observed on
initial bronchoscopy, such as adherent soot or increased friability. For this proposal, we will
collected pooled, leftover BAL fluid from all bronchoscopies that are performed as part of usual
care. We will also collect up to 5 mL of breath exhaled from patients that is normally vented into
the atmosphere and correlate this with the grade of injury.
Blood work that is performed as standard of care in patients with suspected inhalational injury
includes: measurement of arterial blood gas with carboxyhemoglobin quantification, complete blood
count, comprehensive chemistry panel, and lactate. The latter two measurements are performed
to assess for acidosis that may be observed in cyanide poisoning, common in the context of
inhalational injury. When acidosis is present, treatment for cyanide poisoning would be initiated
promptly. While patients are intubated on mechanical ventilation, and their conditions are changing
rapidly, it is standard to obtain an ABG, CBC, and comprehensive chemistry panel at least daily.
Blood work is typically obtained through an indwelling catheter such as an arterial line or central
venous line. While patients are intubated in an intensive care setting, it is also standard to have an
indwelling Foley urinary catheter present to enable monitoring of urine output as a way to assess
the adequacy of volume repletion. Urine is measured every shift and then discarded, unless
patient signs/symptoms suggest a urinary tract infection. For this proposal, we will need to collect
5cc of whole blood on one occasion.
Time sensitive nature of sample collection and analysis:
We are interested in the effects of alcohol and burn injury on the function of alveolar macrophages
and other cells found within the lung, and intend to measure cytokines and growth factors
elaborated by these cellsin an in vitro culture system. To enable us to assess these cells' function
within the clinical context of the patient's illness, we will need to assay the cells as soon as possible
after collection, as delays in processing may affect the type(s) of cytokines/growth factors that are
secreted by these cells. Additionally, freezing or otherwise preserving freshly collected cells would
be expected to affect their viability; as such, it is less likely we would be able to culture
cryopreserved cells successfully to complete the goals of the project. Therefore, collection and
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assay of these cells immediately after collection is preferable, although this might mean delay in
the subject's consent for use of his/her cells in research. Collecting exhaled breath at the time of
bronchoscopy, and measuring elements from both breath and BAL may help to develop the use of
exhaled breath as a non-invasive biomarker.
There are no study specific interventions with the subjects and the study poses no greater than
minimal risk to participants. Since the majority of subjects will not be able to provide their own
consent and sample collection must start immediately upon admission to the Burn ICU, we will
attempt to enroll all eligible patients upon admission. As soon as possible and dependent on the
patient’s ability to consent (as assessed by a standard bedside assessment of patients being alert
and orientated to person, place and time), we will approach the subject for informed consent.
Discussion of the study will stress that participation is voluntary and that declining to participate will
not affect any medical care provided in order to minimize coercion and undue influence. Patients
will be given as much time as necessary to consider participation and will have an opportunity to
ask any questions they might have about participating. Attempts to obtain consent will be
documented in the research record. Samples will be discarded if the subject expires, does not
regain decisional capacity or refuses participation.
Informed consent will be obtained from all patients in accordance with IRB guidelines. We will
enroll patients fitting the inclusion and exclusion criteria listed below.
Aim 2: To correlate the levels of inflammatory mediators in the lungs and blood of burn patients
with and without inhalation injury with outcomes, including pneumonia
The 125 patients enrolled in Aim 1 above will be followed clinically to assess early and late
outcomes as previously described (43). Patient specific data will be managed according to HIPAA
and IRB guidelines to ensure confidentiality; the clinical data listed in appendix A (Case Report
Form) will be collected. ALI/ARDS will be defined by the American-European Consensus
Conference on ARDS and expressed as occurrences per ventilator days. This definition includes
acute onset, bilateral infiltrates on chest radiograph, pulmonary artery occlusion pressure ≤18
mmHg (or the absence of clinical evidence of left atrial hypertension) and a PaO2/FiO2 ratio ≤300
for ALI or ≤200 for ARDS. The incidence of pneumonia and the number of transfusions will be
recorded as these can also lead to ALI and ARDS. Pneumonia will be defined by CDC criteria and
expressed as occurrences per ventilator days. Each of the parameters will be examined for
possible correlation with the measures of pulmonary and systemic inflammation described in Aim 1
above. We will review and collect information from the medical record for up to five years. Some
of the data to be collected includes:
1. Demographics such as age, gender, race
2. Information regarding injury.
3. Medical history, including history of AIDS or HIV, admission blood alcohol level
and previous treatment for Alcohol and Drug Abuse and physical exams.
