Methicillin-resistant Staphylococcus aureus-induced vascular leakage is associated with excessive production of nitric oxide and vascular endothelial growth factor

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S1
Available online http://ccforum.com/supplements/11/S4
Critical Care Volume 11 Suppl 4, 2007
Sepsis 2007
Paris, France, 26–29 September 2007
Published online: 26 September 2007
These abstracts are available online at http://ccforum.com/supplements/11/S4
© 2007 BioMed Central Ltd
P1
Cytokine-mediated regulation of renal urea transporters
during sepsis
Christoph Schmidt, Klaus Hoecherl, Michael Bucher
Anaesthesiology Department, Hospital of the University of
Regensburg, Regensburg, Germany
Critical Care 2007, 11(Suppl 4):P1 (doi: 10.1186/cc5980)
Background The pathogenesis of endotoxemic tubular dys-
function with failure in urine concentration is poorly understood.
Urea plays an important role in the urinary concentrating
mechanism and expression of the urea transporters UT-A1, UT-A2,
UT-A3, UT-A4 and UT-B is essential for tubular urea reabsorption.
The present study attempts to assess the regulation of renal urea
transporters during severe inflammation in vivo.
Materials and methods By agreement of the animal protection
committee C57BL/6J, mice were injected with lipopolysaccharides
(LPS, 10 mg/kg) or proinflammatory cytokines. Hemodynamic,
renal parameters and the expression of renal urea transporters
were investigated. To clarify the role of cytokines and renal
ischemia in the regulation of renal urea transporters, experiments
were performed with cytokine knockout mice, mice treated with
low-dose LPS (1, 5 mg/kg) as a sepsis model without induction of
hypotension, glucocorticoid-treated mice, and mice with renal
artery clipping serving as a model for renal ischemia.
Results and discussion LPS-injected mice (10 mg/kg) presented
with reduced glomerular filtration rate, fractional urea excretion and
inner medulla osmolality associated with a marked decrease in
expression of UT-A1, UT-A2, UT-A3, UT-A4 and UT-B (Figure 1).
Similar alterations were observed after application of TNFα, IL-1β,
IFNγ or IL-6. LPS-induced downregulation of urea transporters was
not affected in knockout mice with deficient TNFα, IL-receptor-1,
IFNγ or IL-6. Glucocorticoid treatment inhibited LPS-induced
increases of tissue TNFα, IL-1β, IFNγ or IL-6 concentration,
diminished LPS-induced renal dysfunction and attenuated the
downregulation of renal urea transporters. Injection of low-dose
LPS (1, 5 mg/kg) also led to renal dysfunction paralleled by a
downregulation of renal urea transporters without alterations in
blood pressure. Renal ischemia induced by renal artery clipping
did not influence the expression of urea transporters.
Conclusion Our findings demonstrate downregulation of renal
urea transporters that probably accounts for tubular dysfunction
during sepsis. Furthermore, they suggest that downregulation of
Figure 1 (abstract P1)
Effect of lipopolysaccharide (LPS) (10 mg/kg), dexamethasone (10 mg/kg) and the combination of both on UT-A1, UT-A2, UT-A3, UT-A4 and UT-B
mRNA in the kidney 6, 12 and 24 hours after intraperitoneal injection. Values are related to signals obtained for β-actin mRNA and presented as
the percentage of vehicle control. Mean ± SEM of six animals per group. *P < 0.05 versus control,
#
P < 0.05 versus LPS treatment.
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Critical Care September 2007 Vol 11 Suppl 4 Sepsis 2007
renal urea transporters during LPS-induced acute renal failure is
mediated by proinflammatory cytokines and is independent from
renal ischemia due to sepsis-induced hypotension.
Acknowledgement Supported by grants from the German
Research Foundation (SFB 699).
P2
The role of regulatory T cells in the resistance of CCR4
knockout mice during severe sepsis
Raphael Molinaro
1
, Alessandra Monteiro de França
1
,
Josi Alves-Filho
2
, Fernando de Queiroz Cunha
2
,
Marcelo Bozza
3
, Steven Kunkel
4
, Claudia Benjamim
1
1
Department of Pharmacology, UFRJ, Rio de Janeiro, RJ, Brazil;
2
Department of Pharmacology, USP, Ribeirão Preto, SP, Brazil;
3
Microbiology Institute, UFRJ, Rio de Janeiro, RJ, Brazil;
4
Department of Pathology, UMMS, Ann Arbor, MI, USA
Critical Care 2007, 11(Suppl 4):P2 (doi: 10.1186/cc5981)
Background Studies reveal that regulatory T (T
reg
) cells control
immune responses; therefore these responses must be controlled
to enable effective protection against infections and cancer. CCR4
knockout (CCR4
–/–
) mice are more resistant to lipopolysaccharide
shock. So, our aim is to study the mechanisms involved in the resis-
tance of CCR4
–/–
mice subjected to severe sepsis by cecal ligation
and puncture (CLP) and how T
reg
cells modulate this effect.
Methods C57/BL6 mice were subjected to a CLP model, whereby
the cecum was partially ligated and punctured nine times with a
21 G needle. Sham-operated mice were used as control. Mice
subjected to CLP and sham surgery were treated with antibiotic
from 6 hours after surgery until 3 days.
Results CCR4
–/–
mice subjected to CLP presented an increase in
the survival rate (78%) compared with wild-type mice (17%), and
presented a marked improvement in the innate response concern-
ing neutrophil migration to the peritoneum and lung, bacterial load
and cytokine levels compared with wild-type mice. Besides, T
reg
cells from CCR4
–/–
CLP mice did not inhibit proliferation of T
effector cells as observed for T
reg
cells from wild CLP mice, at a
proportional ratio of T effector:T
reg
cells. Interesting, T
reg
cells from
CCR4
–/–
CLP mice inhibit 30% of neutrophil migration to broncho-
alveolar lavage when co-injected with fungal challenge as
secondary infection in sham recipient mice, while the T
reg
cells
from wild CLP mice inhibit 80%, much more than expected.
Conclusion These results suggest that T
reg
cells from CCR4
–/–
mice did not present a suppressive response and this could be an
important factor in their survival. These results are strong evidence
for the role of T
reg
cells in immunosuppression following severe
sepsis.
P3
Abdominal sepsis: efficacy of passive immunotherapy
Ruslan Knut, Peter Fomin, Oleg Sydorchuk, Ruslan Sydorchuk,
Olexii Kolomoiets, Larysa Sydorchuk, Igor Sydorchuk,
Mohhamad Daraghmeh
General Surgery Department, Bukovina State Medical University,
Ukraine
Critical Care 2007, 11(Suppl 4):P3 (doi: 10.1186/cc5982)
Background Owing to immune disorders playing a key role in
development of systemic inflammatory response syndrome, passive
immune therapy is considered a method of choice for abdominal
sepsis (AS) patients. Existing remedies (specific hyperimmune
serum, specific antibodies and immunoglobulins) are expensive
and require exact validation of pathogens. The aim of the study was
to evaluate the efficacy of using the AS reconvalescent donor
plasma for passive immunotherapy of AS.
Materials and methods The study was conducted experimentally
on 775 Wistar line rats and 38 inbreed dogs. A total of 296
patients with AS were also involved in the study; 58 formed the
control group; 26 patients were selected as reconvalescent
donors of plasma. Serum concentrations (ELISA) of major anti-
bodies were determined against most significant pathogens
(Escherichia coli, Staphylococcus spp., Staphylococcus aureus,
Bacteroides spp., Klebsiella pneumoniae, Pseudomonas aeruginosa).
Results Changes of serum antibody concentrations were time
dependent and fluctuating during the current of AS forming the
waveform curve. Most remarkable decreases were found during
24–72 hours of AS. Serum antibody titers to the main pathogens
were slightly higher due to antibiotics and detoxification therapy.
Operation by itself decreased titers from 4.42 ± 0.28 to 3.49 ± 0.25
(E. coli), and from 5.41 ± 1.02 to 3.0 ± 0.58 (P. aeruginosa). Anti-
staphylococcal antibody titers decreased from 7.22 ± 0.9 before
surgery to 4.83 ± 0.47 after. Repeated operations alter antibody
concentrations even more significantly. The highest levels of
antibodies were found in patients who underwent successful
treatment of AS 1–2 months prior to investigation. Their plasma
was used in treatment of AS patients. Intravenous administration of
two-dose 100–200 ml hyperimmune plasma per day prevented a
following decrease of antibody levels, and in 98% of cases
increased them (21.39 ± 1.47%). The cost of treatment was
15–37% lower if compared with traditional methods (control group).
Conclusion There is exact evidence of efficacy for using hyper-
immune plasma in patients with abdominal sepsis; it is more cost-
effective if compared with traditional methods of immunotherapy.
P4
The modified light chain of inter-alpha inhibitor/antibody
fusion protein, MR1007, improves survival in the rabbit sepsis
models
Masaki Nakamura, Takashi Takeuchi, Yukiko Yatagai,
Katsuki Naito, Kamon Shirakawa, Yoshitaka Hosaka,
Shoji Furusako
Discovery Research, Mochida Pharmaceutical Co., Ltd, Gotemba,
Japan
Critical Care 2007, 11(Suppl 4):P4 (doi: 10.1186/cc5983)
Background Inter-alpha inhibitor is an endogenous serine protease
inhibitor and is markedly reduced in severe sepsis. Therapeutic
inter-alpha inhibitor replacement showed a survival advantage in
several animal models. The light chain is responsible for the serine
protease inhibitory activity of inter-alpha inhibitor. Since pro-
coagulant and proinflammatory proteases as well as innate immune
cells are activated in sepsis, we genetically engineered a novel
fusion protein, MR1007, which consists of the modified light chain
of inter-alpha inhibitor and the anti-CD14 antibody, and evaluated
the potential of MR1007 as an anti-sepsis agent.
Methods Inhibitory activity against serine proteases was assayed
using purified enzymes and chromogenic substrates. Anticoagulant
activity was measured using human or rabbit plasma. The inhibitory
effect on endothelial cell injury was assessed using human
umbilical vein endothelial cells. Binding to CD14 and leukocytes
was analysed using Biacore or radiolabeling. The survival benefit
was evaluated in the endotoxin shock model and the cecal ligation
and puncture (CLP) model.
Results MR1007 inhibited the thromboplastin-induced thrombin
generation by inhibiting activities of coagulation factors Xa and XIa
at 10–100 μg/ml. It also prevented the contact pathway generation
of bradykinin at 10–30 μg/ml. Additionally, it inhibited the
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leukocyte elastase-induced endothelial cell injury at 10–100 μg/ml.
MR1007 had a high affinity for CD14 and bound to leukocytes, but
did not block lipopolysaccharide binding to CD14. In the rabbit
endotoxin shock model, MR1007 (3 mg/kg, i.v.) even when given
8 hours after the injection of endotoxin improved the survival
(n = 12, P < 0.05), whereas both antithrombin and prednisolone
exhibited less efficacy. Moreover, MR1007 (10 mg/kg, i.v.) given at
2 hours post-CLP improved the survival (n = 9, P < 0.05) in the
CLP model.
Conclusions These results suggest that the modified light chain of
inter-alpha inhibitor fusion protein, MR1007, can effectively
suppress not only the serine protease-mediated coagulation, but
also leukocyte-induced inflammation, so that MR1007 may
become a promising anti-sepsis agent.
P5
Microbial metabolites in the blood of patients with sepsis
Anastasia Khodakova, Natalia Beloborodova
Bakulev Scientific Center for Cardiovascular Surgery, Moscow,
Russia
Critical Care 2007, 11(Suppl 4):P5 (doi: 10.1186/cc5984)
Background Molecular mechanisms of the pathophysiology of
sepsis remain unknown. Preliminary results allow one to suppose
that investigation of biological effects of microbial metabolites,
particularly aromatic acids, is one of the most promising methods
in elucidation of the problem. These compounds can be produced
during microbial fermentation of aromatic amino acids. But little is
known of which microorganisms participate in such processes.
The aim was to assess the comparative level of aromatic acids in
serum of cardiosurgical patients with documentary sepsis and to
clarify the in vitro metabolic profile of aromatic acids in spent
growth medium of main clinical blood isolates.
Materials and methods Serum samples were collected from 83
adult subjects (mean age 52 (42–58) years). The main group of
investigation consisted of 12 cardiosurgical patients with docu-
mentary sepsis, with mortality of 42% (5/12). The comparison
groups were: 16 clinically healthy volunteers, 36 patients with
acquired heart diseases before surgery, nine patients with smooth
recovery on the third day after surgery, 10 patients with pneumonia
after surgery. The cultures (n = 32) of S. aureus, S. epidermidis,
E. faecalis, K. pneumonia, S. marcesceus, E. coli, E. cloacae,
A. baumanii, P. aeruginosa, C. albicans and C. parapsilosis were
isolated from the blood of cardiosurgical patients and identified.
Concentrations of aromatic acids were determined by gas
chromatography–mass spectrometry. Data were compared by
Mann–Whitney U-test, P < 0.05 considered significant.
Results Significant differences were observed among the groups
(Table 1). 3-Phenylpropionic and 1-indolacetic acids were found to
be prevalent in groups of healthy volunteers and patients before
surgery. Increased levels of phenyllactic acid (PLA), p-hydroxy-
phenylacetic acid (HPAA), p-hydroxyphenyllactic acid (HPLA) and
3-indolacetic acid were revealed in the group of sepsis compared
with other groups. Moreover, the highest concentrations of PLA,
HPAA and HPLA were in serum of nonsurvivors (n = 5) compared
with survivors (n = 7): PLA, 1,651 (656–1,959) versus 233
(122–360) ng/ml, P = 0.02; HPAA, 5,976 (2,689–6,667) versus
1,108 (461–2,121) ng/ml, P = 0.02; HPLA, 3,313 (2,409–6,098)
versus 564 (446–718) ng/ml, P = 0.005. Gas chromatography–
mass spectrometry analysis of spent growth medium showed that
Gram-negative enterobacteria produced increased amounts of
PLA and HPLA acids. Particularly, K. pneumonia had the highest
level of acids PLA = 100 r.u. (r.u. – the ratio of substance content
in sample to uninoculated media) and HPLA = 60 r.u., E. coli had
PLA = 35 r.u. and HPLA = 20 r.u., and S. marcesceus and
E. cloacae had PLA = 4 r.u. and HPLA = 6 r.u. The culture of
Gram-positive cocci produced increased level of the same acids,
for S. aureus PLA = 20 r.u. and HPLA = 17 r.u., and for S.
epidermidis PLA = 6 r.u. and HPLA = 3 r.u., except for E. faecalis,
which had the only PLA = 6 r.u. Gram-negative nonfermented
bacteria produced increased levels of 1-indolacetic acid and 3-
indolacetic acid, but no PLA and HPLA. The level of aromatic acids
in the medium after cultivation of fungi was equal to control.
Conclusions Increased levels of PLA, HPAA and HPLA in serum
patients with sepsis, especially with fatal outcome, are
associated with development of infectious complications. These
compounds are produced by clinically important bacteria, such as
K. pneumonia > E. coli > S. aureus > S. marcesceus, E. cloacae,
S. epidermidis, but not by fungi. The results can denote biological
activity of these microbial metabolites and their influence on
pathogenesis of sepsis.
P6
The impact of protocolized sepsis order set on the process of
care in patients with severe sepsis/septic shock
Bekele Afessa, John Mullon, Andrew Badley and Ognjen Gajic
Mayo Clinic College of Medicine, Pulmonary and Critical Care
Division, Rochester, MN, USA
Critical Care 2007, 11(Suppl 4):P6 (doi: 10.1186/cc5985)
Background Based on the available evidence, professional
societies have published practice guidelines on severe sepsis and
septic shock. We started using a paper order set based on the
guidelines in our medical intensive care unit (ICU) in October
Table 1 (abstract P5)
Concentration of aromatic acids in serum sepsis patients and subjects of comparison groups
Patients Patients before Patients with Patients with
with sepsis Volunteers surgery smooth recovery pneumonia
Aromatic acid (ng/ml) (n = 12) (n = 16) (n = 36) (n = 9) (n = 10)
p-Hydroxyphenylacetic acid 2,140 (631–3,516) 72 (62–93)*** 114 (53–220)*** 263 (109–313)** 456 (344–667)*
3-Phenylpropionic acid 0 35 (20–54)** 4 (0–29) 0 0 (0–3)
Phenyllactic acid 367 (217–1,098) 47 (37–64)** 58 (37–93)** 89 (68–126)* 112 (89–177)*
p-Hydroxyphenyllactic acid 1,543 (564–2,731) 195 (159–371)*** 254 (134–373)*** 288 (269–772)* 465 (314–836)
1-Indolacetic acid 0 262 (113–385)*** 47 (0–218)* 275 (228–499)*** 0 (0–27)
3-Indolacetic acid 246 (183–628) 93 (56–141)* 181 (60–542) 57(30–74)* 231 (149–375)
Data presented as median (25th–75th percentile range). *P < 0.05, **P < 0.001, ***P < 0.0001, compared with sepsis patients.
Page 3
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2005. This prospective study aims to describe the impact of the
order set on the process of care.
Materials and methods We included patients with severe sepsis/
septic shock treated in our ICU between October 2005 and April
2007. We collected Acute Physiology and Chronic Health
Evaluation (APACHE) III derived severity data, compliance with six
elements of early goal-directed therapy and hospital mortality.
Compliance with each element was defined as the use of the
following within 6 hours of severe sepsis/septic shock: use of
central venous pressure, central venous oxygen saturation
measurement, adequate fluid resuscitation and appropriate use of
vasopressors, inotropes and transfusion of red blood cells. The
ICU admission severity of illness and sepsis stage (severe or
shock) were entered in a logistic regression model to determine
the independent impact of the order set on mortality. P < 0.05 was
considered significant.
Results Of 561 patients (168 severe sepsis and 373 septic
shock), 31 were excluded for not authorizing research. The order
set was utilized in 328 (61.9%) of 530 patients. There were no
significant differences in gender, age, race, and severity of illness
at ICU admission between the order set and nonorder set groups.
The order set was more likely to be used in patients with septic
shock than in those with severe sepsis (67.3% versus 51.4%;
P = 0.0004). Compliance with all six elements occurred in 130
(39.6%) of the order set group compared with 50 (24.8%) of the
nonorder set group (P = 0.0004). Although mortality did not
change, compliance with five of the six elements improved
significantly with the order set. Logistic regression analysis showed
that shock (odds ratio (OR) = 2.384, 95% confidence interval (CI) =
1.431–3.970; P = 0.0008) and predicted APACHE III mortality
(%) (OR = 1.040, 95% CI = 1.031–1.050; P < 0.0001) were
associated with mortality, not the order set (OR = 0.742, 95% CI =
0.476–1.157; P = 0.1881).
Conclusions This study showed that a protocolized order set
improves compliance with the standard of care in patients with
severe sepsis and septic shock. However, it did not resolve some
of the noncompliance problems and did not improve survival.