4. Admission weight.
5. Presence or absence of inhalation injury. Grade of inhalation injury.
6. Fluid resuscitation requirements; 24, 48 and 72 hour totals.
7. Insulin requirements over 24 hours.
8. Initial base deficit.
9. Length of time on ventilator.
10. Length of stay in the intensive care unit (ICU).
11. Denver and/or Marshal multiple organ failure scale.
a. 24, 48, and 72 hours.
b. Weekly while in the ICU.
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12. Survival.
13. Infectious complications.
a. Pneumonia.
b. Central line infection.
c. Wound infections.
d. Urinary tract infections
14. Date and number of operative interventions.
15. Blood transfusion requirements.
16. Estimated and measured metabolic rate.
17. Initial P:F ratio
18. Presenting CO level
19. BAL culture and cell differential specimen results
20. Daily review of patients condition
Research Procedure 1 - collection of BAL and exhaled breath condensate (EBC)
Bronchoalveolar lavage (BAL) fluid samples will be obtained from intubated patients in the ICU by a
standardized protocol. Initially, the bronchoscope is directed into the left lower lobe and wedged,
the first 20 ml aliquot of saline instilled, and the aspirate discarded. The bronchoscope is then
repositioned into a different sub-segmental bronchus in the same lobe, another 20 ml is instilled,
and the fluid gently aspirated into a sterile Lukens’s fluid trap. Additional one to four 20 ml aliquots
are then collected from the other four lobes as tolerated by the patient. That fluid not required for
routine clinical purposes will then be pooled and saved for research use. The first sample will be
obtained at the time of diagnostic bronchoscopy that is performed at the time of admission for all
injured patients in whom it is clinically indicated. The research BAL specimen will be placed on ice
and transported immediately to the research laboratory. EBC will be collected immediately prior to
bronchoscopy (10 min prior). The collection device will be placed in series with the exhalation port
of the ventilator where it will remain for 10 min to collect breath being exhaled by the patient. It will
then be removed, capped, and placed on ice/transported to lab immediately.
Research Procedure 2 – collection of whole blood
Up to 5 ml of whole blood will be obtained in sodium citrate tubes from patients at the time of their
standard blood draws, as close to the time of the bronchoscopy as possible. This will be performed
by the patient’s nurse in accordance with hospital guidelines from either a central venous catheter
or peripheral arterial catheter previously placed out of clinical necessity or by venipuncture. The
research blood specimen will be placed on ice and transported immediately to the research
laboratory.
Research Procedure 3 – laboratory processing of BAL
A volume of wash buffer (PBS with 5% FBS and 1% PSG) will be added to the BAL sample. This
will then be poured through a screen (BD Falcon 100 micron cell strainer) once into a 50 ml BD
Falcon conical tube. The sample will then be spun in a refrigerated centrifuge at 1200 rpm for 5
minutes, the supernatant harvested, and stored in 300µL aliquots at -80ºC for later analysis as
below.
Research Procedure 4 – BAL leukocyte analysis and culture
Centrifugal fractionation of cells in the BAL fluid will be performed (45), and pulmonary total
leukocyte and differential counts will be obtained as previously described (10). Specifically, BAL
cells will also be cultured as follows. The cell pellet obtained from Research Procedure 3 will be resuspended in wash buffer with the addition of 0.5mg DNase and 5 mg MgCl, and incubated at 37ºC
for 10 min. This will subsequently be spun on a refrigerated centrifuge at 1200 rpm for 5 min, the
supernatant discarded, the cell pellet re-suspended with 5 ml ACK RBC lysis buffer, and the
sample incubated at room temperature for 2 min. Again, the sample will be spun on a refrigerated
centrifuge at 1200 rpm for 5 min, the supernatant discarded, and the cell pellet re-suspended in 5
ml RPMI medium with 5% FBS and 1% PSG. At this time 10 µL of sample will be stained with
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Trypan Blue, the cells counted, and the sample re-suspended to 106 cells/ml in the above medium.