P7
Impact of autologous centrifuged shed mediastinal blood on
procalcitonin, C-reactive protein levels and postoperative
complications during the early period following cardiac
surgery
Judita Andrejaitiene
1
, Audrone Veikutiene
2
,
Edmundas Širvinskas
1
, Rimantas Benetis
3
1
Institute for Biomedical Research, Kaunas Medical University,
Kaunas, Lithuania;
2
Kaunas University of Medicine Hospital,
Department of Cardiothoracic and Vascular Surgery, Kaunas,
Lithuania;
3
Kaunas University of Medicine, Institute for Cardiology,
Kaunas, Lithuania
Critical Care 2007, 11(Suppl 4):P7 (doi: 10.1186/cc5986)
Background Cardiac surgery with cardiopulmonary bypass (CPB)
is associated with a number of adverse effects due to systemic
inflammatory response syndrome, a physiologic reaction to tissue
injury. Because of this response, conventional clinical and
biological signs may be misleading in the diagnosis of post-
operative complications, particularly infections. The aim of the
study was to evaluate the impact of autologous centrifuged shed
mediastinal blood procedures in attitude of infection, estimating
the predictive role of procalcitonin (PCT) and C-reactive protein
(CRP) changes during the postoperative period.
Materials We have analysed data on 90 patients, who had been
subjected to cardiac surgical procedures on CPB: there are 41
patients in Group I, who were reinfused with the centrifuged
autologous mediastinal blood 4 hours after the end of surgery; and
49 patients in Group II, whose shed mediastinal blood was not
reinfused (control group).
Methods We studied the quantity of haemoglobin, haematocrit
and leucocyte counts, and the value of CRP and PCT concen-
trations before the surgery (baseline), 4 and 20 hours after the end
of surgery and during 5 days after surgery. Preoperative patient
conditions, intraoperative and postoperative periods, were recorded.
Statistical significance was accepted at a level of P < 0.05.
Results In Group I, patients who were reinfused with the
centrifuged autologous shed mediastinal blood, requirement for the
allogenic blood transfusion procedures was significantly lower
(14.6% versus 38.8%, P < 0.05). The CRP concentration was
greater, but there were no significant differences between the
groups in all postoperative periods. At 20 hours after the end of
surgery and the second postoperative day, the increase of the PCT
concentration was significant and often observed in group II
(33.3% versus 58.3%), where there were significantly more
complications of infection (2.4% versus 10.2% P < 0.05) and a
significantly longer length of postoperative hospital stay
(9.32 ± 2.55 versus 14.38 ± 4.27 days, P < 0.05).
Conclusions Our data suggest that the early reinfusion of
autologous centrifugated shed mediastinal blood procedures did
not increase bleeding and statistically significantly reduced the
requirement of allogenic blood transfusion procedures, reduced
the number of infection complications, and significantly shortened
the length of postoperative hospital stay. In evaluation of
postoperative infection rates, PCT is highly suggestive as a marker
of postoperative complications.
P8
System approach to the diagnosis and treatment of septic
arthritis in newborns
Gennadiy Khanes
1
, Svitlana Bidnenko
2
,
Volodymyr Grygorovskyy
3
, Iryna Maksakova
1
,
Valeriy Romashko
4
1
Neonatal Surgery Department, Ukrainian Specialized Pediatric
Hospital OkhMatDyt, Kyiv, Ukraine;
2
Bacteriologic Laboratory of
Scientific Research Institute of Traumatology and Orthopedics of
the Academy of Medical Sciences of Ukraine, Kyiv, Ukraine;
3
Morphologic Laboratory of Scientific Research Institute of
Traumatology and Orthopedics of the Academy of Medical
Sciences of Ukraine, Kyiv, Ukraine;
4
Immunologic Laboratory,
Ukraine Specialized Pediatric Hospital OkhMatDyt, Kyiv, Ukraine
Critical Care 2007, 11(Suppl 4):P8 (doi: 10.1186/cc5987)
Background Bone and joint sepsis occurs in 20% of newborns
having perinatal sepsis. The feature of septic arthritis against the
background of age-specific functional immunodeficiency gives us a
reason to consider this decease as the manifestation of immuno-
deficiency. Septic arthritis in newborns is lethal in 10% of cases
and gives orthopedic complications in 20% of cases. The starting
moments of septic arthritis development are: mother’s intrauterine
infection and nosocomial infecting in maternity hospitals and
intensive care departments, and purulent omphalitis.
Materials and methods We present the experience of diagnostics
and treatment of 180 newborns aged 3 days and older having
septic affection of the hip, knee, shoulder and other joints. The
crucial role in diagnostics of septic arthritis is played by: cytology
Critical Care September 2007 Vol 11 Suppl 4 Sepsis 2007
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S5
of smear, ultrasonic examination of a joint, X-ray examination of a
joint, bacteriological, serological, PCR and PCT-Q. The bacterio-
logical monitoring in 30% of newborns among the pathogens
showed CoMRSA, often mixed with fungi and Pseudomonas
aerogenes or Klebsiella pneumonia, and in 10% there was PCR of
Toxoplasma gondi, Chlamidia trachomatis or cytomegalovirus.
Immunological monitoring allowed one to determine the patients
who needed substitute therapy by immunoglobulin and a number
of immunomodulators. The degree of infection process severity
and the adequacy of antibacterial therapy are determined by
serologic investigations, PCR and PCT-Q. The treating complex
included joint lavage, antibiotics, anticytokine, antifungal agents,
probiotics, and magnetic-laser therapy.
Results The lethality was reduced to zero, transition into the
chronic form was up to 2.5%, and orthopedic complications were
presented in 10% of cases.
Conclusion The after-history of treatment was researched and
showed that when the diagnosis was made in less than 3 days,
complications occurred in 3.3% of cases, and after 6–7 days the
complications occurred in 22.4% of patients.
P9
Protease-activated receptor 2 blocking peptide counteracts
endotoxin-induced inflammation and coagulation and
ameliorates glomerular fibrin deposition in a rat model of
acute renal failure
Satoshi Gando, Subrina Jesmin, Sohel Zaedi,
Shamsul Haque Prodhan, Atsushi Sawamura,
Takashi Miyauchi, Naoto Yamaguch
Division of Acute and Critical Care Medicine, Department of
Anesthesiology and Critical Care Medicine, Hokkaido University
Graduate School of Medicine, Sapporo, Japan
Critical Care 2007, 11(Suppl 4):P9 (doi: 10.1186/cc5988)
Background Glomerular and microvascular thrombosis due to the
activation of the inflammation and coagulation pathway contribute
to the occurrence of acute renal failure in sepsis. The protease-
activated receptors (PARs) have been shown to play an important
role in the interplay between the inflammation and coagulation.
Materials and methods We hypothesized that PAR2 blocking
would improve glomerular and vascular thrombosis by attenuating
the inflammation and coagulation, leading to the prevention of
acute renal failure, and we assessed the effects of the PAR2
blocking peptide (PAR2 BP) in a rat model of lipopolysaccharide
(LPS)-induced acute renal failure.
Results Levels of TNFα were significantly expressed 1 hour after
LPS administration, followed by: (i) an increase in levels of tissue
factor, factor VIIa, factor Xa, thrombin and plasminogen activator
inhibitor-1; (ii) unchanged levels of tissue factor pathway inhibitor;
and (iii) subsequent deposition of fibrin in kidney tissues, which led
to the elevation of creatinine and blood urea nitrogen. Time-
dependent PAR2 expression was observed at both the gene and
protein levels. Immunoreactivities of PAR2 and fibrin were co-
localized in the glomerulus and the other kidney tissues. PAR2 BP
suppressed TNFα elevation, and attenuated activation of the
coagulation, thus leading to a decrease in fibrin formation and its
deposition in the glomerulus. However, the levels of creatinine and
blood urea nitrogen remained unchanged.
Conclusions These results show that PAR2 plays a crucial role in
the inflammatory and coagulation process of LPS-induced renal
failure and may in part participate in the pathogenesis of the
disease.
P10
Resistin in severe bacterial infections
Linda Johansson, Anna Linnér, Jonas Sundén-Cullberg,
Carl-Johan Treutiger, Anna Norrby-Teglund
Center for Infectious Medicine, Institutionen för Medicin Huddinge,
F59 Karolinska Institutet, Stockholm, Sweden
Critical Care 2007, 11(Suppl 4):P10 (doi: 10.1186/cc5989)
Background Resistin has recently been recognized to act as a
proinflammatory cytokine in humans. Patients with severe sepsis or
septic shock had significantly elevated systemic levels of resistin,
which correlated with severity of disease. Here we have further
characterized the release of resistin during severe bacterial
infections.
Materials and methods Acute phase sera collected from patients
with septic shock caused by Gram-negative (n = 19) or Gram-
positive (n = 19) bacteria were analyzed for resistin by ELISA. Tissue
biopsies (n = 12) from patients with Streptococcus pyogenes
severe soft tissue infections were stained for resistin and cell
markers, and were analyzed by confocal microscopy. Human
neutrophils were stimulated with lipopolysaccharide or
streptococcal superantigens, and resistin was assessed in the
supernatants.
Results Serum resistin levels were significantly elevated in patients
with Gram-positive, as compared with Gram-negative, septic shock
(P = 0.004). Analyses of tissue biopsies revealed that resistin was
highly expressed at the local site of infection. Dual-staining for cell
markers confirmed published findings that monocytes are a source
of resistin in humans, but importantly the stainings revealed that the
majority of resistin-producing cells were negative for the monocytic
marker CD68. Further analyses identified these cells as
neutrophils. A positive correlation between resistin levels and
neutrophil counts was found in blood of septic shock patients
(P = 0.005). In vitro cell cultures revealed resistin release by
neutrophils stimulated with lipopolysaccharide or superantigens.
Conclusions This study demonstrates that the systemic resistin
levels in septic shock differ depending on the causative
microorganisms. The data also reveal that, at the local tissue site of
infection, resistin is produced mainly by neutrophils, and systemic
resistin strongly correlates with circulating levels of neutrophils.
The systemic and local hyper-resistinemia noted is likely to
contribute to the pathogenesis of acute invasive bacterial infections.
P11
Mortality rate reduction associated with severe sepsis and
septic shock management protocol implementation
Constantino José Fernandes Junior,
Alexandre Gonçalves de Sousa, Gisele de Paula Dias Santos,
Eliezer Silva, Nelson Akamine
Hospital Israelita Albert Einstein Division of Medical Practice –
Department of Protocols, São Paulo, Brazil
Critical Care 2007, 11(Suppl 4):P11 (doi: 10.1186/cc5990)
Background The Surviving Sepsis Campaign is an international
effort to reduce severe sepsis and septic shock associated mortality
by 25% in 5 years. We developed a management protocol in our
institution 2 years ago in order to follow the proposed
recommendations of this campaign, and describe the clinical impact
of assuming this critical pathway on the mortality rate.
Materials The study was conducted within the emergency
department and intensive care unit of a tertiary hospital. A manage-
ment protocol for severe sepsis and septic shock was based on
the Surviving Sepsis Campaign guidelines and was implemented
Available online http://ccforum.com/supplements/11/S4
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by a ‘sepsis’ team including emergency department and critical
care physicians, intensive care nurses and pharmacists, chaired by
a full-time coordinator.
Methods We performed a ‘before and after’ evaluation of the
critical pathway concerning 184 critically ill patients sequentially
admitted throughout a 16-month period.
Results A total of 184 patients with severe sepsis or septic shock
entered the study. Ninety-four patients had their analysis performed
before the implementation of the standardized protocol (the
‘before’ group), and 90 patients were managed following the
implementation of the protocol (the ‘after’ group). Basal demo-
graphic variables and the severity of illness score (APACHE II)
were similar for both groups.
Patients in the ‘after’ group had statistically more cultures obtained
before institution of antibiotics and more patients received
antibiotics in a due time (2 hours from diagnosis). In addition, those
patients received more corticosteroids and activated protein C
(Table 1).
The intensive care unit length of stay and the hospital length of stay
were similar in both groups. Remarkably the hospital mortality rate
was significantly lower (34.4%) in the ‘after’ group in septic shock
patients (67.7% versus 44.4%, P < 0.04) (Figure 1).
Conclusions The implementation of the Surviving Sepsis
Campaign guidelines through a standardized protocol was
associated with a 34% reduction in septic shock-related hospital
mortality.
P12
Neonatal sepsis due to multidrug-resistant Klebsiella
terrigena in the neonatal intensive care unit in Gaza, Palestine
Farid Abu Elamreen
Medical Microbiology Department, AlShifa Hospital Central
Laboratory & Blood Bank, Ministry of Health, Gaza, Palestine
Critical Care 2007, 11(Suppl 4):P12 (doi: 10.1186/cc5991)
Background Bloodstream infection (BSI) is a major cause of
morbidity and death encountered in the neonatal intensive care
units (NICUs). The rates of BSIs vary significantly in NICUs across
the nation. Klebsiella spp. led to serious concern about
septicaemic neonates in NICUs due to high resistance against
commonly used antimicrobial agents. The objective of the study
was to report the prevalence and resistance pattern of Klebsiella
terrigena isolated from cases of neonatal septicaemia at Alshifa
Hospital, the largest tertiary hospital in Palestine.
Methods Blood taken from newborn babies admitted to the NICU
at Alshifa Hospital, Gaza, Palestine with a clinical diagnosis of
neonatal sepsis was cultured. A total of 355 positive blood
cultures isolated from January to December 2005 were studied.
Antimicrobial susceptibility was determined by disc diffusion
method.
Results A total of 355 blood cultures positive were studied; the
most common organism found were Klebsiella spp. 202/355
(56.9%), and 56/202 (25.5%) were K. terrigena. K. terrigena
showed a high degree of resistance to commonly used antibiotics
(Ampicillin, Piperacillin, Cephalexin, Cefuroxime, Cefaclor and
Gentamicin), a moderate degree of resistance to Cefotaxime,
Ceftazidim, Ceftriaxone and Amikacin, and most of the isolates
were sensitive to Meropenem.
Conclusion Neonatal sepsis remains one of the leading causes of
neonatal admission, morbidity, and mortality in developing
countries. Klebsiella spp. were the major cause of neonatal sepsis
in Gaza in 2005. The rare Klebsiella species (K. terrigena) have
developed multidrug resistance, and management of patients
infected with them is becoming a problem in developing countries.
There is a need to carefully formulate therapeutic strategies to
control infections in NICUs.
P13
The identification and use of common physiologic monitoring
parameters in the care of critically ill patients at risk for sepsis
Karen Giuliano, Erica Cummings
Philips Medical Systems, Andover, MA, USA
Critical Care 2007, 11(Suppl 4):P13 (doi: 10.1186/cc5992)
Background Sepsis is a common source of morbidity and
mortality among critically ill patients. Targeting measures to reduce
the incidence of and to promote early recognition and treatment of
sepsis is at the forefront of many critical care initiatives. Advances
in the management of severe sepsis have evolved over recent
years in an attempt to combat the spiraling mortality trends. The
Surviving Sepsis Campaign (SSC) is a worldwide initiative
promoting the evidence-based treatment of sepsis, with the explicit
goal of reducing both the morbidity and mortality associated with
sepsis. This study was conducted to assess the clinical relevance
of the early physiologic screening criteria advocated by early goal-
directed therapy for sepsis, and the Surviving Sepsis Campaign
guidelines.
Materials and methods The Project IMPACT
®
database was
used to obtain a sample of patients (n = 363) with an ICU admis-
sion diagnosis of sepsis and a random acuity-matched comparison
sample of patients with an admission diagnosis (n = 364) other
than sepsis.
Results Significant group differences were found on all physio-
logic monitoring variables tested (high temperature, P = 0.000; low
Critical Care September 2007 Vol 11 Suppl 4 Sepsis 2007
Figure 1 (abstract P11)
Mortality rate reduction in septic shock patients (42 deaths in ‘before’
group versus 32 deaths in ‘after’ group). P < 0.04.
Table 1 (abstract P11)
Proceedings concerning ‘before’ versus ‘after’ groups
‘Before’ ‘After’
group group
Proceedings (%) (%) P value
Cultures obtained before antibiotics 68.1 84.3 0.01
Antibiotics in a due time (2-hour interval) 61.7 80.0 0.009
Corticosteroids 56.4 74.4 0.01
Activated protein C 1.0 12.2 0.002
Page 6
S7
temperature, P = 0.001; heart rate, P = 0.004; respiratory rate,
P = 0.005; and mean arterial pressure, P = 0.000). In the logistic
regression model, high temperature and mean arterial pressure
functioned as significant predictors, with odds ratios of 2.12 for
temperature at or above 38°C and 3.87 for MAP less than
70 mmHg. The odds ratio of having sepsis was 4.63 if both of
these predictors were present.
Conclusions It is important to understand the value of common
monitoring parameters in the early identification of sepsis, since
those parameters are continuously monitored and readily available.
It is the responsibility of bedside clinicians to assure that the
parameters chosen for monitoring provide the most accurate
reflection of the patient’s clinical status. These results provide
some support for the use of the currently recommended criteria for
physiologic monitoring in the early identification of patients at risk
for developing sepsis. Furthermore, if this could be done
automatically, it would probably shorten the recognition time and
thus speed up the initiation of sepsis treatment. ProtocolWatch is a
tool that offers an electronic version of the SSC guidelines,
screens the physiologic criteria automatically, and is resident on a
bedside patient monitor. Development of tools such as
ProtocolWatch will probably be an important adjunct to sepsis
identification and treatment in the future.
P14
The role of CC-chemokine receptor 4 in murine polymicrobial
sepsis
Stefan Maier
1
, Tobias Traeger
1
, Wolfram Keßler
1
, Pia Körner
1
,
Hendrik Mehmcke
1
, Yolande Chvatchko
2
, Marlene Mikulczak
1
,
Tobias Ebker
1
, Claus-Dieter Heidecke
1
1
Department of Surgery, Universität Greifswald, Germany;
2
Serono
Pharma, Geneva, Switzerland
Critical Care 2007, 11(Suppl 4):P14 (doi: 10.1186/cc5993)
Background Chemokines and chemokine receptors are crucially
involved in the mechanisms leading to septic shock after severe
systemic infections. The CC-chemokine receptor 4 (CCR4) is
predominantly expressed on T cells driving the immune response in
a Th2 direction. Interestingly, CCR4 knockout (KO) mice show no
phenotype in allergy models. In contrast, they are highly protected
in the lipopolysaccharide shock model. We analyzed the role of
CCR4 in a murine model or polymicrobial sepsis, colon ascendens
stent peritonitis (CASP).
Materials and methods In the CASP model, a stent is surgically
inserted into the ascending colon of experimental mice. This leads
to a persistent leakage of the gut with defined size and –
depending on the stent size – to a lethal or sublethal polymicrobial
sepsis. We performed 16 G CASP operations in CCR4 KO mice
or wild-type (WT) controls. For ex vivo analysis, organ expression
of CCR4 and its ligands, CCL17 and CCL21 were detected by
real-time PCR. The bacterial loads of various organs were
analyzed. Additionally, tissue cytokine levels were detected by
cytometric bead array. Finally, adoptive transfer experiments from
CCR4 KO mice to WT animals with or without CASP-induced
peritonitis were performed.