The resultant sample will then be cultured separately in 96 well culture plates (100 µL per well) t
37ºC and 5% CO2 in either the presence of LPS (100 ng/ml), TNF-α (300 pg/ml), or IL-6 (300
pg/ml) for 20 hours. The plates will then spun at 1080 RPI for 5 min, the supernatants harvested,
vortexed, and stored in 110 µL aliquots at -80ºC for later analysis as below.
Research Procedure 5 – Laboratory processing of whole blood
The whole blood will be spun in a refrigerated centrifuge at 600xG for 10 min. The plasma will then
be separated and stored in 110 µL aliquots at -80ºC for later analysis as below.
Research Procedure 6 - assessment of cytokines
Cytokine concentrations in BAL fluid, plasma, EBC and cell culture supernatant will be measured
by multiplex analysis (Bio-Rad Bio-Plex Assays, Hercules, CA) per the manufacturer’s instructions.
Briefly, 50 μl of each sample will be added to a 96 well filter-bottom plate containing 50 μl of
antibody coated fluorescent beads. In combination with several incubation and washing steps,
biotinylated detection and streptavidin-PE antibodies are added to the plate and after resuspension
of the beads the plate is read on a Luminex 100 reader using a high throughput fluidics system. All
samples will be assayed in duplicate and the results averaged, and the data will be analyzed using
the Bio-Plex manager software, version 5.0.
Research Procedure 7 - Measurement of neutrophil elastase and alpha(1)-antitrypsin (A1AT) levels
in BAL fluid:
A1AT and neutrophil elastase will be measured in BAL fluid and EBC using ELISA kits from ICL,
Inc. (Newberg, OR) and Cell Sciences (Claton, MA), respectively, per the manufacturer’s
instructions.
Assessment of ubiquitin and proteosomes in BAL samples: Quantification of ubiquitin, and 20S
and 26S concentrations in BAL will be performed with newly developed ELISAs (42, 46).
Proteosome activities will be assessed using specific fluorogenic peptide substrates for the main
proteolytic activities of the proteosomes in combination with the highly specific proteosome inhibitor
epoxomicin as described previously (41, 42)
Collection of clinical data will begin soon after the samples are obtained. Information will be
entered onto a case report form (see attached) and saved with a unique identifying number in order
to de-identify the data. We will collect outcomes data until hospital discharge.
C. Description of Population to be Enrolled:
Inclusion Criteria:
1. Patients with burn injury, with or without smoke inhalation injury, who are intubated.
2. Patients undergoing diagnostic bronchoscopy for assessment of the level of smoke
inhalation injury within the first 24 hours after injury.
Exclusion Criteria:
1. Age less than 18.
2. Patients with co-morbid malignancy.
3. Patients taking immunosuppressive medications (i.e., corticosteroids).
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4. Patients with known autoimmune or chronic inflammatory diseases.
5. Pregnant or lactating women
6. Decisionally challenged
7. Prisoners
SUBJECT RECRUITMENT & SCREENING
Dr.’s Burnham and Lindberg along with the research coordinators will monitor patients entering the
Burn Unit with appropriate inclusion/exclusion criteria.
Study subjects will be intubated and sedated and therefore will not initially be approached for the
consent process. Due to the time sensitive nature of specimen collection and analysis, we will not
obtain consent until after the subject is extubated and regains decisional capacity. Should a subject
expire, not regain decisional capacity, or expire, all study samples and data collected with be
destroyed.
A master enrollment log will be kept of subjects enrolled and the date of enrollment. Information
recorded will include: name, medical record number, date enrolled, and participant numbers in
sequence {01, 02, etc.}. This information will be kept separate from the case report form and will
not be included in specimen labeling. Since this information is PHI, it will be kept on a secure,
password protected computer in the PIs office.
D. Description, Risks and Justification of Procedures and Data Collection Tools:
Bronchoalveolar lavage (BAL) - The potential side effects or problems that may be associated with
this study procedure include infection, bleeding, irregular heart beats, mucous plugging and
temporary decrease in the amount of oxygen in the blood. Because the procedure is being
performed as part of the usual care, there are no additional risks from the research.
Exhaled breath condensate (EBC)—There are no known potential side effects or problems
associated with collecting exhaled breath from patients. Breath would normally be vented into the
atmosphere.