Results Similarly to the lipopolysaccharide shock, CCR4 KO mice
are protected in the CASP model. After sepsis induction, CR4 is
massively downregulated, whereas expression of the ligands
seems to be not severely affected. The absence of CCR4 signals
improves bacterial clearance in the investigated organs. In organs
of septic CCR4 KO mice significantly reduced IL-6 and monocyte
chemoattractant protein-1 levels were found as compared with the
WT controls. Astonishingly, adoptive transfer of CCR4 KO
splenocytes from CASP mice in WT animals resulted in a strongly
reduced susceptibility of these mice to the CASP procedure,
whereas transfer of WT splenocytes did not affect the outcome.
Conclusion We report a significant role of CCR4 signals in a
clinically relevant polymicrobial sepsis model.
P15
LL-37 at the local site of streptococcal skin and soft-tissue
infections
Pontus Thulin
1
, Linda Johansson
1
, Parham Sendi
1
,
Erika Hertzén
1
, Adam Linder
2
, Per Åkesson
2
,
Bertil Christensson
2
, Malak Kotb
3
, Donald Low
4
,
Birgitta Agerberth
1
, Anna Norrby-Teglund
1
1
Karolinska Institutet, Stockholm, Sweden;
2
Lund University, Lund,
Sweden;
3
Veterans Affairs Medical Center, Memphis, TN, USA;
4
University of Toronto, Toronto, ON, Canada
Critical Care 2007, 11(Suppl 4):P15 (doi: 10.1186/cc5994)
Background As part of the innate immune system, antimicrobial
peptides (defensins and cathelicidins) are produced by both
circulating and epithelial cells. Cathelicidins have been reported as
an essential component against group A streptococcal (GAS) skin
infections. However, bacterial factors such as streptococcal
pyrogenic exotoxin B (SpeB) may inactivate these peptides. We
have studied the interaction between GAS and the human
cathelicidin LL-37, by use of patient tissue material.
Methods and materials Thirty-seven biopsies from 17 patients
suffering from GAS skin and soft-tissue infection were obtained
and graded according to disease severity (erysipelas, cellulitis,
necrotizing fasciitis). Three additional biopsies served as negative
controls. Tissue sections were immunostained for LL-37, GAS,
SpeB and specific cell markers. Sections were investigated by
light and confocal microscopy, and results were quantified by in
situ imaging.
Results High expression of LL-37 was detected in erysipelas and
severe soft-tissue infections, and showed a significant positive
correlation to bacterial load (P < 0.001 and P = 0.042, respec-
tively). Confocal microscopy identified neutrophils as the main
source of LL-37 at the epicenter of infection, and the degree of
neutrophil infiltration showed a significant positive correlation to
LL-37 levels (P < 0.001). LL-37 and SpeB were detected in the
same biopsy areas, and colocalization was confirmed by confocal
microscopy.
Conclusions Despite the high expression of LL-37 in close proximity
to streptococci at the local site of infection, there seems to be a
significant lack of antimicrobial effect, as evident by the bacterial
load. The colocalization of SpeB and LL-37 suggests that this
streptococcal factor probably contributes significantly to a resistance
mechanism towards antimicrobial peptides at the local tissue site.
P16
HMGB1 expression in streptococcal soft-tissue infections
correlates with disease severity
Parham Sendi
1
, Linda Johansson
1
, Pontus Thulin
1
,
Adam Linder
2,
Per Åkesson
2
, Bertil Christensson
2
,
Donald Low
3
, Anna Norrby-Teglund
1
, Carl-Johan Treutiger
1
1
Karolinska Institutet, Stockholm, Sweden;
2
Lund University, Lund,
Sweden;
3
University of Toronto, Toronto, ON, Canada
Critical Care 2007, 11(Suppl 4):P16 (doi: 10.1186/cc5995)
Background High mobility group box-1 (HMGB1) is an intra-
cellular protein that is secreted by activated immune cells during
inflammation, or is passively released by cells undergoing necrosis.
HMGB1 acts both as a proinflammatory cytokine and a chemokine,
Available online http://ccforum.com/supplements/11/S4
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S8
and has been identified as a late mediator of sepsis. We have
studied HMGB1 expression in streptococcal skin and soft-tissue
infections, and its relation to bacterial load as well as to infiltration
of inflammatory cells.
Materials and methods Thirty-seven biopsies from 17 patients
suffering from streptococcal skin and soft-tissue infection were
obtained and graded according to disease severity (erysipelas,
cellulitis, necrotizing fasciitis). Three additional biopsies served as
negative controls. Tissue sections were immunostained for HMGB1,
group A streptococcus, and specific cell markers. Sections were
investigated by light microscopy, and results were quantified by in
situ imaging.
Results HMGB1 was found both intracellularly and secreted in the
tissue. Its expression increased in parallel to disease severity and
was significantly higher in necrotizing fasciitis than in erysipelas
(P = 0.023). HMGB1 showed a positive correlation to neutrophils
(P < 0.01) in erysipelas, but not in severe infections. A correlation
to bacterial load was not found.
Conclusion In contrast to erysipelas, large amounts of necrotic
tissue are present in severe skin infections, which probably
contribute considerably to the expression of HMGB1. The high
values may disturb a statistical correlation to the degree of
inflammatory cell infiltration in the tissue. However, our results
suggest that the massive HMGB1 expression at the local site of
infection is probably an important mediator and enhancer of
inflammation in skin tissue and soft-tissue infections, as evident by
its expression in correlation to disease severity.
P17
Can procalcitonin reflect the etiology of the bacteremia?
Ekaterina Chernevskaya, Natalia Beloborodova,
Tatyana Vostrikova
Bakoulev Scientific Center for Cardiovascular Surgery, Moscow,
Russia
Critical Care 2007, 11(Suppl 4):P17 (doi: 10.1186/cc5996)
Background An early diagnosis of bacteremia is crucial to
facilitate adequate treatment of severe infections. We analyzed
5,564 blood cultures for a 3-year period (2004, 2005, 2006) with
a 20% rate of positive blood culture, and observed the increasing
prevalence of Gram-positive bacteremia (45%/48%/63%, respec-
tively). Classical clinical inflammatory signs of Gram-negative and
Gram-positive infection are often similar, while some biomarkers
may help in early diagnostic of the nature of pathogen before
obtaining the blood culture results. The objective of the study was
to estimate the value of procalcitonin (PCT) as a discriminate
marker of Gram-positive and Gram-negative infection in suspected
bacteremia patients.
Materials and methods During 3 years monitoring of PCT and
blood culture in a total of 150 episodes (113 cardiac patients with
postoperative complication (systemic inflammatory response syn-
drome)) of positive blood culture with simultaneous PCT-test,
results were registered and retrospectively analyzed. For blood
culture we used the BacT/ALERT 3D system (bioMerieux, France)
and the BBL CRYSTAL Identification Systems Enteric/
Nonfermenter ID Kit (Becton Dickinson, USA). PCT concentrations
were measured by immunoluminometric method (PCT LIA;
B.R.A.H.M.S Aktiengesellschaft GmbH, Germany). The data were
compared by Mann–Whitney U-test, and P < 0.05 was considered
statistically significant. The data are expressed as the median and
25th and 75th percentiles.
Results In our study, 101/150 (67%) clinically important
bacteremia were caused by Gram-negative bacteria and 49 (33%)
by Gram-positive pathogens. The serum PCT concentration
(median) was significantly higher in the group of Gram-negative
bacteremia patients than in the group of patients with Gram-
positive bacteremia (5.40 versus 0.86 ng/ml, P < 0.001) (Table 1).
A PCT level > 2 ng/ml was reported in 72/101 (71%) cases in
Gram-negative bacteremia patients, whereas in patients with
Gram-positive bacteremia this level of PCT was reported twofold
lower (16/49 (32.6%) cases). The analysis of mortality in patients
with systemic infection (PCT > 2ng/ml + bacteremia) has shown
comparable data in groups of patients with Gram-positive and
Gram-negative bacteremia (8/14 (57.1%) and 31/55 (56.3%),
respectively).
Conclusion A high PCT level in patients with suspected infection
may be indicative of Gram-negative infection before obtaining the
culture results.
P18
Control of hyperglycemia among septic and nonseptic
patients in the general intensive care unit
Alexander Samokhvalov, Fabio Zveibil, Nicola Makhoul
General and Respiratory Intensive Care Units, Western Galilee
Medical Center, Nahariya, Israel
Critical Care 2007, 11(Suppl 4):P18 (doi: 10.1186/cc5997)
Background Hyperglycemia is common among patients admitted
to intensive care units (ICUs) and carries risk for various complica-
tions, especially sepsis, and prolonged ICU stay. Some studies
have demonstrated that intensive insulin control of blood glucose
reduced morbidity and mortality. The objective was to determine
whether intensive or conventional insulin control of blood glucose
in hyperglycemic septic and nonseptic ICU patients is correlated
with the prognosis.
Materials and methods Septic and nonseptic ICU patients with
hyperglycemia were randomly assigned to a group treated
intensively with insulin targeting glucose at 6.6–8.4 mmol/l or to a
conventional insulin therapy group where glucose upon exceeding
12 mmol/l was controlled at 8.4–12 mmol/l. Rates of morbidity and
mortality, hypoglycemic episodes and required insulin dosage were
compared.
Results A total of 89 patients were enrolled, including 27 patients
with sepsis: 11 patients were treated with insulin intensively with a
Critical Care September 2007 Vol 11 Suppl 4 Sepsis 2007
Table 1 (abstract P17)
Procalcitonin and clinical inflammatory signs in groups of patients with Gram-negative and Gram-positive bacteremia
Gram-negative bacteremia patients Gram-positive bacteremia patients P
Number of cases 101 49
Procalcitonin (ng/ml) 5.4 (1.78–12.21) 0.86 (0.28–2.19) <0.001
White blood cell count (× 10
9
/l) 15.4 (11.1–23.8) 14.2 (11–22) Not significant
Temperature (°C) 37.5 (37–38) 37 (37–38) Not significant
Multiple organ failure (number of patients (%)) 16/80 (20%) 7/32 (21.8%) Not significant
Page 8
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mean glucose of 8.3 mmol/l, while 16 patients received conven-
tional insulin treatment with a mean of 10.3 mmol/l. Thirty non-
septic patients received intensive insulin treatment with a mean of
8.46 mmol/l and 32 nonseptic patients were treated conventionally
with a mean of 10.4 mmol/l. Among septic patients, both groups
were similar with respect to age and Acute Physiology and Chronic
Health Evaluation scores. There was no significant difference
between groups in the morbidity, including rates of new infection,
renal and hepatic damage. There was a somewhat shorter ICU stay
in the intensive treatment group. Both groups had similar ICU,
inhospital and 28-day follow-up mortalities and similar rates of
hypoglycemic episodes. The daily dosage of insulin was higher
with the conventional treatment. Similar results were obtained
among nonseptic patients between both groups, but septic
patients had a longer total ICU stay and higher mortality.
Conclusions Intensive insulin control of blood glucose at 8.4 mmol/l
does not affect the mortality or morbidity of septic and nonseptic
patients in intensive care, except for a somewhat shorter ICU stay.
An increased insulin dosage in the conventional treatment group
was attributed to the group’s higher initial blood glucose, probably
due to a higher prevalence of diabetes and associated insulin
resistance and toxicity hyperglycemia.
P19
Effects of vasopressin and terlipressin in ovine septic shock
on mesenteric blood flow and survival
Sebastian Rehberg
1
, Christian Ertmer
1
, Matthias Lange
1
,
Andrea Morelli
2
, Hugo Van Aken
1
Martin Westphal
1
1
Department of Anaesthesiology and Intensive Care, University of
Münster, Münster, Germany;
2
Department of Anesthesiology and
Intensive Care, University of Rome, ‘La Sapienza’, Rome, Italy
Critical Care 2007, 11(Suppl 4):P19 (doi: 10.1186/cc5998)
Background Vasopressin agonists, such as arginine vasopressin
(AVP) and terlipressin (TP), are increasingly used to stabilize
haemodynamics in catecholamine refractory hyperdynamic septic
shock. However, it is still not fully understood if and how low-dose
infusion of both drugs impacts on mesenteric blood flow (Qma)
and outcome. The present study was conducted as a prospective,
randomized, controlled laboratory experiment to compare the
effects of AVP and TP on Qma and mortality in an established
model of ovine septic shock.
Materials and methods Fifteen ewes were anaesthetized and
instrumented for chronic haemodynamic monitoring. A median
laparotomy was performed to place a flow-probe around the
superior mesenteric artery and to take faeces from the caecum
under sterile conditions. After the gut and abdomen had been
closed and baseline measurements (BL1) taken, the faeces were
injected into the peritoneal cavity. After the onset of septic shock
(defined as mean arterial pressure (MAP) < 60 mmHg), a second
set of measurements (BL2) was taken. The animals were then
randomly assigned to receive either AVP (0.5 mU/kg/min) or TP
(1 μg/kg/hour). The control group received only the vehicle (normal
saline). Norepinephrine was titrated to maintain MAP at 70 ± 5 mmHg
in all groups. Systemic haemodynamics, global oxygen transport
including arterial lactate concentrations, gas exchange, electrolytes
and Qma were determined at baseline and following every hour
after the onset of septic shock. Animals surviving the 12-hour study
period were deeply anaesthetized and killed by an overdose of
saturated potassium solution. Mortality was analyzed by the Kaplan–
Meier survival analysis. All the other variables were compared using
two-way analysis of variance with appropriate post-hoc comparisons.
Results The Qma and electrolytes were similar between groups.
However, systemic haemodynamics and global oxygen transport
were stabilized more effectively in both treatment groups versus
control animals. Notably, continuous infusion of AVP and TP
significantly prolonged survival as compared with the control group
(P < 0.05 each; Figure 1). There were no differences between the
treatment groups.
Conclusions In this clinically relevant large animal model of septic
shock, low-dose infusion of AVP and TP did not impair the Qma,
but stabilized systemic haemodynamics and prolonged survival.
Our data suggest that early infusion of AVP or TP may be
beneficial in catecholamine-refractory septic shock.
P20
Neutrophil CD64 expression, a marker of sepsis/infection, can
be performed on a hematology blood counter and has variable
correlation to C-reactive protein, procalcitonin and soluble
CD163
Bruce Davis
1
, Karen Becker
1
, Henry Rinder
2
, Kathleen Davis
1
1
Trillium Diagnostics, Brewer, ME, USA;
2
Department of Laboratory
Medicine, Yale University, New Haven, CT, USA
Critical Care 2007, 11(Suppl 4):P20 (doi: 10.1186/cc5999)
Background Neutrophil CD64 expression (PMN CD64) has been
proposed as an improved laboratory indicator of severe infection
and sepsis. Little is published on the interrelationship between
PMN CD64 and the soluble phase indicators of inflammation, such
as C-reactive protein (CRP), procalcitonin (PCT), and soluble
CD163.
Methods We studied PMN CD64 in three clinical groups:
neonates (390 specimens), hospitalized patients (236 specimens),
and ambulatory outpatients (96 specimens). PMN CD64 was
measured as an index using the Leuko64 (Trillium Diagnostics).
Samples were also processed in parallel for the measurement of
CD64 using the Leuko64 assays for flow cytometry on a FACScan
(Becton Dickinson) and the blood cell counter Cell Dyn Sapphire
(Abbott Diagnostics). Data were analyzed using Leuko64 software
(Trillium Diagnostics). Results from both platforms were expressed
as the PMN CD64 index, the monocyte CD64 index, and the
monocyte CD163 index. CRP was measured in parallel. Plasma
samples were stored at –70°C for subsequent measurement of
procalcitonin (B.R.A.H.M.S.) and soluble CD163 (Trillium Diagnostics).
Results PMN CD64 correlated best with CRP, closely followed by
PCT, but the degree of correlation varied among the clinical
groups. The correlation was best in neonates (r = 0.592 for CRP
and r = 0.391 for PCT), followed by hospitalized patients
Available online http://ccforum.com/supplements/11/S4
Figure 1 (abstract P19)
Survival of animals over time.
Page 9
S10
(r = 0.345 for CRP and r = 0.349 for PCT), and less so in
outpatients (r = 0.251 for CRP and r = 0.257 for PCT). The
correlation between PMN CD64 and the soluble markers was
higher than that between CRP and PCT (r = 0.331 for hospitalized
patients, r = 0.305 for neonates, and r = 0.196 for ambulatory
patients). Soluble CD163 levels only weakly correlated with PMN
CD64, CRP and PCT. The Sapphire results were highly correlated
with flow cytometry (r = 0.99). The measured level of imprecision
of both assays was <12% CV for PMN CD64, monocyte CD64,
and monocyte CD163 indices. The assay results were available in
<1 hour.
Conclusions This study shows a moderate correlation of PMN
CD64 with the ‘acute phase reactants’ CRP and PCT. Soluble
CD163 is only weakly correlated with the other parameters and
may independently define further subsets of patients based upon
different anti-inflammatory responses to the clinical condition. The
interrelationship of these parameters varies in different clinical
situations. We demonstrate it is feasible to automate cellular
assays for infection/sepsis in a routine hematology laboratory
providing access to a larger patient population.
P21
Tolerance to lipopolysaccharide regulates apoptosis in
B lymphocytes
Edielle de Sant Anna Melo, Renata Gorjao, Ruy Curi,
Irineu Tadeu Velasco, Francisco Garcia Soriano
University of São Paulo, Medicine School, Medical Clinical
Department, Clinical Emergency – LIM 51, São Paulo, Brazil
Critical Care 2007, 11(Suppl 4):P21 (doi: 10.1186/cc6000)
Background The most important event determining sepsis
evolution is immune system cell apoptosis, the immune cell
elimination compromises the host effective response, and preven-
tion of apoptosis events improve survival in sepsis models. Our
objective was to identify whether lipopolysaccharide (LPS)
tolerance regulates apoptotic genes and caspase pathway.
Materials and methods Male Balb-C mice received LPS
(1 mg/kg), a tolerant dose, and controls received 0.9% physio-
logical serum during 5 days, both receiving on day 7 a LPS lethal
dose (20 mg/kg). Control, 2 and 4 hours after lethal dose, IL-10,
IL-6, IL-1β, TNFα and MIP2 were measured by ELISA. Splenic B
lymphocytes were separated through magnetic beads and genes
were analyzed by microarray, comparing control and tolerant
groups. The tolerant and control groups were followed during
5 days to analyze survival.
Results See Table 1. The mRNA of caspases 2, 7, 8 and 11, Bid,
Apaf-1 and FAS genes were reduced in the tolerant mice. The IL-6
levels reduced in the tolerant mice (724 ± 15 pg/ml) versus control
mice (1,488 ± 96 pg/ml) in 2 hours. IL-1β was reduced at 0 hours
and at 4 hours in the tolerant group (657 ± 25 pg/ml) versus
control (1,117 ± 20 pg/ml). MIP2 also showed a reduction at
4 hours in tolerant (1,803 ± 159 pg/ml) versus control mice
(2,173 ± 252 pg/ml). The tolerant animals had 100% survival,
controls had zero survival. In all mentioned data, P < 0.05.