5 ml of blood (about 1 teaspoons) will be collected for the study. The blood will be drawn from an
existing central venous line or arterial line and therefore the common risks of venipuncture will not
apply.
There is a risk of the potential loss of confidentiality
If the subjects are injured or have side effects as a result of participating in this research project,
their doctor will take the necessary steps to treat the problem. The subject will be responsible for
the cost of care of any problems that occur while they are participating in this research project. No
other compensation is available from the University of Colorado Denver or the sponsor of this
research.
CONFIDENTIALITY
All patient health information (PHI), demographics and sensitive data will be collected prospectively
in a master database at which point identifying data will be removed. Patients will be referred to by
their assigned study number only. Research records will be stored in a confidential manner so as to
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protect the confidentiality of subjects’ information. Paper records linking the patient and his study
id will be kept locked in the offices of principal investigator. No sensitive patient information will be
distributed via unencrypted email.
Access to the PHI will be limited to the principal investigator, co-investigators, and clinical research
coordinators.
Data will be collected prospectively, pooled, statistically analyzed and formatted for presentation at
region or national critical care meetings and/or publication in peer reviewed surgical/medical
journals for the purpose of advancing medical knowledge. The patient will not be identified by
name or by any other identifying information in any publication or report about this research..
F. Data Collection and Data Analysis Plan:
Each sample will be labeled with the subject code number (01, 02, etc). These samples will
be delivered to Dr. Burnham’s lab in Research Complex 2 Building for processing
(according to the protocol). Medical records will be reviewed daily to assess for routine care
done on the subject and laboratory values and events will be recorded. By reviewing the
medical records the study team can obtain samples necessary during procedures done as
routine care for this population. BAL samples will be obtained as per usual care and a one
time blood sample of 5cc whole blood will be collected.
Analysis for Aim 1
The analysis for Aim 1 will be descriptive in nature. Means, standard deviations, minimum and
maximum values for inflammation biomarkers (e.g., TNF-) will be tabled as a function of source
(lung lavage, peripheral blood), body mass index (using categories of underweight, normal, and
overweight developed by the Centers of Disease Control), sex, and inhalation injury grade (scored
as 0, 1, 2, 3, 4).
Analysis for Aim 2
The analyses for Aim 2 also will be descriptive, but the focus will be on quantifying relationships
between inflammatory biomarkers (from pulmonary lavage and peripheral blood) and clinical
outcomes. The p values that are generated from these analyses will be examined, but they will not
be relied upon in the classical sense as indicating statistically significant findings. The proposed
research is novel and highly exploratory. As such, many measures of association will be computed
and the investigators will focus more on the magnitude of the associations, as opposed to their
statistical significance. The findings of these analyses will be reported to the burn trauma
community and will be used to plan future studies in this area.
One group of analyses will be to measure the association of the levels of inflammation biomarkers
(TNF-α, IL-1β, IL-6, IL-8, MIP-1α, MCP-1, PDGF, IL-10, and TGF-1) with the grade of inhalation
injury (scored 0, 1, 2, 3, 4). This will be done with a one-way analysis of variance (ANOVA). The
means of the biomarkers will be observed across the grade of inhalation injury and the eta-square
coefficient will be computed (= sum of squares between groups / total sum of squares).
Obviously, it would be of greater sophistication to analyze the biomarker data by way of a multifactor ANOVA (i.e., grade of inhalation injury by time). However, unknown rates of patient drop out
due to death and withdrawal, as well as unknown numbers of patients within the different grades of
inhalation injury, make the use of higher order designs tenuous.
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Other analyses correlating inflammation biomarkers with clinical variables (e.g., multiple organ
failure, insulin requirements) and outcomes (e.g., pneumonia, length of stay) will be conducted with
the Pearson Correlation Coefficient. Given the availability of data, multiple regression and multiple
logistic regression analyses will be considered. For example, an analysis could be structured to
predict pneumonia (yes, no) from variables such as an inflammation biomarker (e.g., TNF-α), burn
size, and time on a ventilator.
LABELING & STORAGE OF DATA & SPECIMENS
The informed consent document states that the samples taken for study purposes will be stored
(frozen) and may be used for future studies. The patient may choose on the consent document to
participate in the study but not allow the samples to be stored. The patient must sign for sample
storage or they will be discarded after analysis. Consenting samples will be stored in locked
laboratories with limited access (personnel in that laboratory) and may be kept for up to 15 years
after the last participant completes the study. The study staff may destroy the samples before
then. If the subject withdraws consent to participate, the samples will be destroyed, and Dr.