Conclusion Tolerance was able to reduce cytokine plasma levels,
immune cell apoptosis and mortality to LPS lethal doses.
P22
Physiological parameters, location of infection and organ
failure are significant predictors of misdiagnosing severe
sepsis
Diane Chamberlain
1
, Christine Wilson
1
, Tamara Hunt
2
1
Flinders University, Faculty of Health Sciences, Adelaide,
Australia;
2
Flinders Medical Centre, Adelaide, Australia
Critical Care 2007, 11(Suppl 4):P22 (doi: 10.1186/cc6001)
Background Severe sepsis and septic shock are common disease
processes in the critically ill and are associated with substantial
morbidity and mortality. The importance of the early identification
and diagnosis of severe sepsis has been highlighted by the
Surviving Sepsis Guidelines with the aim to provide early and
aggressive management in order to improve outcome. In
contemporary practice, all clinicians have the responsibility of
identifying severe sepsis. Therefore the objectives of this study
were to determine whether emergency department and intensive
care clinicians could identify and diagnose severe sepsis in those
patients in their care within the first 24 hours of admission, and to
identify predictors of failing to diagnose sepsis.
Methods The patient cohort were prospectively screened and
enrolled on admission to intensive care within the first 24 hours.
Severe sepsis was defined as new-onset acute organ dysfunction,
using consensus criteria. Clinical data and physiological
parameters were collected prospectively. Diagnosis was based on
microbiologically confirmed clinical findings. Clinicians caring for
each patient were prospectively surveyed.
Results All 402 subjects had infection. Infection sites included
52% pneumonia, 17% urinary, 15% abdominal, 6% wound and
skin, and 10% isolated organs and bone. Single-organ failure was
evident in 21%, 42% had two-organ failure, 29% had three-organ
failure and 8% had four-organ failure. Nurses identified sepsis in
141 of the 402 patients (P < 0.001) whereas physicians did so in
265 of the 402 patients (P < 0.05). Misdiagnosis of severe sepsis
by the attending nurse or physician was more likely to be
associated with pneumonia (odds ratio (OR) = 4.2 (95% confi-
dence interval (CI) = 3.6–4.2), P < 0.01), urinary sepsis (OR = 2.9
(95% CI = 2.6–3.4), P < 0.5), less than three-organ failure (OR =
3.1 (95% CI = 2.4–3.7), P < 0.01), Gram-negative infection (OR =
2.3 (95% CI = 1.6–3.5), P < 0.5) and presenting without fever
(OR = 3.5 (95% CI = 3.1–3.9), P < 0.05). Thirty-two percent of
clinicians did not know the criteria for severe sepsis and 57%
missed the patient diagnosis in their care at that time.
Conclusion In this study, misdiagnosis of severe sepsis is still an
acknowledged problem in meeting the goals of early resuscitation.
Critical Care September 2007 Vol 11 Suppl 4 Sepsis 2007
Table 1 (abstract P21)
Genic expression of apoptosis in tolerance to lipopolysaccharide
Genebank
analysis
number Name Ratio
a
X92346 TNF receptor-associated factor 4 (TRAF4) 3.8
U06948 Fas antigen ligand (FASL) 4.5
U37522 TNF-related apoptosis-inducing ligand (TRAIL) 3.9
M83649 Fas I receptor 4.8
U88990 Inhibitor of apoptosis protein 3 Down
D28492 Caspase 2 precursor Up
U39613 Caspase 7, apoptosis-related cysteine protease Up
U59463 Caspase 11 3.4
U13021 Caspase 2, apoptosis-related cysteine protease Up
AF064071 Apoptotic protease activating factor 1 Up
U39643 Fas-associated factor 1 Up
X67914 Programmed cell death 1 protein precursor Up
NM022684 Bid, apoptotic protease 4.6
XM232860 Caspase 8, apoptosis-related cysteine protease Up
a
Ratio = controls/tolerants: down, down to 0.05; up, up to 5.0.
Page 10
S11
Protocols and monitoring tools may assist the early identification of
severe sepsis so appropriate care can be prioritised and resusci-
tation implemented early in their admission.
P23
In vitro and in vivo determination of anti-TNF
αα
activity in canine
plasma from donors subject to preconditioning with endotoxin
Michael Kotiw
1
, Michael Morgan
2
, Jesus Lopez
1
,
Steven Taylor
2
, Ian Shiels
2
1
Centre for Systems Biology, University of Southern Queensland
Toowoomba, Queensland, Australia;
2
School Biomedical Sciences,
University of Queensland, St Lucia, Australia
Critical Care 2007, 11(Suppl 4):P23 (doi: 10.1186/cc6002)
Background Septic shock is characterized by cardiovascular and
vasomotor failure that is induced by an uncontrolled cascade of
inflammatory mediators such as TNFα, IL-1β and IL-6. In dogs,
systemic bacterial infections, haemorrhage, trauma, gastric
dilatation/volvulus and pancreatitis are the major causes of septic
shock. While endotoxin is a recognized effector molecule that can
initiate an inflammatory cascade, it has been reported that
preconditioning with endotoxin can downregulate inflammatory
cytokine responses to subsequent endotoxin challenge. This study
reports the effect of endotoxin preconditioning on anti-TNFα
activity present in plasma from canine donors.
Materials and methods Plasma from preconditioned (Caniplas
®
)
and normal dogs (FFP) was provided blind to the study by a
commercial supplier (Plasvacc Pty Ltd). In vitro anti-TNFα activity
in canine donor plasma was determined by a L929 murine cell
TNFα inhibition bioassay using recombinant murine TNFα. In vivo
effects were tested by a rat subcutaneous skin pouch model. Rats
were pretreated for 3 days with either Caniplas
®
, FFP, saline
(2 ml/day, s.c.) or carprofen (5 mg/kg, s.c.) and inflammation was
induced by injecting monosodium urate crystals into the pouch
(5 mg/ml in 5 ml saline). Fluid was taken from pouches at specified
intervals for cell count, TNFα and IL-6 analysis. Data analysis:
normalized data were fitted to a four-parameter logistic curve. The
fitted midpoints were compared statistically for datasets using an
F-test and calculated fitted hill slopes.
Results In the rat skin pouch model, both Caniplas
®
and FFP
reduced TNFα levels and Caniplas
®
was a more potent antagonist.
The heightened anti-TNFα activity of Caniplas
®
compared with
FFP was confirmed in the in vitro cell bioassay (Figure 1). Neither
Caniplas
®
nor FFP reduced inflammatory cell infiltration or the
levels of IL-6.
Conclusion While we need to confirm the mechanism, we report
that preconditioning with endotoxin does illicit specific anti-TNFα
activity and that this observation has been confirmed in both in
vitro testing and in vivo animal models. It is plausible that
preconditioning animals with endotoxin induces an increase in the
concentration of soluble TNFα receptors I and II in donor plasma,
and that this is the probable source of TNFα antagonism. This
report suggests that preconditioned plasma may be a beneficial
treatment where inflammation causes increased expression of
TNFα.
P24
The human antimicrobial peptide LL-37 induces endothelium-
dependent vascular smooth muscle relaxation mediated via
the lipoxin A4 receptor
Ingrid Berkestedt, Axel Nelson, Mikael Bodelsson
Section of Anaesthesiology and Intensive Care, Department of
Clinical Sciences, Lund University, Lund, Sweden
Critical Care 2007, 11(Suppl 4):P24 (doi: 10.1186/cc6003)
Background Septic shock includes blood vessel dilatation and
activation of innate immunity. The activation causes release of
antimicrobial peptides such as LL-37. It has been shown that LL-
37 can attract leukocytes via the lipoxin A4 receptor (ALX). ALX is
also present in vascular endothelial cells. To explore possible ways
of pharmacological intervention in septic shock, we investigated
whether LL-37 can affect vascular tone.
Materials and methods Human omental arteries and veins were
obtained during abdominal surgery. The circular smooth muscle
activity, in the wall of the vessel segments, was studied in organ
baths. Gene expression was studied using reverse transcriptase
PCR.
Results LL-37, at micromolar concentrations, induced a
concentration-dependent and endothelium-dependent relaxation in
vein segments but not in artery segments precontracted by
endothelin-1. The relaxation was profoundly reduced by potassium
chloride (30 mM) to inhibit endothelium-derived hyperpolarizing
factor (EDHF), while it was less affected by the NOS inhibitor
L-NAME and not at all by indomethacin. The ALX agonist,
WKYMVm, did also induce a relaxation, and both the relaxations
induced by LL-37 and WKYMVm were inhibited by the ALX
antagonist WRWWWW. ALX was expressed in the endothelium.
Conclusion We demonstrate for the first time that the human
antimicrobial peptide LL-37 induces endothelium-dependent
Available online http://ccforum.com/supplements/11/S4
Figure 1 (abstract P23)
In vitro TNFα dose-response to canine sera.
Page 11
S12
relaxation in human omental veins mediated via an effect on
endothelial ALX. The relaxation involves the release of nitric oxide
and EDHF but not prostanoids. LL-37 released from white blood
cells could contribute to the blood vessel dilatation during sepsis
and treatment with ALX antagonists such as WRWWWW might
be successful.
P25
Human protein C concentrate in the treatment of hemolytic
uremic syndrome
Burkhard Wermter
1
, Harald Koeditz
1
, Kathrin Seidemann
1
,
Thomas Jack
1
, Bernadette Brent
1
,
Armin Wessel
1
,
Michael Sasse
1
, Lars Pape
2
1
PICU Interdisciplinary Paediatric Intensive Care Unit and
2
Paediatric Nephrology, MHH Medizinische Hochschule Hannover,
Germany
Critical Care 2007, 11(Suppl 4):P25 (doi: 10.1186/cc6004)
Background Human protein C (PC) concentrate may anticipate
thrombotic microangiopathy and facilitate fibrinolysis in the severe
hemolytic uremic syndrome (HUS).
Materials and methods We report the effects of PC in six HUS
patients. HUS is characterized by a simultaneous occurrence of
hemolytic anemia, thrombocytopenia and acute renal failure. Post-
diarrheal HUS is often based on an infection with enterohemor-
rhagic Escherichia coli producing Shiga toxins. Our current patho-
genetic understanding is that Shiga toxins cause endothelial injury,
leading to thrombotic microangiopathy. There is still a 5% rate of
mortality particularly caused by cerebral involvement. We treated
six children with a severe cerebral manifestation, five of them
suffered from a multiple organ dysfunction syndrome (MODS), of
HUS with PC over 7–10 days. All patients suffered peritoneal
dialysis, one patient a plasmapheresis. In addition to the treatment
of the MODS, all of the patients received 100–200 U/day PC.
Results All of the patients showed signs of disseminated
intravascular coagulation. We found typical hypodense lesions in
basal ganglia and edema of the brain in computed tomography.
During the therapy with PC, MODS was remarkably improved and
abnormal D-dimer and plasminogen activator inhibitor 1 levels
could be normalized. All of the patients recovered a nearly normal
kidney function. Two patients persisted in a severe reduced neuro-
logical status. The others showed only slight or no neurological
disabilities on discharge. No adverse effects were observed with
the PC concentrate administration.
Conclusion There is no generally accepted therapy regimen to
treat HUS in case of neurological involvement. Mortality in HUS
accompanied with cerebral microangiopathy is high and difficult to
alter. This is the first trial of human PC concentrate administration
to anticipate thrombotic microangiopathy in HUS. All of our
patients showed rapid clinical improvement under PC adminis-
tration. Four of six patients were discharged in a healthy condition
despite their severe disease. The containment of the severe
neurological involvement and the lack of side effects in the
treatment with human PC concentrate administration in our
patients yield hope that PC treatment may be an effective therapy
regimen in the treatment of severe HUS.
P26
Biofilm forming P. aeruginosa induces an enhanced
inflammatory response in human monocytes
Cristina Ciornei
1
, Christophe Beloin
2
, Alexey Novikov
3
,
Martine Caroff
3
, Catherine Fitting
1
, Jean-Marc Ghigo
2
,
Jean-Marc Cavaillon
1
, Minou Adib-Conquy
1
1
Pasteur Institut, Cytokines & Inflammation, Paris, France;
2
Pasteur
Institut, Genetics of Biofilms, Paris, France;
3
Equipe ‘endotoxines’,
CNRS UMR 8619, Paris-Sud University, Orsay, France
Critical Care 2007, 11(Suppl 4):P26 (doi: 10.1186/cc6005)
Background The clinical picture of sepsis is varying, and the
severity of the disease is influenced by numerous factors including
the infectious agent and the host genetics. P. aeruginosa and
S. aureus, the frequently sepsis-causing agents, can switch from a
planktonic to a biofilm lifestyle. The cell wall of P. aeruginosa
contains lipopolysaccharide (LPS), which can detach and trigger
the immune response in the patient. We investigated the effects of
live planktonic or biofilm bacteria on human monocytes in terms of
the production of proinflammatory cytokines, such as TNF, in order
to determine whether these two bacterial lifestyles influenced the
immune response during sepsis.
Materials and methods Clinical isolates of P. aeruginosa and S.
aureus, bacterial culture plates and media (Luria-Bertani and tryptic
soy broth), RPMI media, ELISA kits, a spectrophotometer and
biofermentors were used. The same bacterial concentration of
planktonic and biofilm forming P. aeruginosa and S. aureus strains
was obtained by optical density measurements and confirmed by
colony counting. Blood samples were collected from healthy
donors and monocytes were isolated by adherence and further
incubated together with live planktonic or biofilm forming strains of
P. aeruginosa or S. aureus for 5 hours; the cytokine content was
measured by ELISA. LPS was extracted from P. aeruginosa in
order to investigate the structural differences between planktonic
and biofilm derived LPS. The limulus amebocyte lysate test, SDS-
PAGE gel electrophoresis and mass spectrometry of LPS were
performed to analyze the LPS structure.
Results The production of TNF, IL-6 and IL-1α was increased in
monocytes incubated with biofilm forming P. aeruginosa as
compared with planktonic ones, whereas no difference between
cytokine responses was observed in monocytes incubated
together with planktonic or biofilm S. aureus. Two predominant
forms of rough LPS were detected in planktonic P. aeruginosa by
SDS-PAGE and one of the rough LPS bands was absent in the
biofilm. Fatty acids differed by their level of hydroxylation in the two
bacterial growth conditions as seen by mass spectrometry.
Conclusions Biofilm forming P. aeruginosa induces an enhanced
inflammatory response in human monocytes compared with the
planktonic bacteria, and LPS structures were found to be different.
No difference was seen in response to S. aureus planktonic or
biofilm bacteria. The biofilm P. aeruginosa was more immun-
ostimulatory than the planktonic form. LPS from biofilm forming
bacteria may increase the immune response during sepsis.
Critical Care September 2007 Vol 11 Suppl 4 Sepsis 2007
Page 12
S13
P27
Laboratory markers to determine clinical significance of
coagulase-negative staphylococci in blood cultures
Piret Mitt
1
, Siiri Kõljalg
2
, Krista Lõivukene
2
, Epp Sepp
2
,
Irja Lutsar
2
, Matti Maimets
1
, Paul Naaber
2
1
Tartu University Hospital, Department of Infection Control, Tartu,
Estonia;
2
University of Tartu, Departmentt of Microbiology, Tartu,
Estonia
Critical Care 2007, 11(Suppl 4):P27 (doi: 10.1186/cc6006)
Background Coagulase-negative staphylococci (CoNS) are
important causes of bloodstream infection, especially in immuno-
compromised patients, but they are also the most common
contaminants of blood cultures; discrimination between these two
is often difficult. The time necessary for microbial growth to appear
(time to positive) has been used as a laboratory marker for
assessing clinical significance of CoNS bacteremia. However, with
the use of continuously monitoring blood culture systems, the
previous study results are controversial. The aim of the present
study was to assess microbiological laboratory markers that are
suggestive of true CoNS bacteremia.
Materials and methods All blood cultures positive for CoNS
between 1 October 2006 and 30 April 2007 in Tartu University
Hospital were included in this analysis. Blood specimens were
monitored with the BACTEC 9240 system. Microbes were
identified using the VITEK 2 system and antibacterial susceptibility
pattern was tested according to CLSI standards. The CDC
definition for bloodstream infection to determine the clinical
significance of CoNS was used.
Results A total of 109 CoNS blood isolates from 86 patients (51
male; median age 22 years, range 0–94 years) were identified.
According to the CDC criteria, 81 isolates were contaminants and
28 were true causes of bacteremia. Fifteen of the patients with
infection were from the intensive care unit. The time to positive for
blood cultures in infected patients was shorter than that in
contaminated subjects: median 30 hours (range 20–68 hours)
versus 42 hours (range 16–116 hours), respectively (P = 0.029).
A total of 10 different CoNS species were identified;
S. epidermidis was most commonly isolated in both groups –
21/28 in infection and 49/81 in contamination (OR, 1.87; 95% CI,
0.77–4.56). S. hominis comprised a higher proportion in the
contamination group than in the infection group (17/81 versus
2/28 respectively; OR, 2.59; 95% CI, 0.84–8.01).
Conclusions The majority of CoNS isolated from blood cultures
were contaminants. The time necessary for microbial growth is an
important laboratory marker in differentiating between true
bacteremia and contamination.
P28
Inhibition by telithromycin of systemic and respiratory
inflammation induced by endotoxin in mice
Magdalena Leiva, Alfonso Ruiz-Bravo, Maria Jimenez Valera
University of Granada, Department of Microbiology, Granada, Spain
Critical Care 2007, 11(Suppl 4):P28 (doi: 10.1186/cc6007)
Background Lower respiratory tract infections are the cause of
septic shock in 25% of patients. Telithromycin (TEL), the first
ketolide antibiotic, is used for treatment of respiratory infections.
TEL is a semisynthetic derivative of the macrolide erythromycin.
Beyond their antimicrobial activity, macrolides display immuno-
modulatory effects, including the inhibition of inflammatory
reactions. In the present study, we demonstrate the anti-inflam-
matory effects of TEL on a septic shock model and a respiratory
inflammation model in mice.
Materials and methods TEL was a gift from Aventis Pharma
(Neuville-sur-Saonc, France). Lipopolysaccharide (LPS) from
Escherichia coli O26:B6 was purchased from Sigma Chemical
Co. (St Louis, MO, USA). BALB/c female mice (10–12 weeks old)
were maintained in the facilities of the University of Granada. To
induce septic shock, mice were injected intraperitoneally with a
dose of 50 mg LPS/kg body weight. To induce respiratory inflam-
mation, mice were exposed for 20 minutes to aerosolized LPS
(500 μg/ml saline) in a chamber connected to an air nebulizer
(Miko, CA-MI s.n.c., Italy). To investigate the effects of TEL, mice
received a single dose of 20 mg ketolide/kg body weight by
intraperitoneal injection, 1 hour prior to the administration of LPS.