Burnham will only keep and study information collected/generated up to that point if approved by
the subject.
Any samples processed at the University of Colorado laboratory will have the results stored in the
subject’s electronic medical record (EPIC).
As described above, once consent has been obtained, the original signed copy will be stored in a
locked cabinet in the research team’s office.
After each subject completes the study, the Case Report Forms will be entered into RedCap with
the original paper record being destroyed. Only de-identified data will be stored in electronic
format.
G. Summarize Knowledge to be Gained:
The results of this study may provide insight into the inflammatory response to injury
in burn patients with inhalation injury and future patients may benefit from the
knowledge gained from this study
H. References:
1.
2.
3.
4.
5.
Faunce, D. E., Gregory, M. S., and Kovacs, E. J. (1997) Effects of acute ethanol exposure
on cellular immune responses in a murine model of thermal injury. J Leukoc Biol 62, 733740
Faunce, D. E., Llanas, J. N., Patel, P. J., Gregory, M. S., Duffner, L. A., and Kovacs, E. J.
(1999) Neutrophil chemokine production in the skin following scald injury. Burns 25, 403410
Messingham, K. A., Heinrich, S. A., and Kovacs, E. J. (2001) Estrogen restores cellular
immunity in injured male mice via suppression of interleukin-6 production. J Leukoc Biol
70, 887-895
Gregory, M. S., Duffner, L. A., Faunce, D. E., and Kovacs, E. J. (2000) Estrogen mediates
the sex difference in post-burn immunosuppression. J Endocrinol 164, 129-138
Gregory, M. S., Faunce, D. E., Duffner, L. A., and Kovacs, E. J. (2000) Gender difference
in cell-mediated immunity after thermal injury is mediated, in part, by elevated levels of
interleukin-6. J Leukoc Biol 67, 319-326
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6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
Messingham, K. A., Messingham, K. A., Shirazi, M., Duffner, L. A., Duffner, L. A.,
Emanuele, M. A., Kovacs, E. J., Kovacs, E. J., Kovacs, E. J., and Kovacs, E. J. (2001)
Testosterone receptor blockade restores cellular immunity in male mice after burn injury. J
Endocrinol 169, 299-308
Endorf, F. W., and Gamelli, R. L. (2007) Inhalation injury, pulmonary perturbations, and
fluid resuscitation. J Burn Care Res 28, 80-83
Mosier, M. J., Gamelli, R. L., Halerz, M. M., and Silver, G. (2008) Microbial contamination
in burn patients undergoing urgent intubation as part of their early airway management. J
Burn Care Res 29, 304-310
Davis, K. A., Santaniello, J. M., He, L. K., Muthu, K., Sen, S., Jones, S. B., Gamelli, R. L.,
and Shankar, R. (2004) Burn injury and pulmonary sepsis: development of a clinically
relevant model. J Trauma 56, 272-278
Murdoch, E. L., Brown, H. G., Gamelli, R. L., and Kovacs, E. J. (2008) Effects of ethanol on
pulmonary inflammation in postburn intratracheal infection. J Burn Care Res 29, 323-330
Patel, P. J., Faunce, D. E., Gregory, M. S., Duffner, L. A., and Kovacs, E. J. (1999)
Elevation in pulmonary neutrophils and prolonged production of pulmonary macrophage
inflammatory protein-2 after burn injury with prior alcohol exposure. Am J Respir Cell Mol
Biol 20, 1229-1237
Herndon, D. N., Barrow, R. E., Linares, H. A., Rutan, R. L., Prien, T., Traber, L. D., and
Traber, D. L. (1988) Inhalation injury in burned patients: effects and treatment. Burns Incl
Therm Inj 14, 349-356
Moore, F. A., Sauaia, A., Moore, E. E., Haenel, J. B., Burch, J. M., and Lezotte, D. C.