Heparinized blood samples were centrifuged, and plasma was
stored at –20ºC until cytokine determination. To obtain broncho-
alveolar lavage (BAL) fluids, the lungs were lavaged with 1 ml
phosphate-buffered saline through an intratracheal catheter.
Enzyme immunoassays kits were use to determine TNFα (Pierce
Endogen, Rockford, IL, USA) and macrophage inflammatory
peptide 2 (MIP-2) (R&D Systems, Minneapolis, MN, USA). The
differences in cytokine levels were analyzed using Student’s t test.
P < 0.05 was considered significant.
Results When mice were intraperitoneally challenged with a lethal
dose of LPS, TEL protected 50% of animals and significantly
reduced the plasma levels of TNFα at 2 hours after LPS
administration. In the respiratory inflammation model, the treatment
with TEL significantly reduced the BAL levels of TNFα and MIP-2
at 4 hours post endotoxin (Table 1).
Conclusions TEL exerts anti-inflammatory activity in vivo that may
contribute to its pharmacological effectiveness in the treatment of
respiratory infections and their possible progression to septic
shock.
P29
Study of procalcitonin as a marker of sepsis in patients of a
general hospital during a 2-year period
Caterina Petrochilou, Myrianthi Pouyouka, Vasiliki Skandami,
Charika Kontou, Melpomeni Kalderi, Alkipi Nitsa,
Marina Toutouza
Department of Microbiology, ‘Hippokration’ General Hospital,
Athens, Greece
Critical Care 2007, 11(Suppl 4):P29 (doi: 10.1186/cc6008)
Background The aim of this study was to determine the number of
specimens with abnormal procalcitonin (PCT) values in the total of
specimens that were examined during a 2-year period.
Materials and methods We tested 1,114 patients who were
hospitalized in various clinics of Hippokration General Hospital of
Available online http://ccforum.com/supplements/11/S4
Table 1 (abstract P28)
Effect of telithromycin on cytokine levels in bronchoalveolar lavage
and plasma in a mouse respiratory inflammation model
TNFα in BAL MIP-2 in BAL MIP-2 in plasma
Treatment (pg/ml) (pg/ml) (pg/ml)
None 11 ± 6 41 ± 3 1 ± 4
TEL 9 ± 4 28 ± 2 0 ± 0
LPS 2846 ± 369 1637 ± 254 40 ± 10
TEL + LPS 1279 ± 285
a
1020 ± 240
b
24 ± 5
c
a
P < 0.005, significantly different from mice treated with LPS alone.
b
P < 0.001, significantly different from mice treated with LPS alone.
c
P < 0.05, significantly different from mice treated with LPS alone.
Page 13
S14
Athens during the period 2005–2006. PCT was measured
because there was clinical suspicion of sepsis. The PCT value in
serum was determined using the immunoluminometric assay method
(Liaison-BRAHMS PCT) with a normal range of 0.10–0.50 ng/ml.
Results In the total of 1,114 specimens, which corresponded to
the same number of patients, 178 patients (15.98%) had abnormal
PCT values that ranged between 1 and 167 ng/ml. These patients
were a posteriori clinically and by laboratory examination proven to
be in systemic inflammatory response syndrome and sepsis.
Conclusion According to the results, PCT is a useful marker for
the early diagnosis of systemic bacterial infection when clinical and
laboratory signs are nonspecific for this determination.
P30
Lung-derived macrophage migration inhibitory factor in adult
patients with septic shock, and its role in cardiocirculatory
depression
Hans Lee
1
, Kaie Ojamaa
1
, Greg Ruskin
1
, Erfan Hussain
1
,
Maowen Hu
1
, Xinchun Lin
1
, Christine Metz
1
, Yousef Al-Abed
1
,
Edmund Miller
1,2
1
Laboratory of Cardiopulmonary Research, The Feinstein Institute,
Manhasset, NY, USA;
2
Albert Einstein College of Medicine Bronx,
NY, USA
Critical Care 2007, 11(Suppl 4):P30 (doi: 10.1186/cc6009)
Background Migration inhibitory factor (MIF), a critical proinflam-
matory mediator in sepsis, has a profound affect on cardiovascular
function. Our animal studies show that the lungs release MIF into
the systemic circulation during late sepsis. MIF released in this way
has direct and immediate access to cardiac cells. The purpose of
our study was to assess the lung as a source of MIF in human
septic shock patients and to further study the MIF-associated
pathways involved in cardiovascular depression.
Materials and methods Nine adult patients with septic shock in
the medical intensive care unit were studied. Blood samples were
collected pre-lung (pulmonary artery or central venous catheter)
and post-lung (arterial line catheter) at 12, 24 and 48 hours from
the time of diagnosis. MIF was measured using an ELISA, and
differences in plasma MIF concentration pre-lung versus post-lung
were assessed using a paired t test on signed ranks. Furthermore,
since inhibition of caspase 3 activity during sepsis reduces
myocardial apoptosis and cardiac dysfunction, we examined in
vitro the effect of MIF on cardiomyocyte apoptosis.
Results The mean age of patients was 57.6 years (range 25–82
years). Bloods from six patients were culture positive. There was a
wide variation in plasma MIF concentrations in both pre-lung
(0.2–64.7 ng/ml) and post-lung (0.2–76.4 ng/ml). However, there
was a significant increase in the median MIF level of the post-lung
blood (3.9 ng/ml) compared with pre-lung blood samples
(2.9 ng/ml, P = 0.005). At 48 hours post diagnosis, eight out of
nine individuals had increased MIF concentration post-lung, with a
mean increase of 64 ± 49%. These findings were independent of
the source or nature of the infection. The basal level of apoptosis in
primary cardiomyocytes cultured in defined, serum-free medium
was 3.7 ± 0.9% (% TUNEL positive of total nuclei). This increased
significantly (P < 0.001) to 15.5 ± 3.9% and 26.0 ± 7.1% when
treated with MIF 20 and 30 ng/ml, respectively. Simultaneous
treatment of cardiomyocytes with MIF and specific MIF inhibitor
ISO1 (50 mM) resulted in significant attenuation of the apoptotic
effect, returning apoptosis to a basal level at 4.1 ± 0.6%.
Conclusion The study demonstrates for the first time in humans
that the lung is a major source of MIF in septic shock. The study
further suggests that MIF released from the lung may reduce
cardiac function by increasing cardiomyocyte apoptosis, and that
blocking MIF released from the lung to prevent cardiocirculatory
deterioration may provide new strategies for the treatment of sepsis.
P31
Inhibition of central leukotrienes and nitric oxide production
affects vasopressin secretion induced by polymicrobial sepsis
Maria José Alves da Rocha, Letícia Antunes Athayde,
Gabriela Ravanelli de Oliveira-Pellegrin
Departmento de Morfologia, Estomatologia e Fisiologia, Faculdade
de Odontologia e de Ciências Farmacêuticas de Ribeirão Preto,
Universidade de São Paulo, Ribeirão Preto, SP, Brazil
Critical Care 2007, 11(Suppl 4):P31 (doi: 10.1186/cc6010)
Background Sepsis induces an increase in vasopressin (AVP)
secretion and massive production of inflammatory mediators,
including leukotrienes (LTs) and nitric oxide (NO), which are also
produced in the brain and may affect the neuroendocrine system.
Our aim was to study the role of central LTs and NO in AVP
secretion during polymicrobial sepsis induced by cecal ligation and
puncture (CLP).
Methods Male Wistar rats (250–280 g) received an intracerebro-
ventricular injection of MK-886 (1 μg/kg), an LT biosynthesis
inhibitor, or L-NAME (250 μg), an NO synthase inhibitor. Controls
received a carrier injection before CLP, or were only sham
operated. In one group of animals, survival was monitored for
5 days. In another group, the animals were decapitated at 4, 6 or
24 hours after surgery and blood was collected for measurements
of hematocrit, plasma nitrate (NOx) and AVP levels.
Results CLP caused an increase in plasma AVP levels in the initial
phase of sepsis, which was blocked by the central administration
of MK-886 and was elevated by intracerebroventricular-injected L-
NAME (P < 0.05). This contrasted with the final phase of sepsis,
when plasma AVP remained at basal levels and was not altered by
the administration of LT and NO blockers. The gradual increase in
NOx levels was reduced by MK-886 and blocked by L-NAME. The
increase in hematocrit caused by CLP was diminished (P < 0.05)
by L-NAME injection, but was not modified by central adminis-
tration of MK-886. The low survival rates observed after CLP were
improved by the central administration of both drugs (P < 0.05).
Conclusion These results suggest that central LTs and NO
differentially affect AVP secretion during CLP-induced experi-
mental sepsis.
Acknowledgement Supported by FAPESP.
P32
Real-time, low-cost detection of individual fungal cells in blood
using Fountain Flow™ cytometry
Paul E Johnson
1,2
, Christopher M Havens
1,2
,
Joseph F Johnson
1,2
1
Department of Physics & Astronomy, University of Wyoming,
Laramie, WY, USA;
2
SoftRay, Inc., Laramie, WY, USA
Critical Care 2007, 11(Suppl 4):P32 (doi: 10.1186/cc6011)
Background As fungi are considerably more slow growing than
bacteria and are not affected by broad-spectrum antibiotics, too
often conventional diagnostics fail to identify fungal bloodstream
infection and confirmation is made postmortem. We describe the
initial tests of a Fountain Flow™ cytometer (FFC) to detect
pathogenic fungi in human blood, rapidly and cost-effectively. This
innovative technique is based on fluorescence in which a stream of
solution containing the cells, labeled with a fluorescent stain, is
illuminated with an LED (Figure 1). The resulting fluorescence is
detected with a CMOS imager.
Critical Care September 2007 Vol 11 Suppl 4 Sepsis 2007
Page 14
S15
Materials and methods The FFC used here was used by us in
previous published research to detect individual bacteria and
amoebae in river water at concentrations 200 bacteria/ml and
0.06 amoebae/ml. An optical stimulant of blood was made by
mixing red blood cells (RBCs) in saline to obtain a RBC
concentration comparable with that of human blood. This was
further diluted 1:20 in distilled water to reduce the opacity. Then
the solution was inoculated with Candida albicans from the
American Type Culture Collection to obtain samples of varying
concentration. To label the Candida, FUN1, a fluorescent, fungus-
specific dye (Invitrogen), was used. The dye Trypan was used to
suppress background, especially from RBCs, which absorb small
amounts of FUN1.
Three 0.1 ml samples at each concentration were flowed into the
FFC in 200 seconds (although a much greater rate was possible),
while 500 images were taken continuously. The images were then
analyzed (in minutes) with custom software to count the
fluorescent spots in the images corresponding to Candida. Plate
counts of Candida on YM agar (18 hours incubation) were used
for comparison.
Results Figure 2 shows the comparison of the FFC and plate
counts.
Conclusions The FFC has potential as a system for low-cost, real-
time detection of low concentrations of fungi in human blood.
Further work is required to lower the false detection rate, to
increase the detection efficiency, and to perform tests on human
blood with its additional complexity.
P33
Recurrence of Gram-negative nosocomial pneumonia in the
critically ill patient following short-course antibiotic therapy
Richard Pugh, Richard Cooke, Ged Dempsey
University Hospital Aintree, Department of Critical Care, Liverpool,
UK
Critical Care 2007, 11(Suppl 4):P33 (doi: 10.1186/cc6012)
Background The optimal duration of antibiotic therapy for hospital-
acquired pneumonia (HAP) and ventilator-associated pneumonia
(VAP) is not clear. A multicentre randomised controlled trial
indicated similar clinical efficacy for 8 versus 15 days of antibiotic
therapy for VAP, with less emergence of multiresistant organisms
following the shorter course [1]. Pseudomonas aeruginosa is
difficult to eradicate, however, and American Thoracic Society
guidelines for treatment of HAP due to P. aeruginosa recommend
14 days of therapy [2]. Our aim was to study the rate of recurrence
following treatment of Gram-negative HAP in a critically ill
population with short-course (typically 5 days) antibiotic therapy.
Materials and methods We retrospectively reviewed 50 patients
treated consecutively with short-course (typically 5 days) antibiotic
therapy for Gram-negative HAP in a UK teaching hospital critical
care unit from 2004 to 2007. Pneumonia was defined as semi-
quantitative respiratory culture (2+) of a single Gram-negative
isolate, clinical pulmonary infection score 6 and initiation of
antibiotic therapy. Recurrence of HAP was defined either as
relapse (pure growth of the organism causing the initial infection)
or reinfection (due to a different organism). Patients were studied
until hospital discharge or death.
Results Demographic and outcome data are summarised in
Table 1. The commonest causative organisms were P. aeruginosa
Available online http://ccforum.com/supplements/11/S4
Figure 1 (abstract P32)
The Fountain Flow™ cytometer. Fluorescently stained cells flow
through the flow cell toward the CMOS camera, illuminated by an LED
or laser. Cell(s) in the CMOS camera focal plane are imaged by the
camera through the flow cell window at the wavelength of
fluorescence.
Figure 2 (abstract P32)
Fountain Flow™ cytometer counts of C. albicans in 0.1 ml red blood
cells. Comparison of Fountain Flow™ cytometer (FFC) counts and
plate counts of C. albicans spiked into 1:20 diluted red blood cells
(RBCs). The line of best fit gives a 52% counting efficiency.
Page 15
S16
(42%), Enterobacter species (14%) and Klebsiella species (14%).
Two patients died before completing the initial course of antibiotics.
Conclusions Treatment of Gram-negative HAP in the critically ill
patient with short-course antibiotic therapy is associated with a low
rate of recurrence (10.4%). This compares favourably with
reported recurrence rates of 18–26% following 2 weeks of
antibiotic therapy for VAP [1,3,4].
References
1. Chastre J, Wolff M, Fagon JY, et al.: Comparison of 8 vs 15 days
of antibiotic therapy for ventilator-associated pneumonia in
adults: a randomized trial. JAMA 2003, 290:2588-2598.
2. American Thoracic Society: Guidelines for the management of
adults with hospital-acquired, ventilator-associated, and
healthcare-associated pneumonia. Am J Respir Crit Care Med
2005, 171:388-416.
3. Combes A, Figliolini C, Trouillet JL, et al.: Recurrent
Pseudomonas aeruginosa pneumonia in ventilated patients:
relapse or reinfection? Crit Care Med 2003, 31:1102-1107.
4. Rello J, Mariscal D, March F, et al.: Factors predicting ventilator-
associated pneumonia recurrence. Am J Respir Crit Care Med
1998, 157:912-916.
P34
Fibrinolysis in early recognition of sepsis in patients with
severe trauma
Lev Gerasimov, Viktor Moroz
Research Institute of General Reanimatology, Russian Academy of
Medical Sciences, Moscow, Russia
Critical Care 2007, 11(Suppl 4):P34 (doi: 10.1186/cc6013)
Background Early diagnosis of sepsis improves outcomes for
trauma patients. The aim of this study was to detect additional
markers that help to recognize sepsis before manifestation in
patients with severe multiple trauma and blood loss. It is known
that depletion of fibrinolysis promotes microvascular alterations
after massive blood loss. During inflammation, C-reactive protein
may inhibit fibrinolysis by inducing plasminogen activator inhibitor 1.
These alterations are correlated with an increased risk of multiple
organ failure and death. In accordance with the above-mentioned
purpose, we investigated fibrinolysis at the early post-traumatic
period in patients with and without sepsis.
Materials and methods We examined a database of 58 intensive
care unit patients with trauma and shock, Acute Physiology and
Chronic Health Evaluation II score (first day in intensive care unit)
of 27.4 ± 0.5. Group 1 consisted of 23 patients who developed
sepsis (day after admission, when severe sepsis or septic shock
manifested, was 6.1 ± 0.28). Group 2 consisted of 35 patients
without sepsis within the early post-traumatic period (1–14 days
after injury). The plasma fibrinolytic potential was evaluated by the
euglobulin lysis time, determined by a semiautomatic method.
Results Data are presented as the mean ± median (Table 1). It
was established that fibrinolytic potential was significantly lower in
patients with sepsis versus patients without sepsis since the fifth
day after admission. At the same time, for white blood cells, a
significant difference was observed just after the seventh day
following admission. Platelets did not differ between groups.
Conclusion Plasma fibrinolytic potential is closely related to
inflammation and may be used as a marker of sepsis. Moreover,
plasma fibrinolytic potential may be a useful test for early
identification of the high-risk sepsis patient.
P35
A functional microsatellite in the macrophage migration
inhibitory factor gene influences susceptibility to
meningococcal sepsis
Pascal Renner
1
, Thierry Roger
1
, Saul Faust
2
,
Anne-Laure Schiesser
1
, Marlies Knaup-Reymond
1
,
Helen Betts
3
, Pierre-Yves Bochud
4
, Murielle Bochud
5
,
Robert Read
6
, Michael Levin
3
, Thierry Calandra
1
1
Infectious Diseases Service, CHUV, Lausanne, Switzerland;
2
Division of Infection, Inflammation and Repair, University of
Southampton, Southampton, UK;
3
Department of Paediatrics, St
Mary’s Hospital, Imperial College, London, UK;
4
Institute for
Systems Biology, Seattle, USA;
5
Division of Genetic Epidemiology,
Case Western Reserve University, Cleveland, USA;
6
Division of
Genomic Medicine, Royal Hallamshire Hospital, Sheffield, UK
Critical Care 2007, 11(Suppl 4):P3 (doi: 10.1186/cc6014)
Background The cytokine migration inhibitory factor (MIF) has
recently emerged as an important effector molecule of innate
immune responses and sepsis. Two functional MIF promoter poly-
morphisms, a 5–8 CATT tetranucleotide repeat at –794 (CATT5–8)
and a –173*G/C single nucleotide polymorphism, have been
associated with susceptibility to and/or severity of rheumatoid
arthritis, atopy and ulcerative colitis. Our objective was to study the
impact of MIF gene polymorphisms on susceptibility to Neisseria
meningitidis sepsis and to analyze the functional and biological
effects of MIF polymorphisms in vitro.
Critical Care September 2007 Vol 11 Suppl 4 Sepsis 2007
Table 1 (abstract P33)
Demographic and outcome data
Demographic data at initial diagnosis
Median age (range) (years) 69.5 (19–84)
Sex (% male) 78
Mechanical respiratory support (%) 78
Median APACHE II score 19
Outcome of patients surviving initial course of antibiotics
Nonresolution (%) 2.1
Recurrence (%) 10.4 (all relapse)
Intensive care unit mortality (%) 41.7
Hospital mortality (%) 60.4
Table 1 (abstract P34)
Fibrinolysis, white blood cells and platelets in groups during the early post-traumatic period
Group 1 (with sepsis, n = 23) Group 2 (without sepsis, n = 35)
Days after admission 3 5 7 3 5 7
Fibrinolysis (6–12 min) 76.2 ± 2.8 86.5 ± 2.9* 90.3 ± 3.9** 74.8 ± 3.2 76.3 ± 3.2 62.5 ± 4.5
White blood cells 9.2 ± 0.32 9.9 ± 0.28 10.9 ± 0.45* 9.3 ± 0.19 9.6 ± 0.25 9.5 ± 0.36
Platelets 218 ± 14.4 192 ± 16 184 ± 15.5 209 ± 15.1 208 ± 14.6 218 ± 14.5
*P
w
< 0.05 versus Group 2. **P
w
< 0.001 versus Group 2.