(1996) Postinjury multiple organ failure: a bimodal phenomenon. J Trauma 40, 501-510;
discussion 510-502
ABA (2002) National Burn Repository: 2002 Report, American Burn Association, Chicago,
IL
Fitzwater, J., Purdue, G. F., Hunt, J. L., and O'Keefe, G. E. (2003) The risk factors and
time course of sepsis and organ dysfunction after burn trauma. J Trauma 54, 959-966
Deitch, E. A., and Goodman, E. R. (1999) Prevention of multiple organ failure. Surg Clin
North Am 79, 1471-1488
Bulger E, J. G., LM Gentillelo, RV. Maier (2000) Current clinical options for the treatment
and management of acute respiratory distress syndrome. J Trauma 48, 562
Demling, R. H. (2008) Smoke inhalation lung injury: an update. Eplasty 8, e27
Munoz-Price, L. S., and Weinstein, R. A. (2008) Acinetobacter infection. N Engl J Med 358,
1271-1281
Blanchard, J. S. (2005) Old approach yields new antibiotic. Nat Med 11, 1045-1046
Falagas, M. E., Fragoulis, K. N., Kasiakou, S. K., Sermaidis, G. J., and Michalopoulos, A.
(2005) Nephrotoxicity of intravenous colistin: a prospective evaluation. Int J Antimicrob
Agents 26, 504-507
Falagas, M. E., and Kasiakou, S. K. (2006) Toxicity of polymyxins: a systematic review of
the evidence from old and recent studies. Crit Care 10, R27
Bregeon, F., Papazian, L., Visconti, A., Gregoire, R., Thirion, X., and Gouin, F. (1997)
Relationship of microbiologic diagnostic criteria to morbidity and mortality in patients with
ventilator-associated pneumonia. Jama 277, 655-662
Hassett, D. J., Alsabbagh, E., Parvatiyar, K., Howell, M. L., Wilmott, R. W., and Ochsner,
U. A. (2000) A protease-resistant catalase, KatA, released upon cell lysis during stationary
phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low
cell densities. J Bacteriol 182, 4557-4563
Hassett, D. J., and Cohen, M. S. (1989) Bacterial adaptation to oxidative stress:
implications for pathogenesis and interaction with phagocytic cells. Faseb J 3, 2574-2582
Hassett, D. J., Ma, J. F., Elkins, J. G., McDermott, T. R., Ochsner, U. A., West, S. E.,
Huang, C. T., Fredericks, J., Burnett, S., Stewart, P. S., McFeters, G., Passador, L., and
Iglewski, B. H. (1999) Quorum sensing in Pseudomonas aeruginosa controls expression of
catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen
peroxide. Mol Microbiol 34, 1082-1093
Protocol Template
CF-146, Effective 7/10/11
Version 10-6-11
Page 10
On the Web at http://comirbweb.uchsc.edu
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
Mulligan, M. S., Till, G. O., Smith, C. W., Anderson, D. C., Miyasaka, M., Tamatani, T.,
Todd, R. F., 3rd, Issekutz, T. B., and Ward, P. A. (1994) Role of leukocyte adhesion
molecules in lung and dermal vascular injury after thermal trauma of skin. Am J Pathol 144,
1008-1015
Cox, R. A., Burke, A. S., Traber, D. L., Herndon, D. N., and Hawkins, H. K. (2007)
Production of pro-inflammatory polypeptides by airway mucous glands and its potential
significance. Pulm Pharmacol Ther 20, 172-177
Furie, M. B., and Randolph, G. J. (1995) Chemokines and tissue injury. Am J Pathol 146,
1287-1301
Schall, T. J., and Bacon, K. B. (1994) Chemokines, leukocyte trafficking, and inflammation.
Curr Opin Immunol 6, 865-873
Sikora, J. P., Chlebna-Sokol, D., Andrzejewska, E., Chrul, S., Polakowska, E., Wysocka,
A., and Sikora, A. (2008) Clinical evaluation of proinflammatory cytokine inhibitors (sTNFR
I, sTNFR II, IL-1 ra), anti-inflammatory cytokines (IL-10, IL-13) and activation of neutrophils
after burn-induced inflammation. Scand J Immunol 68, 145-152
Ogle, C., Mao, JX, Wu, JZ, Ogle, JD, and Alexander, JW. (1994) The production of tumor
necrosis factor, interleukin-1, interleukin-6, and prostaglandin E2 by isolated enterocytes
and gut macrophages: Effect of lipopolysaccharide and thermal injury. J Burn Care Rehab
15, 470
Kowal-Vern, A., Walenga, J. M., Hoppensteadt, D., Sharp-Pucci, M., and Gamelli, R. L.