Page 16
S17
Methods A case–control study of 1,106 children and adults with
meningococcal sepsis and 626 control subjects and a family-
based study (106 families with one afflicted child) to analyze
transmission of MIF alleles using the transmission disequilibrium
test. The –794 CATT5–8 microsatellite and –173*G/C single
nucleotide polymorphism were detected by PCR. MIF promoter
alleles cloned into a luciferase reporter construct were tested in
resting and stimulated THP-1 human monocytic cells. DNA binding
activity was assessed by EMSA.
Results Compared with control subjects, the frequency of the
CATT5–5 genotype was markedly reduced in meningococcal
sepsis patients (5.3% versus 8.8%; OR = 0.6, 95% CI = 0.4–0.9,
P = 0.01 for CATT5–5 versus all other CATTxx). The frequencies
of the –173*G/G, –173*G/C or –173*C/C genotypes was
comparable in meningococcal sepsis patients and control subjects
(P = 0.75). The transmission disequilibrium test in families with one
afflicted child revealed that the CATT5 allele was preferentially not
transmitted (P = 0.002) to meninogoccal sepsis offspring, while
the opposite situation occurred for the CATT6 allele (P = 0.024).
In vitro studies showed lower activity of CATT5 promoter compared
with all other CATT promoters in resting and N. meningitidis-
stimulated THP-1 cells. Consistently, DNA binding activity to the
CATT region of the MIF promoter increased with an increasing
number of the CATT repeats.
Conclusion The frequency of the –794 CATT5–5 genotype was
reduced in children with meningococcal sepsis and was less
frequently transmitted from parents to affected offspring,
suggesting that it may confer protection against severe sepsis due
to N. meningitidis. Reduced transcription factor DNA-binding activity
to CATT5 and weaker CATT5 promoter transcriptional activity
provided a functional relevance of the CATT5 polymorphism. MIF
polymorphisms might help to identify patients who may benefit
from anti-MIF treatment strategies.
P36
Methicillin-resistant Staphylococcus aureus-induced vascular
leakage is associated with excessive production of nitric oxide
and vascular endothelial growth factor
Perenlei Enkhbaatar, Esechie Aimalohi, Lillian Traber,
Collette Joncam, Rhykka Connelly, Matthias Lange,
Atsumori Hamahata, D Herndon, Daniel Traber
Department of Anesthesiology, University of Texas Medical Branch,
and Shiners Hospital for Children, Galveston, TX, USA
Critical Care 2007, 11(Suppl 4):P36 (doi: 10.1186/cc6015)
Background Methicillin-resistant Staphylococcus aureus (MRSA)-
related pneumonia and/or sepsis are a frequent serious menace.
The aim of the study was to establish a standardized and repro-
ducible model of MRSA-related ovine septic pneumonia and to
compare the pathophysiological responses to those seen in a
previously established Pseudomonas aeruginosa-induced ovine
sepsis model.
Materials and methods Twenty-four sheep were operatively
prepared for chronic study. After 5 days recovery, tracheostomy
was performed under anesthesia and injury was given. Sheep were
randomly allocated into four groups: (1) Sham group, no injury
(n = 6); (2) Smoke group, exposed to smoke inhalation (n = 6); (3)
PS group, exposed to smoke inhalation and instilled with
P. aeruginosa (2.5 × 10
11
CFU) into the lungs by bronchoscope
(n = 6); and (4) MRSA group, exposed to smoke inhalation and
instilled with MRSA (2.5 × 10
11
CFU) into the lungs by broncho-
scope (n = 6). Smoke inhalation injury was induced by inhalation of
cotton smoke (48 breaths, <40°C). After the injury, animals were
awakened and maintained on mechanical ventilation by 100% O
2
for first 3 hours, and thereafter the O
2
concentration was adjusted
according to blood gases. The sheep, including the Sham group,
were resuscitated by lactated Ringer’s solution with an initial rate
of 2 ml/kg/hour that was further adjusted according to hematocrit
and filling pressure. The experiment lasted 24 hours.
Results The mean arterial pressure was severely depleted in the
MRSA and PS groups, while it was stable in the Sham and Smoke
groups. The fluid net balance was significantly higher in the MRSA
group compared with the other groups, including the PS group.
Plasma nitrite/nitrate was unchanged in the Sham and Smoke
groups compared with baseline values. There was an approxi-
mately sevenfold increase in Plasma nitrite/nitrate in the MRSA
group versus an approximately 2.5-fold increase in the PS group
compared with the Sham group 12 hours post-injury (Table 1). The
excessive nitric oxide in the MRSA group was associated with a
significant increase in lung tissue vascular endothelial growth
factor mRNA and its protein expression.
Conclusion The severe vascular leakage syndrome seen in the
MRSA group may be due to excessive production of nitric oxide
and vascular endothelial growth factor, a potent permeability
factor. MRSA is largely responsible for these pathological changes
rather than smoke inhalation alone.
P37
Epidemiology of community-acquired bacteremia patients
admitted to the intensive care unit
Guiomar Castro
1
, Rui Carneiro
1
, Orquidea Ribeiro
2
,
Altamiro Ribeiro
2
, António Carneiro
1
, Teresa Cardoso
1
1
Unidade de Cuidados Intensivos Polivalente, Hospital Geral de
Santo António, Porto, Portugal;
2
Serviço de Bioestatística e
Informática da Faculdade de Medicina do Porto, Porto, Portugal
Critical Care 2007, 11(Suppl 4):P37 (doi: 10.1186/cc6016)
Background Community-acquired bacteremia (CAB) is a common
cause of hospital and intensive care admission, with a case fatality
rate of 20–30%. Its early identification and association with the
probable source of infection and agents will permit an early and
effective antibiotic therapy, and would probably contribute to a
decrease in the morbidity and mortality.
Materials and methods A prospective, observational study of all
the patients with community-acquired sepsis (CAS) admitted to a
tertiary, mixed, 12-bed, intensive care unit (ICU), at a University
Hospital, between 1 December 2004 and 30 November 2005. In
this study the CAB subgroup was analyzed. CAB was defined by
an infection present at hospital admission or within the first
48 hours with a positive blood culture obtained in the same period.
Results Throughout the study period, 347 patients were admitted;
149 (43%) with CAS. Blood cultures were obtained in 137
Available online http://ccforum.com/supplements/11/S4
Table 1 (abstract P36)
Sham group Smoke group PS group MRSA group
Fluid net balance (ml/kg), 24 hours –0.01 ± 6.0 12.7 ± 8.8 64.3 ± 14.8*
167.0 ± 7.0*
†#
Plasma nitrite/nitrate (µM), 24 hours 5.12 ± 0.7 4.85 ± 0.5 12.09 ± 0.7*
22.3 ± 3.4*
†#
Data expressed ±SEM.*P < 0.05 versus Sham group;
P < 0.05 versus Smoke group;
#
P < 0.05 versus PS group.
Page 17
S18
patients (93%), and 24 patients (17.5%) had CAB. From these,
67% were male, with a median age of 57 years (interquartile range =
47–71). The median SAPS II score was 51. Distribution by focus
of infection was as follows: respiratory (35%); endovascular
(21%); intraabdominal (17%); urinary (13%); central nervous
system (8%) and skin and soft tissue (8%). All patients admitted
with skin and soft-tissue CAS had CAB. Gram-positive
microorganisms represented 50% of all isolations, followed by
Gram-negative (33%). No fungus was isolated. Streptoccocus
pneumoniae and Escherichia coli were the more frequent,
representing 45% of all isolations. Patients with CAB had more
septic shock than patients with negative blood cultures (75%
versus 49%, P = 0.039) and higher ICU (50% versus 28%,
P = 0.049), 28-day (54% versus 28%, P = 0.014) and hospital
(57% versus 33%, P = 0.041) mortalities, although the median
SAPS II score was not significantly different between both groups
(51 versus 45, P = 0.112).
Conclusions CAB represented 7% of all ICU admissions, with an
associated ICU crude mortality of 50%. Respiratory and endo-
vascular focuses of infection were the two most frequent, with
Gram-positive microorganisms representing one-half of all isolations.
P38
Time course of granulocyte–macrophage colony-stimulating
factor and IL-8 in severe sepsis: are the initial low levels of
IL-8 a consequence of immunodepression?
Simona Mera, Doina Tatulescu, Cristina Cismaru,
Mirela Flonta, Dumitru Carstina
Clinic of Infectious Diseases, Cluj-Napoca, Romania
Critical Care 2007, 11(Suppl 4):P38 (doi: 10.1186/cc6017)
Background Neutrophil proliferation, activation and recruitment
are key events in the pathogenesis of sepsis. Granulocyte–
macrophage colony-stimulating factor (GM-CSF) and IL-8 are
cytokines regulating the proliferation, differentiation and maturation
of polymorphonuclear cells and mononuclear phagocytic pro-
genitors, enhancing their functions, adhesion and chemotaxis.
Materials and methods In 11 patients fulfilling the ACCP/SCCM
criteria for severe sepsis, we analyzed serum levels of GM-CSF
and IL-8 at days 1, 2 and 7 after admission. Patients (10 males/one
female) had a median age of 68 years (range 22–87 years). The
primary infection had urinary (seven patients) and respiratory (four
patients) origins. Seven healthy volunteers served as controls.
Results Four out of 11 patients had positive blood cultures (Gram-
negative). At admission, the median procalcitonin was 2 ng/ml
(0.5–10 ng/ml). All patients survived except one patient that died
5 days after admission following acute myocardial infarction.
GM-CSF was always below the detection limit (7.8 pg/ml) in septic
patients and controls. In seven patients, serum IL-8 was below the
detection limit (31.2 pg/ml) at all time points. The median value for
IL-8 for the group of patients was 0 pg/ml (0–264 pg/ml), but
while IL-8 was absent in the sepsis of respiratory origin it had a
median value of 32 pg/ml in urinary sepsis. Surprisingly, IL-8 was
detectable in five out of seven controls, with a median of
68.7 pg/ml (0–145.2 pg/ml).
Conclusions We failed to identify detectable circulating levels of
GM-CSF in healthy individuals or in severe sepsis. The circulating
levels of IL-8 were lower in septic patients than in healthy controls.
As previously suggested, locally produced GM-CSF might have a
role in this condition. Initial low levels of IL-8 found might be a
component of the immunodepression seen in severe sepsis. Also,
it is possible that the IL-8 levels correlate with the primary site of
infection and the severity of the immunodepression related to that.
P39
Severe sepsis and septic shock in pregnancy and puerperium:
an 11-year review in a maternity intensive care unit
Spyridon Pactitis, Grammati Koutra,
Charalambos Charalambidis, Zoi Psomadaki,
Athanasia Karouzou, Charalambos Ladakis
IASO Maternity Hospital, ICU, Athens, Greece
Critical Care 2007, 11(Suppl 4):P39 (doi: 10.1186/cc6018)
Background To estimate the incidence, the etiology and the out-
come of severe sepsis and septic shock during pregnancy and
puerperium.
Materials and methods Retrospective collection of data for all
obstetric patients with severe sepsis and septic shock admitted to
the intensive care unit over an 11-year period (May 1996–May
2007) in a maternity hospital (IASO). Data collected include the
characteristics of severe sepsis and septic shock [1], the source of
infection, the responsible infectious organisms, and the outcome.
Results In the 11-year period, 1,321 women required intensive
care unit admission (0.80% of all deliveries) and 52 of them (4%)
had severe sepsis or septic shock. The most common infection
was chorioamnionitis (23 patients, 41%) and endomyometritis
(primarily after cesarean delivery) (11 patients, 21%). Other
common infections were septic abortion, pneumonia and pyelo-
nephritis. The most common etiologic agents were Gram-negative
rods, followed by Gram-positive bacteria. One patient had fungal
(Candida albicans) infection and another Clostridium spp. No
deaths were recorded.
Conclusions Sepsis is an infrequent yet important cause of
morbidity and possible death in gravitas. Early recognition of
sepsis may prevent maternal complications. The factors that
contribute to a decrease rate of severe sepsis, septic shock and
death in pregnant women are young age and few comorbid
conditions, and the organisms responsible for infections in these
women are usually responsive to common broad-spectrum
antimicrobial agents.
Reference
1. Levy MM, Fink MP, Marshall JC, et al.: 2001
SCCM/ESICM/ACCP/ATS/SIS International Sepsis Defini-
tions Conference. Crit Care Med 2003, 31:1250-1256.
P40
The kinetics of IL-17 production in the lungs and plasma of
mice after intratracheal infection with Klebsiella pneumoniae
Vanja Vasiljev Marchesi
1
, Tomislav Rukavina
1,2
1
Department of Microbiology and Parasitology, Medical Faculty,
University in Rijeka, Croatia;
2
Department of Microbiology, Public
Health Institute, Rijeka, Croatia
Critical Care 2007, 11(Suppl 4):P40 (doi: 10.1186/cc6019)
Background IL-17 is a proinflammatory cytokine predominantly
produced by T cells, which is involved in the innate immune
responses to various physiologic and pathophysiologic processes
including bacterial host defense. The neutralisation experiments
showed that the lack of IL-17 leads to decreased neutrophil
emigration and systemic granulopoietic responses to pulmonary
bacterial pathogens and allergens. The aim of our study was to
determine the kinetics of IL-17 in plasma and lungs of animals
intratracheally infected with Klebsiella pneumoniae.
Materials and methods In our experiments we used 8–12-week-
old BALB/c male mice. Mice were intratracheally inoculated with
150 CFU K. pneumoniae strain Caroli. At different time points,
mice were sacrificed and the lungs and blood were aseptically
Critical Care September 2007 Vol 11 Suppl 4 Sepsis 2007
Page 18
S19
removed and prepared for the cytokine determination. Cytokine
determination was performed by commercial ELISA kit (Bender-
Med Systems, Vienna, Austria).
Results The IL-17 concentration in lung homogenates slightly
increased in the first 2 hours of infection. Then it slightly decreased
and again started to increase 24 hours after the infection. The
concentration in the lungs reached the maximal value 48 hours
post infection. These results are consistent with data previously
published by others. On the other hand, we also found increased
plasma values of IL-17. Its concentration in plasma started to
increase 12 hours after the infection and reached the peak value
24 hours post infection. These results are in contrast with the
results of others who reported no changes in systemic IL-17
production after the intratracheal K. pneumoniae challenge.
Conclusions IL-17 in local host defenses against the Gram-
negative pathogens is undoubtedly important for the clearance of
microorganisms, but its importance in the systemic host response
is still not resolved. Its maximal concentration in plasma correlates
with the appearance of the bacteria in the blood after 24 hours, so
we speculate that its role is also to stimulate systemic
proinflammatory cytokines to combat release of bacteria and/or
their toxic products into the blood system.
P41
Microbiological diagnosis of sepsis: comparison between real-
time polymerase chain reaction and blood culture techniques
Simona Barnini, Carlotta Dodi, Mario Campa
Dipartimento di Patologia Sperimentale, Biotecnologie Mediche,
Infectivologia ed Epidemiologia, Unità Operativa di Microbiologia,
Università di Pisa, Pisa, Italy
Critical Care 2007, 11(Suppl 4):P41 (doi: 10.1186/cc6020)
Background A rapid microbiological diagnosis permits one to
undertake an appropriate therapy against bacterial sepsis and
contributes both to improve the healing possibilities and to lower
the high costs of hospitalisation.
Materials and methods Blood samples from hospitalised patients
were inoculated into Bactec Plus Aerobic/F and Anaerobic bottles
(standard inoculum; Becton Dickinson) and into an EDTA tube
(3 ml). The Septifast Lys kit, Septifast Prep kit and LightCycler
Septifast kit (Roche) were used in disrupting bacterial walls, and in
extracting and amplifying bacterial DNA, respectively. Blood agar
plates were from Becton Dickinson. Strains were identified by
Vitek2 (Biomerieux) panels. Real-time PCR results were analysed
by Septifast identification software. Blood cultures were incubated
into a Bactec 9240 instrument and, if positive, aliquots were taken,
plated onto blood agar plates and identified according to routine
procedures. EDTA–blood samples were processed according to
the LightCycler Septifast test.
Results Among 147 samples from 51 patients with sepsis clinical
signs, 30 were positive by Bactec and 27 by Septifast (which did
not contain probes for three species isolated by cultural method).
Concordance between the two methods was 76% for species
identification. Different results between the two methods included
10 Bactec-positive and Septifast-negative samples (Acinetobacter
baumannii was not amplified seven times, Gram-positive cocci
were not determined by Septifast identification software three
times) and 12 Bactec-negative and Septifast-positive samples (five
Gram-positive cocci, three Pseudomonas aeruginosa, three
Escherichia coli, one Klebsiella spp.), while four samples were
positive by both methods but gave different species. The time
response was evaluated: Septifast was faster than the traditional
method in 97% cases.
Conclusions Although the impossibility to determine antibiotic
susceptibility represents a great limit of the Septifast technique,
the short response time may contribute to undertake a rapidly
tuned therapeutic regimen. Septifast Test appears to be a valid
help in an early assessment of sepsis, along with cultural method.
P42
Community-acquired bacteraemia with sepsis in adults
admitted to the intensive care unit: a prospective multicentre
study
Teresa Cardoso
1
, António Carneiro
1
, Orquídea Ribeiro
2
,
Altamiro Pereira
2
1
Unidade de Cuidados Intensivos Polivalente do Hospital Geral de
Santo António, Porto, Portugal;
2
Serviço de Bioestatística e
Informática Médica da Faculdade de Medicina da Universidade do
Porto, Porto, Portugal
Critical Care 2007, 11(Suppl 4):P42 (doi: 10.1186/cc6021)
Background The main objectives of this study were to describe
the epidemiology and microbiology of community-acquired bac-
teraemia (CAB) in patients with sepsis and to determine the
associated crude mortality.
Materials and methods A prospective, multicentre, cohort, study,
was performed on community-acquired bloodstream infections
with sepsis admitted to Portuguese intensive care units (ICUs),
from 1 December 2004 to 30 November 2005, with a follow-up
until discharge. CAB was defined as an infection that was present
on admission or within the first 48 hours, with positive blood
cultures drawn in the same period. Bacteraemia was considered to
be healthcare related (HCRB) if: (a) patients had wound dressing
or intravenous treatments in the previous 30 days, (b) patients
were observed at a hospital or haemodialysis centre or had
chemotherapy in the previous 30 days, (c) patients were admitted
to an acute care hospital for 2 days or more in the previous 90
days, (d) patient lives at a nursing home or institution.
Results Seventeen units entered the study from north to south
Portugal, corresponding to 41% of all mixed national ICU beds.