(1994) Interleukin-2 and interleukin-6 in relation to burn wound size in the acute phase of
thermal injury. J Am Coll Surg 178, 357-362
Myrianthefs, P. M., and Baltopoulos, G. J. (2008) Circulating cytokines and outcome
prediction of burned children with concomitant inhalation injury. Crit Care 12, 155
Gauglitz, G. G., Finnerty, C. C., Herndon, D. N., Mlcak, R. P., and Jeschke, M. G. (2008)
Are serum cytokines early predictors for the outcome of burn patients with inhalation
injuries who do not survive? Crit Care 12, R81
Kovacs, E. J., and DiPietro, L. A. (1994) Fibrogenic cytokines and connective tissue
production. Faseb J 8, 854-861
Gorbach, S. L., Neale, G., Levitan, R., and Hepner, G. W. (1970) Alterations in human
intestinal microflora during experimental diarrhoea. Gut 11, 1-6
van den Bogaard, A. E., Weidema, W. F., van Boven, C. P., and van der Waay, D. (1986)
Recolonization and colonization resistance of the large bowel after three methods of
preoperative preparation of the gastrointestinal tract for elective colorectal surgery. J Hyg
(Lond) 97, 49-59
Mai, V., Greenwald, B., Morris, J. G., Jr., Raufman, J. P., and Stine, O. C. (2006) Effect of
bowel preparation and colonoscopy on post-procedure intestinal microbiota composition.
Gut 55, 1822-1823
Majetschak, M., Krehmeier, U., Bardenheuer, M., Denz, C., Quintel, M., Voggenreiter, G.,
and Obertacke, U. (2003) Extracellular ubiquitin inhibits the TNF-alpha response to
endotoxin in peripheral blood mononuclear cells and regulates endotoxin
hyporesponsiveness in critical illness. Blood 101, 1882-1890
Majetschak, M., Sorell, L. T., Patricelli, T., Seitz, D. H., and Knoferl, M. W. (2008) Detection
and possible role of proteasomes in the bronchoalveolar space of the injured lung. Physiol
Res
Majetschak, M., Patel, M. B., Sorell, L. T., Liotta, C., Li, S., and Pham, S. M. (2008)
Cardiac proteasome dysfunction during cold ischemic storage and reperfusion in a murine
heart transplantation model. Biochem Biophys Res Commun 365, 882-888
Silver, G. M., Albright, J. M., Schermer, C. R., Halerz, M., Conrad, P., Ackerman, P. D.,
Lau, L., Emanuele, M. A., Kovacs, E. J., and Gamelli, R. L. (2008) Adverse clinical
outcomes associated with elevated blood alcohol levels at the time of burn injury. J Burn
Care Res 29, 784-789
Walker, H. L., and Mason, A. D., Jr. (1968) A standard animal burn. J Trauma 8, 10491051
Protocol Template
CF-146, Effective 7/10/11
Version 10-6-11
Page 11
On the Web at http://comirbweb.uchsc.edu
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
Lambert, S. B., Frazier-Jessen, M. R., VanStedum, S., Hahn, E. L., Filkins, J. P., Garrity,
E. R., Jr., and Kovacs, E. J. (1995) Bronchoalveolar lavage fluid endotoxin elevation in
human single lung transplant recipients during rejection. Transpl Immunol 3, 81-85
Majetschak, M., and Sorell, L. T. (2008) Immunological methods to quantify and
characterize proteasome complexes: development and application. J Immunol Methods
334, 91-103
Baker, G. C., Smith, J. J., and Cowan, D. A. (2003) Review and re-analysis of domainspecific 16S primers. J Microbiol Methods 55, 541-555
Dethlefsen, L., Huse, S., Sogin, M. L., and Relman, D. A. (2008) The pervasive effects of
an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing.