Over this period 4,142 patients were admitted to the study – 897
(22%) had community-acquired sepsis, and of these 804 (90%)
had blood cultures done in the first 48 hours. Significant isolates
were grown from samples of 160 patients (20%). The following
focus of infection was identified: respiratory (37%), intra-abdominal
(19%), urinary (16%), endovascular (14%), skin and soft tissue
(7%), central nervous system (5%) and other (2%). Of these 31%
were health-care related (HCRB). Comparing patients without
HCRB with those who had HCRB, an inversion in the micro-
biological profile was found with Gram-positive dominating the first
(55% versus 29%) and Gram-negative dominating in the latter
(29% versus 54%). When compared with patients without
bacteraemia, the 28-day crude mortality was higher for patients
with positive blood cultures (39% versus 29%, P = 0.014);
however, when adjusted to other variables, it was not found to be
an independent determinant of 28-day mortality.
Conclusion CAB with severe sepsis accounts for 4% of all ICU
admissions with a crude 28-day mortality rate of 39%, although not
representing an independent risk factor for 28-day mortality. The
microbiological profile is quite different in healthcare-associated
bacteraemia, making their identification extremely important to
prompt an initial adequate antibiotic approach.
Acknowledgement Presented on behalf of the SACiUCI study
group.
Available online http://ccforum.com/supplements/11/S4
Page 19
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P43
Clinical experience with lipopolysaccharide adsorber in
cardiac surgery
Sten Blomquist, Victoria Gustafsson, Teo Manolopolos,
Leif Pierre
Department of Cardiothoracic Anaesthesia, Heart–Lung Division,
Lund University Hospital, Lund, Sweden
Critical Care 2007, 11(Suppl 4):P43 (doi: 10.1186/cc6022)
Background Endotoxemia is common in cardiac surgery using
extracorporeal circulation (ECC) and is correlated to the time of
bypass, aortic clamping and postoperative complications. Adsorp-
tion of lipopolysaccharide (LPS) has been used in septic patients
with positive results.
Materials and methods The Alteco LPS Adsorber contains
polyethylene discs with a specific polypeptide that binds LPS. The
priming volume is 80 ml and the recommended blood flow is
100–150 ml/min. Heparin is used for anticoagulation. Fifteen
patients scheduled for elective surgery for coronary artery disease
and/or valvular surgery were included in the study. Nine patients
had the LPS adsorber included in the ECC circuit whereas six
patients served as controls with no adsorber. Blood flow through
the adsorber was initiated at the time of aortic clamping, adjusted
to 150 ml/min, and terminated when weaning from ECC started.
Samples for LPS, TNFα, IL-6, and IL-1β were taken before
anaesthesia, 10 minutes after aortic clamping, at skin suture and
6 hours after skin suture.
Results There were no differences between the groups in age,
perfusion time and time of aortic clamping (Table 1). The time of
adsorber treatment is also shown. The use of the adsorber was
uneventful and flow was easily maintained at 100–150 ml/min.
LPS was found in two patients, one in each group at skin suture.
Both patients had a long clamping time, 113 versus 123 min.
Cytokines and complement results are presented in Tables 2 and
3. There were no significant changes in TNFα or IL-1β while IL-6
increased in both groups. Complement factors C3 and C4
decreased in the control group while C1q increased in the
adsorber group.
In vitro studies have shown that the Alteco LPS adsorber can reduce
LPS in whole blood. LPS was found in only two patients in our study,
probably due to the short duration of perfusion and clamping. Thus
the possibility to evaluate the efficacy of the adsorber in this setting
was limited. Only IL-6 increased during the study but no difference
between the groups was seen. The decrease in C3 and C4 seen in
the control group may indicate activation of inflammatory system,
more pronounced in the control group.
Conclusion The adsorber can be used safely in the extracorporeal
circulation. Further studies in patients with more pronounced
endotoxic response are needed to evaluate the effects of the
adsorber on clinical outcome.
P44
Oxidant-induced TLR4 translocation in murine macrophages is
Src kinase dependent
Patrick Tawadros, Zhanfei Wang, Simone Birch, Katalin Szaszi,
Andras Kapus, John Marshall, Ori Rotstein
St Michael’s Hospital, Department of Surgery, University of Toronto,
Toronto, ON, Canada
Critical Care 2007, 11(Suppl 4):P44 (doi: 10.1186/cc6023)
Background Multiorgan failure is a major cause of late mortality
following trauma. Oxidative stress generated during shock/resusci-
tation (S/R) contributes to tissue injury by priming the immune
Critical Care September 2007 Vol 11 Suppl 4 Sepsis 2007
Table 1 (abstract P43)
Demographics and technical data
Age Perfusion Clamping Adsorption
(years) time (min) time (min) time (min)
Adsorber 104 (35–80) 104 (46–198) 71 (30–139) 85 (41–160)
Controls 73 (59–86) 118 (50–239) 83 (24–167)
Data presented as the mean (range).
Table 2 (abstract P43)
Concentrations of cytokines
Preanaesthesia 10 min declamp Skin suture 6 hours postoperative ANOVA P value
TNFα (pg/ml)
Adsorber 7.18 ± 4.3 11.6 ± 4.4 11.9 ± 4.7 5.3 ± 1.6 Not significant
Controls 11.88 ± 10.1 4.0 ± 0.5 5.4 ± 0.9 5.4 ± 1.2 Not significant
IL-6 (pg/ml)
Adsorber 8.88 ± 2.9 46.0 ± 19.6 38.6 ± 16.1 87.3 ± 19.0 <0.05
Controls 2.47 ± 0.6 38.7 ± 20.0 51.4 ± 25.3 117.6 ± 36.9 <0.05
IL-1β (pg/ml)
Adsorber 0.19 ± 0.1 1.10 ± 0.6 1.50 ± 0.9 0.36 ± 0.31 Not significant
Controls 0.20 ± 0.1 0.02 ± 0.02 0.11 ± 0.01 0.01 ± 0.01 Not significant
Data presented as the mean ± SEM.
Table 3 (abstract P43)
Concentrations of complement factors
6 hours t-Test
Preanaesthesia postoperative P value
C3 (g/l)
Adsorber 1.45 ± 0.06 1.11 ± 0.15 Not significant
Controls 1.16 ± 0.05 0.97 ± 0.16 <0.05
C4 (g/l)
Adsorber 0.32 ± 0.03 0.26 ± 0.05 Not significant
Controls 0.30 ± 0.03 0.25 ± 0.02 <0.05
C1q (%)
Adsorber 113.9 ± 7.4 92.3 ± 7.4 <0.05
Controls 94.8 ± 5.0 83.1 ± 9.2 Not significant
Data presented as the mean ± SEM.
Page 20
S21
system for an exaggerated response to subsequent inflammatory
stimuli such as lipopolysaccharide (LPS): the so-called ‘two-hit
hypothesis’. The mechanisms of oxidant-induced cell priming,
however, remain poorly elucidated. Our group has previously
reported a role in this priming process for translocation of the LPS
receptor TLR4 to the plasma membrane [1]. We have also
previously shown that oxidant priming reprograms LPS signaling in
macrophages to a Src-dependent pathway leading to PI3k
activation [2]. Taken together, we hypothesized that Src activation
may play a role in oxidant-induced TLR4 translocation.
Materials and methods Wild-type (WT) and triple Src
(hck/fgr/lyn
–/–
) knockout (KO) mice were subjected to hemorrhagic
shock and resuscitation to generate oxidative stress in vivo.
Alveolar macrophages (AMs) were then retrieved by broncho-
alveolar lavage and analyzed for TLR4 translocation by immuno-
fluorescence staining and flow cytometry. In a separate in vitro
experiment, AMs from WT and KO mice were exposed to 200 μM
hydrogen peroxide for 60 minutes and similarly analyzed by flow
cytometry for surface expression of TLR4.
Results In vivo, S/R induced visible translocation of TLR4 to the
plasma membrane of AMs from WT mice as seen by immuno-
fluorescence. This effect was completely inhibited in the Src KO
animals, and the observation was corroborated quantitatively by
flow cytometry (see Figure 1). In order to confirm the role of
oxidative stress in S/R-induced TLR4 translocation, AMs from WT
and KO mice were exposed to hydrogen peroxide in vitro.
Hydrogen peroxide caused an increase in surface expression of
TLR4 in WT AMs by a ratio of 1.23 compared with WT control.
This effect was once again inhibited in the Src KO AMs, where the
ratio was 0.95 compared with WT control.
Conclusion Oxidative stress induces TLR4 translocation to the cell
surface of macrophages in a Src-dependent manner. Since cellular
responsiveness to LPS is known to correlate with surface levels of
TLR4, this novel finding may direct future therapies in modulating
oxidant-induced cellular priming and subsequent organ failure.
References
1. Powers KA, Szaszi K, Khadaroo RG, et al.: Oxidative stress gen-
erated by hemorrhagic shock recruits Toll-like receptor 4 to
the plasma membrane in macrophages. J Exp Med 2006,
203:1951-1961.
2. Khadaroo RG, Kapus A, Powers KA, et al.: Oxidative stress
reprograms lipopolysaccharide signaling via Src kinase-
dependent pathway in RAW 264.7 macrophage cell line. J Biol
Chem 2003, 278:47834-47841.
P45
Reduction of gap junction proteins and intercalated disc
structural remodeling in the hearts of mice submitted to
sepsis
Mara Rubia Nunes Celes, Diego Torrez-Dueñas,
Jose Carlos Alves-Filho, Djane Braz Duarte,
Fernando Queiroz Cunha, Marcos Antonio Rossi
Departments of Pathology and Pharmacology, Faculty of Medicine
of Ribeirao Preto, University of São Paulo, Ribeirao Preto, São
Paulo, Brazil
Critical Care 2007, 11(Suppl 4):P45 (doi: 10.1186/cc6024)
Background Cardiac dysfunction due to impaired myocardial
contractility has been recognized as an important factor contribu-
ting to high mortality in septic patients. A recent study from our
laboratory gives support to the opinion that myocardial structural
change, classifiable as inflammatory cardiomyopathy, could be
responsible for sepsis-induced myocardial dysfunction. The
present study describes intercalated disc remodeling under both
protein expression and structural features in experimental severe
sepsis induced by cecal ligation and puncture (CLP) in mice.
Materials and methods Male C57Bl/6 mice weighing between
18 and 22 g were subjected to sham-operated surgery, moderate
or severe septic injury, both induced by CLP.
Results Severe septic injury was accompanied by large number of
bacteria in the peritoneal cavity and blood, high levels of TNFα and
MIP-1α in the septic focus and serum, marked hypotension, and
high mortality rate. Western blot analysis and immunofluorescence
showed a marked decrease of key gap and adherens junction
proteins (connexin43 and N-cadherin, respectively) in mice
subjected to severe septic injury. These changes may result in the
loss of intercalated disc structural integrity characterized in the
electron microscopic study by partial separation or dehiscence of
the gap junction and adherens junctions.
Conclusion Our data provide important insight regarding the
alterations in intercalated disc components resulting from severe
septic injury. The intercalated disc remodeling under both protein
expression and structural features in experimental severe sepsis
induced by CLP may be partly responsible for myocardial
depression in sepsis/septic shock. The abnormal parameters may
emerge as therapeutic targets and their modulation might provide
beneficial effects on future cardiovascular outcomes and mortality
in sepsis.
Acknowledgements Supported by FAPESP (04/01777-0 and
04/14578-5) and FAEPA.
P46
The critical role of heme oxygenase in neutrophil migration
impairment in polymicrobial sepsis
Andressa Freitas, Jose Carlos Alves-Filho,
Marina Moreira Suavinha, Fernando Spiller, Daniela Dal-Secco,
Fernando Queiroz Cunha
Department of Pharmacology, Faculty of Medicine of Ribeirao
Preto, University of São Paulo, Ribeirao Preto, São Paulo, Brazil
Critical Care 2007, 11(Suppl 4):P46 (doi: 10.1186/cc6025)
Background Sepsis is a systemic inflammatory response resulting
from the inability of the host to restrict local infection. During
severe sepsis occurs a marked failure of neutrophil migration into
the infectious focus, which is associated with dissemination of
infection resulting in high mortality. Recently, we showed that heme
oxygenase (HO) products, carbon monoxide and biliverdin,
downregulate neutrophil recruitment by reducing the neutrophil/
Available online http://ccforum.com/supplements/11/S4
Figure 1 (abstract P44)
Shock/resuscitation (S/R)-induced TLR4 translocation is inhibited in
Src knockout (KO) mice. WT, wild type.
Page 21
S22
endothelium rolling and adhesion in a noninfectious inflammatory
model. This study aimed to investigate a possible role of the HO-1
pathway on the failure of neutrophil recruitment in mice subjected
to severe (S-CLP) polymicrobial sepsis induced by cecal ligation
and puncture (CLP).
Materials and methods To evaluate the role of HO-1 in S-CLP,
the Balb/c mice were pretreated with a specific HO-1 inhibitor,
zinc protoporphyirin IX (ZnPPIX 30 mg/kg, subcutaneously) 30
minutes before severe sepsis induction. Mice were killed 6 hours
after CLP and HO-1 expression in mesenteric tissue and in blood
neutrophils were determined. In another set of experiments, mice
were killed 6 hours and 12 hours after CLP, and neutrophil
migration to the peritoneal cavity, bacteremia, lung neutrophil
sequestration assessed by myeloperoxidase activity and lung
histology, cytokines, liver, kidney and cardiac function and mean
arterial pressure were determined. The survival rate of animals was
assessed every 12 hours up to 120 hours after surgery.
Results A significant increase in HO-1 expression was observed in
mesenteric tissue and in blood neutrophils after S-CLP. The
ZnPPIX pretreatment prevents the failure of neutrophil endothelium
rolling, adhesion and migration observed in vehicle-pretreated mice
subjected to S-CLP. As consequence, these mice presented
reduced bacteremia, low levels of seric TNFα and lung neutrophil
sequestration, reduced liver, kidney and cardiac injury and
improved mean arterial pressure, resulting in an increase of the
survival rate.
Conclusions These data suggest that during an infectious process
HO-1 displays a crucial role on the failure of neutrophil migration to
infectious focus and consequently on the susceptibility in severe
sepsis.
Acknowledgement Supported by FAPESP/CAPES/FAEPA.
P47
TLR2 signaling downregulates chemokine receptor CXCR2
and impairs neutrophil migration in severe polymicrobial
sepsis
Jose Carlos Alves-Filho, Andressa Freitas, Fernando Spiller,
Fabricio Souto, Heitor Paula-Neto, Joao Santana Silva,
Fernando Queiroz Cunha
Department of Pharmacology and Immunology, School of Medicine
of Ribeirao Preto, University of São Paulo, São Paulo, Brazil
Critical Care 2007, 11(Suppl 4):P47 (doi: 10.1186/cc6026)
Background There is a marked defect in neutrophil migration into
the infectious focus during severe sepsis, which is associated with
the severity of disease. We recently demonstrated that this pheno-
menon is a consequence of the downregulation of chemokine
receptor CXCR2 on the surface of circulating neutrophils. Toll-like
receptors (TLRs) are pattern-recognition receptors that are impor-
tant in innate immune responses to bacterial infection. TLR
activation in phagocytes produces proinflammatory cytokines and
chemokines that contribute directly to elimination of infectious
agents. However, a sustained inflammatory response can result in
tissue damage and sepsis. Here, we address the role of TLR2 in
the downregulation of CXCR2 and establishment of neutrophil
migration impairment in severe sepsis.
Materials and methods TLR2-deficient (TLR2
–/–
) and C57BL/6
(WT) mice were subjected to severe polymicrobial sepsis by the
cecal ligation and puncture (CLP) model, and neutrophil migration,
bacteremia, CXCR2 expression and cytokines levels were evaluated.
Results It was observed that TLR2 is critical for downregulation of
CXCR2 expression on circulating neutrophils during severe sepsis,
since this event was prevented in TLR2
–/–
mice. In accordance,
TLR2
–/–
mice did not present failure of neutrophil migration into the
infectious focus and, consequently, they presented lower
bacteremia and did not display systemic inflammation determined
by reduced levels of circulating cytokines, showing an improved
survival rate. Furthermore, in vitro, TLR2 agonist (lipoteichoic acid)
was able to downregulate CXCR2 expression and to markedly
inhibit neutrophil chemotaxis induced by CXCR2-ligand. The
downregulation of CXCR2 was associated with enhanced
expression of G-protein-coupled receptor kinase-2 (GRK-2), which
is known to play an important role in the desensitization and
internalization of this chemokine receptor. Finally, we showed that
in vitro lipoteichoic-acid-stimulated neutrophils adoptively transferred
into naïve WT mice display a significantly reduced competence to
migrate into the perinoneal cavity in response to thioglycolate.
Conclusion Altogether, these findings suggest that TLR2 through
GRK-2 signaling downregulates CXCR2 expression on the surface
of circulating neutrophils, which is a critical determinant of
impairment of neutrophil migration into the infection focus during
severe sepsis.
Acknowledgements Supported by FAPESP and FAEPA.
P48
The activity of the endothelial Tie-2 receptor modulates
ventilation-induced lung injury in septic mice
Shirley Mei
1,2
, Jack Haitsma
3
, Daniel James
3
, Colleen Parker
2
,
Arthur Slutsky
3
, Duncan Stewart
1,2
1
Institute of Medical Science, University of Toronto, Toronto, ON,
Canada;
2
Terrence Donnelly Heart Centre, St Michael’s Hospital,
Toronto, ON, Canada;
3
Interdepartmental Division of Critical Care
Medicine, University of Toronto, Toronto, ON, Canada
Critical Care 2007, 11(Suppl 4):P48 (doi: 10.1186/cc6027)
Background Patients requiring mechanical ventilation, particularly
in the presence of sepsis, are at risk for ventilation-induced lung
injury (VILI), which is characterized by increased pulmonary
vascular permeability and inflammation. Inflammation in VILI is
initiated through interactions between endothelial and inflammatory
cells. The vasoprotective protein angiopoietin-1, a ligand for the
endothelial-selective tyrosine kinase receptor Tie-2, has recently
been recognized to play a critical role in regulating endothelial
inflammation and vascular permeability. In contrast, angiopoietin-2,
which binds with equal affinity, has been described as a Tie-2
antagonist, inhibiting receptor activation in response to angio-
poietin-1. We therefore examined the role of the angiopoietins/Tie-2
pathway in mechanically ventilated, septic mice.
Materials and methods Wild-type (WT) or Tie-2
+/–
mice were
anaesthetized and sepsis was induced by cecum ligation and
puncture (CLP). After 24 hours, animals were ventilated for 6 hours
to induce VILI. Animals were ventilated in a pressure-controlled
mode, at a fractional inspired oxygen concentration of 0.5, an
inspiration to expiration ratio of 1:2, a peak inspiratory pressure of
14 cmH
2
O and a positive end-expiratory pressure of 2 cmH
2
O.
Animals were sacrificed and bronchoalveolar lavage (BAL) was
performed for total cell count, differential, and permeability
parameters. Inflammatory cytokine IL-6 levels were measured in
BAL and plasma.