PLoS Biol 6, e280
Margulies, M., Egholm, M., Altman, W. E., Attiya, S., Bader, J. S., Bemben, L. A., Berka, J.,
Braverman, M. S., Chen, Y. J., Chen, Z., Dewell, S. B., Du, L., Fierro, J. M., Gomes, X. V.,
Godwin, B. C., He, W., Helgesen, S., Ho, C. H., Irzyk, G. P., Jando, S. C., Alenquer, M. L.,
Jarvie, T. P., Jirage, K. B., Kim, J. B., Knight, J. R., Lanza, J. R., Leamon, J. H., Lefkowitz,
S. M., Lei, M., Li, J., Lohman, K. L., Lu, H., Makhijani, V. B., McDade, K. E., McKenna, M.
P., Myers, E. W., Nickerson, E., Nobile, J. R., Plant, R., Puc, B. P., Ronan, M. T., Roth, G.
T., Sarkis, G. J., Simons, J. F., Simpson, J. W., Srinivasan, M., Tartaro, K. R., Tomasz, A.,
Vogt, K. A., Volkmer, G. A., Wang, S. H., Wang, Y., Weiner, M. P., Yu, P., Begley, R. F.,
and Rothberg, J. M. (2005) Genome sequencing in microfabricated high-density picolitre
reactors. Nature 437, 376-380
Gill, S. R., Pop, M., Deboy, R. T., Eckburg, P. B., Turnbaugh, P. J., Samuel, B. S., Gordon,
J. I., Relman, D. A., Fraser-Liggett, C. M., and Nelson, K. E. (2006) Metagenomic analysis
of the human distal gut microbiome. Science 312, 1355-1359
Huse, S. M., Dethlefsen, L., Huber, J. A., Welch, D. M., Relman, D. A., and Sogin, M. L.
(2008) Exploring microbial diversity and taxonomy using SSU rRNA hypervariable tag
sequencing. PLoS Genet 4, e1000255
Huse, S. M., Huber, J. A., Morrison, H. G., Sogin, M. L., and Welch, D. M. (2007) Accuracy
and quality of massively parallel DNA pyrosequencing. Genome Biol 8, R143
Schloss, P. D., and Handelsman, J. (2005) Introducing DOTUR, a computer program for
defining operational taxonomic units and estimating species richness. Appl Environ
Microbiol 71, 1501-1506
Schloss, P. D., and Handelsman, J. (2006) Introducing SONS, a tool for operational
taxonomic unit-based comparisons of microbial community memberships and structures.
Appl Environ Microbiol 72, 6773-6779
Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai,
T., Steppi, S., Jobb, G., Forster, W., Brettske, I., Gerber, S., Ginhart, A. W., Gross, O.,
Grumann, S., Hermann, S., Jost, R., Konig, A., Liss, T., Lussmann, R., May, M., Nonhoff,
B., Reichel, B., Strehlow, R., Stamatakis, A., Stuckmann, N., Vilbig, A., Lenke, M., Ludwig,
T., Bode, A., and Schleifer, K. H. (2004) ARB: a software environment for sequence data.
Nucleic Acids Res 32, 1363-1371
Olsen, G. J., Matsuda, H., Hagstrom, R., and Overbeek, R. (1994) fastDNAmL: a tool for
construction of phylogenetic trees of DNA sequences using maximum likelihood. Comput
Appl Biosci 10, 41-48
Saitou, N., and Nei, M. (1987) The neighbor-joining method: a new method for
reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425
Ludwig, W., and Schleifer, K. H. (1994) Bacterial phylogeny based on 16S and 23S rRNA
sequence analysis. FEMS Microbiol Rev 15, 155-173
Cole, J. R., Chai, B., Farris, R. J., Wang, Q., Kulam-Syed-Mohideen, A. S., McGarrell, D.
M., Bandela, A. M., Cardenas, E., Garrity, G. M., and Tiedje, J. M. (2007) The ribosomal
database project (RDP-II): introducing myRDP space and quality controlled public data.
Nucleic Acids Res 35, D169-172
Vanderwarf, C: Procedure for Blind BAL using CombiCath, Policy
#407.00, Department of Respiratory Care, Policy and Procedures, Loyola
University Health System. Loyola University of Chicago, 2003 July
Protocol Template
CF-146, Effective 7/10/11
Version 10-6-11
Page 12
On the Web at http://comirbweb.uchsc.edu
61.
McGill V, Kowal-Vern A, Fisher SG, Kahn S, Gamelli RL. (1995) The impact
of substance use on mortality and morbidity from thermal injury. J Trauma. 38(6), 931934.
Protocol Template
CF-146, Effective 7/10/11
Version 10-6-11
Page 13