Results There was no difference between sham-operated or CLP-
operated WT mice in total cell numbers in BAL. Sham operated
Tie-2
+/–
mice had similar cell numbers, while ventilation in CLP-
operated Tie-2
+/–
mice resulted in higher cell numbers (P = 0.06
versus WT), with an increase mainly in macrophages. IL-6 plasma
levels were increased in septic Tie-2
+/–
mice subjected to
mechanical ventilation compared with the WT mice, whereas IL-6
levels in BAL were not significantly different. Despite the higher
total cell counts, permeability parameters measured in Tie-2
+/–
Critical Care September 2007 Vol 11 Suppl 4 Sepsis 2007
Page 22
S23
mice were lower than in WT mice (albumin, P = 0.08; total
proteins, not significant; IgM, P = 0.05).
Conclusions Pulmonary vascular permeability in mice with
knockdown of endothelial Tie-2 receptor is preserved in ventilated
septic mice. However, there was an increase in the number of
inflammatory cells and systemic inflammation (IL-6) in these
animals. These results further emphasize the importance of Tie-2 in
regulating vascular permeability in the lungs.
P49
Pharmacological implications of the spleen in sepsis
Bolin Cai, Fei Chen, Jared Huston, Luis Ulloa
Laboratory of Surgical Immunology, University of Medicine and
Dentistry of New Jersey, Newark, NJ, USA
Critical Care 2007, 11(Suppl 4):P49 (doi: 10.1186/cc6028)
Background Our previous studies indicated that nicotine
attenuated systemic inflammation, and improved survival in both
lethal endotoxemia and cecal ligation and puncture [1].
Materials and methods Here we analyzed the pathophysiological
and pharmacological implications of the spleen in experimental
sepsis. Acetylcholine and nicotine inhibit the production of TNF
and HMGB1 from lipopolysaccharide-treated RAW264.7 macro-
phage-like cells in a concentration-dependent manner. Nicotine
inhibits the production of these cytokine macrophage-like cells
through a mechanism dependent on the α
7
-nicotine acetylcholine
receptor (nAChR). From a physiological perspective, our studies
indicate that the spleen has a significant expression of the
α
7
nAChR. To study the implications of the splenic α
7
nAChR in the
therapeutic potential of nicotine in sepsis, we analyzed the effects
of treatment with nicotine in splenectomized mice. Adult male
8–12-week-old BALB/c mice (25–30 g) were splenectomized and
7 days later subjected to cecal ligation and puncture to induce
polymicrobial peritonitis. Control sham animals underwent
laparotomy without splenectomy. Administration of nicotine
(400 mg/kg, i.p.) or vehicle injection (1 × PBS, i.p.) began
24 hours after surgery, and thereafter twice a day for 3 days.
Animals were euthanized after 44 hours for serum cytokine analysis.
Results Treatment with nicotine improved survival in sham mice
but it worsened the survival of splenectomized mice. Nicotine
inhibited the production of TNF and HMGB1 from lipopoly-
saccharide-treated macrophages differentiated from peripheral
blood mononuclear cells. However, in vivo, nicotine attenuated
serum TNF levels in sham mice but not in splenectomized mice
with polymicrobial peritonitis. There were no differences in serum
TNF, IL-12, IFNγ or IL-10 levels between vehicle-treated or
nicotine-treated splenectomized animals. Unlike systemic TNF,
circulating HMGB1 levels correlated with the increased mortality
induced by nicotine in splenectomized mice. Treatment with
nicotine increased circulating serum HMGB1 levels in
splenectomized mice with experimental sepsis.
Conclusion These results suggest that the spleen has major
pharmacological implications for the treatment of sepsis.
Acknowledgements Supported by grants of the AHA and the
USAMRMC Log#05308004 to LU.
Reference
1. Wang H, Liao H, Ochani M, et al.: Cholinergic agonists inhibit
HMGB1 release and improve survival in experimental sepsis.
Nat Med 2004, 10:1216-1221.
P50
Peroxynitrite mediates neutrophil migration failure in severe
polymicrobial sepsis
Daniela Dal-Secco, Diego Torres-Dueñas, Andressa Freitas,
Mara Rubia Nunes Celes, José Carlos Alves-Filho,
Fernando Spiller, Vitor Faeda Dalto, Marcos Antonio Rossi,
Sérgio Henrique Ferreira, Fernando Queiroz Cunha
Departments of Pharmacology and Pathology, Faculty of Medicine
of Ribeirao Preto, University of São Paulo, Ribeirao Preto, São
Paulo, Brazil
Critical Care 2007, 11(Suppl 4):P50 (doi: 10.1186/cc6029)
Background Sepsis is a systemic inflammatory response resulting
from the host inability to restrict local infection. The failure of
neutrophil migration to the infectious focus is one of the mecha-
nisms involved in this process. Recently, it was demonstrated that
this event is mediated by nitric oxide. The present study addressed
the possibility that peroxynitrite (ONOO
), a nitric oxide-derived
powerful oxidizing and nitrating compound, can be also involved in
the neutrophil migration failure.
Materials and methods Male C57Bl/6 mice were pretreated,
subcutaneously, with saline or uric acid (UA; 100 mg/kg) 1 hour
before induction of severe sepsis injury (SSI) or were treated with
Tetrakis (FeTPPS; 5.0 mg/kg) 15 minutes after SSI, induced by
cecal ligation and puncture. Intravital microscopy was used to
evaluate leukocyte rolling and adhesion in the mesenteric micro-
circulation. TNFα and MIP-1α levels were evaluated by ELISA, lung
neutrophil influx by myeloperoxidase activity and nitrotyrosine by
immunofluorescence.
Results The mice pretreated with UA (an ONOO
scavenger) and
subjected to SSI did not present failure of neutrophil rolling,
adhesion and migration to the infectious focus. As a consequence,
they presented low bacteremia, diminished TNFα and MIP-1α
levels in circulation and reduced myeloperoxidase activity. More-
over, increased nitrotyrosine labeling detected in leukocytes
present in mesenteric tissues and neutrophils obtained from SSI
peritoneal exudate were reduced by UA pretreatment. Finally, the
UA pretreatment enhanced significantly the survival rate of the SSI
mice. Similarly, treatment with Tetrakis (FeTPPs), a more specific
ONOO
scavenger, reestablished neutrophil migration and
increased the survival rate.
Conclusion Together, the results indicate that ONOO
partially
mediates the reduction of neutrophil/endothelium cell interaction
and consequently the neutrophil migration failure to the infectious
focus and susceptibility in severe sepsis. Therefore, these results
identify ONOO
inhibitors as potential targets for novel sepsis
therapies.
Acknowledgement Supported by CAPES, FAPESP and FAEPA.
P51
Selective V1a receptor agonists in experimental septic shock
Daniel Traber
Department of Anesthesiology, University of Texas Medical Branch,
Galveston, TX, USA
Critical Care 2007, 11(Suppl 4):P51 (doi: 10.1186/cc6030)
Background Septic shock is caused by hypotension secondary to
vasodilation. This hypotension is resistant to fluid resuscitation and
requires the use of vasopressor agents. Recently, it was shown
that it may be due to a deficiency in the vasopressor hormone
arginine–vasopressin (AVP), a mixed V1a/V2 receptor agonist,
leading to the clinical use of AVP in septic shock. We tested the
hypothesis that the selective V1a receptor agonist FE 202158 is at
Available online http://ccforum.com/supplements/11/S4
Page 23
S24
least as effective as AVP in a sheep model of pneumonia-induced
septic shock.
Materials and methods Sheep were surgically instrumented
ahead of the study. After obtaining baseline data, they were
anesthetized and insufflated with 48 breaths of cotton smoke and
1,011 colony forming units of Pseudomonas aeruginosa instilled
into their airways via a tracheostomy. The sheep were then placed
on a ventilator and awakened for the study. They were resuscitated
with Ringer’s solution to maintain left atrial and central venous
pressures and hematocrit at baseline levels. If, despite fluid
management, the mean arterial pressure (MAP) fell by 10 mmHg
from baseline, a continuous intravenous infusion of AVP or FE
202158 was initiated and titrated to keep MAP within this limit,
except in a septic control group and in a nonseptic sham group.
Results In the septic control group, MAP fell by ~30 mmHg and
the systemic vascular resistance index (SVRI) by ~50% over the
24-hour study. In the AVP group, although the MAP and SVRI were
maintained above septic control group levels, they could not be
brought back to sham group levels. In contrast, in the FE 202158
group, the MAP and SVRI were maintained at sham group levels.
The septic control group accumulated ~7 l fluid over the 24-hour
study (~20% body weight), while this was reduced by ~50% in the
AVP group, and was totally prevented in the FE 202158 group.
The fluid accumulation was associated with a constant hematocrit,
suggesting that it was extravascular. It was also correlated with a
fall in plasma total protein and oncotic pressure, suggesting
protein leakage from the vasculature and probably reflecting
sepsis-induced vascular leak syndrome. The full prevention of this
fluid and protein leakage with FE 202158 was reversed to AVP
group levels by infusing the selective V2 receptor agonist
desmopressin together with FE 202158.
Conclusion FE 202158 was not only fully effective at preventing a
sepsis-induced decrease in MAP and SVRI, but was superior to
AVP at reducing vascular leak syndrome because of its V1a
receptor selectivity.
P52
Gender, infection, critical illness, and death: superior survival
for female trauma patients admitted to the intensive care unit
treated for infection in a dedicated, prospective, multicenter
study
Brian Swenson, Hugo Bonatti, Lesly Dossett, Addison May,
Robert Sawyer
University of Virginia Health System, Department of Surgery,
Charlottesville, VA, USA
Critical Care 2007, 11(Suppl 4):P52 (doi: 10.1186/cc6031)
Background Multiple studies have suggested gender-based
differences exist in infection incidence and outcome in critically ill
patients, but these relationships have not been clearly defined. We
present the largest prospective multicenter study designed to
measure the impact of gender on infection incidence and outcome
in critically ill surgical and trauma patients to date.
Methods Patients admitted for at least 3 days to surgical or trauma
intensive care units (ICUs) at two academic tertiary care centers
from October 2001 to May 2006 were prospectively followed for
the occurrence of infection and clinical outcomes. Logistic
regressions were performed to assess independent risk of gender
on the acquisition of an ICU infection and inhouse mortality in
patients with ICU-acquired infections. Demographic and clinical
variables that were significant by univariate analyses were included
in the models.
Results The final cohort followed 2,291 patients (trauma n = 1,407,
nontrauma n = 884) with 383 (27.2%) female trauma patients and
413 (46.7%) female nontrauma patients. The proportion of
Critical Care September 2007 Vol 11 Suppl 4 Sepsis 2007
Table 1 (abstract P52)
Logistic regression analysis assessing predictors of mortality in patients with ICU-acquired infections
Infected trauma Infected nontrauma
patients (n = 652) patients (n = 397)
Odds 95% confidence P Odds 95% confidence P
Variable ratio interval value ratio interval value
Female gender 0.45 0.22–0.95 0.036 1.15 0.6961.911 0.58
Age, per year 1.02 0.101.04 0.087 1.04 1.019–1.062 <0.001
White race 1.00 1.00 ––
Black race 0.62 0.241.62 0.33 1.08 0.442.59 0.87
Other race 1.03 0.293.67 0.97 3.61 1.08–12.1 0.037
Any comorbidity 0.89 0.402.00 0.77 0.76 0.29–1.98 0.57
Chronic corticosteroid use 0.48 0.055.0 0.54 1.35 0.66–2.74 0.41
Hepatic insufficiency 4.25 1.33–13.6 0.015 1.61 0.78–3.33 0.20
Cardiac disease 1.18 0.492.82 0.72
Baseline serum creatinine 2.0 (units) 7.46 1.11–50.1 0.039
Malignancy 0.17 0.018–1.56 0.12 1.39 0.80–2.44 0.24
Body mass index, per kg/m
2
0.98 0.96–1.01 0.24
Admission from home 1.03 0.41–2.60 0.95
McCabe score at ICU admission, per point 1.21 0.60–2.44 0.59 1.35 0.91–2.01 0.14
APACHE II score at ICU admission, per point 1.07 1.021.12 0.0081 1.08 1.041.12 <0.001
WHO functional status at ICU admission, per point 1.46 0.89–2.39 0.14
Multiple Organ Dysfunction score at ICU admission, per point 1.12 1.041.22 0.0040
Probability of trauma survival 0.49 0.23–1.02 0.055
ICU length of stay, per day 1.02 0.97–1.07 0.42
Hospital length of stay, per day 0.95 0.910.98 0.032
Bold data are statistically significant.
Page 24
S25
patients with at least one ICU-acquired infection was 47.6% for
trauma males and 43.1% for trauma females (P = 0.13); and was
44.2% for nontrauma male patients and 45.8% for nontrauma female
patients (P = 0.63). The inhospital mortality of patients with at least
one ICU-acquired infection was 12.5% for trauma males and 7.3%
for trauma females (P = 0.06); and was 30.8% for nontrauma male
patients and 28.0% for nontrauma female patients (P = 0.55).
Logistic regression analysis did not show gender to be a significant
variable in the acquisition of an ICU infection. The results of a logistic
regression analysis of predictors of inhospital mortality in patients
with ICU-acquired infections are presented in Table 1. Only variables
significant by univariate analysis were entered into logistic models.
Empty cells represent variables that were significant for only one
group (for example – only trauma, but not nontrauma).
Conclusion Gender does not appear to play a role in acquisition of
an ICU infection in either trauma or nontrauma patients. In contrast,
female gender appears to provide a strong survival advantage in
trauma patients with ICU-acquired infections. This phenomenon
was not observed in the nontrauma group. Further investigation
into the hormonal and cytokine differences between the genders in
critically ill trauma patients and their response to infection may
prove beneficial.
P53
Effects of antifungal agents on the production of cytokines
from macrophages stimulated by Aspergillus fumigatus conidia
Jung-Hyun Choi
1
, Eun-Young Kwon
2
, Chulmin Park
2
,
Sun Hee Park
1
, Su-Mi Choi
1
, Dong-Gun Lee
1
, Jin-Hong Yoo
1
,
Wan-Shik Shin
1
1
Department of Internal Medicine, Division of Infectious Diseases,
College of Medicine, Catholic University of Korea, Korea;
2
Catholic
Research Institutes of Medical Science, Catholic University of
Korea, Korea
Critical Care 2007, 11(Suppl 4):P53 (doi: 10.1186/cc6032)
Background We evaluated the immunomodulatory effect of three
antifungal agents, amphotericin B (AmB), micafungin (MF) and
itraconazole (ITZ), in the aspect of cytokine production and
expression of NF-κB in Aspergillus fumigatus conidia-treated
RAW264.7 cells, a murine alveolar macrophage cell line.
Materials and methods We evaluated the cytotoxic effect of
antifungal agents using a commercial cell proliferation assay. TNFα
and IL-10 production according to stimulation (control, A. fumigatus
conidia only, conidia + antifungal drug, conidia + antifungals +
GM-CSF) was evaluated and each compared using a commercial
ELISA method. NF-κB activation was evaluated by western blot
analysis.
Results AmB, MF and ITZ showed a dose-dependent cytotoxic
effect on the tested cells. Stimulation of cells by A. fumigatus
conidia induced TNFα production. Pretreatment of AmB at
concentrations not affecting cellular survival did not change the
production of TNFα compared with conidia-treated cells, but
pretreatment of MF or ITZ showed a reduced amount of TNFα
production compared with conidia-treated cells. AmB also showed
a synergistic effect on TNFα production when simultaneously
treated with GM-CSF. IL-10 production was markedly increased
when the cells were treated with AmB with conidia. MF and ITZ
induced a smaller increase of IL-10 production. AmB also showed
a synergistic effect on the production of IL-10 when treated with
GM-CSF simultaneously. A. fumigatus conidia enhanced expres-
sion of NF-κB. The degree of NF-κB expression was associated
with the amount of TNFα and IL-10 produced.
Conclusion The antifungal agents we used in this experiment
showed decreased TNFα production and increased IL-10
production from the RAW264.7 cells stimulated by A. fumigatus
conidia after pretreatment of antifungal agents. But more studies
such as the association between the immunomodulatory effect and
antifungal activity and the difference of the signal pathway of
cellular activation according to drugs should be performed.
P54
Time to positivity as a novel predictor of outcome in intensive
care unit patients with sepsis
Susie Jerwood
1
, Sonia Hudson
1
, Matthew Hankins
2
,
John Paul
1
, Owen Boyd
1
, Jonathan Cohen
2
1
Brighton Sussex County Hospital, Eastern Road, Brighton,
Sussex, UK;
2
Brighton and Sussex Medical, Falmer, Sussex, UK
Critical Care 2007, 11(Suppl 4):P54 (doi: 10.1186/cc6033)
Background Inadequate antibiotic therapy predicts poor outcome
from sepsis, but there is no simple test of adequacy. We sugges-
ted that a time-to-positivity assay (T
pos
) might act as a surrogate for
antimicrobial activity and predict outcome from sepsis in the
intensive care unit (ICU) [1]. We are conducting a prospective
clinical trial to test this hypothesis.
Methods We studied 35 sequential ICU patients with onset of
sepsis who had not had antibiotics for at least 3 days. Sepsis was
defined as clinical evidence of infection plus at least three criteria
for systemic inflammatory response syndrome. All patients received
standard empiric therapy. Sera taken at 24 hours post antibiotics
were inoculated into blood culture bottles containing standardised
bacteria, incubated in an automated microbial detection system
and the time to positivity noted. The primary clinical endpoint was
days in the ICU.
Results Cultures that are negative after 5 days of incubation
indicate adequate antimicrobial therapy; cultures that become
positive in <5 days indicate inadequate antimicrobial therapy.
Patients with negative T
pos
(n = 11) were associated with a stay in
the ICU of less than 6.5 days (P = 0.052). See Figure 1.
Conclusion These data show that some septic patients on
standard antimicrobial regimens are receiving inadequate therapy.
The T
pos
is a new, simple assay that might be used effectively to
monitor antibiotic use.
Reference
1. Kaltsas P, Want S, Cohen J: Development of a time-to-positiv-
ity assay as a tool in the antibiotic management of septic
patients. Clin Microbiol Infect 2005, 11:109-114.
Available online http://ccforum.com/supplements/11/S4
Figure 1 (abstract P54)
Average length of stay for patients with negative and positive time-to-
positivity 1 (taken at 24 hours after first dose of antibiotics) with 95%
confidence intervals.
Page 25
  • [Show abstract] [Hide abstract] ABSTRACT: Oxidative stress generated by ischemia/reperfusion is known to prime inflammatory cells for increased responsiveness to subsequent stimuli, such as lipopolysaccharide (LPS). The mechanism(s) underlying this effect remains poorly elucidated. These studies show that alveolar macrophages recovered from rodents subjected to hemorrhagic shock/resuscitation expressed increased surface levels of Toll-like receptor 4 (TLR4), an effect inhibited by adding the antioxidant N-acetylcysteine to the resuscitation fluid. Consistent with a role for oxidative stress in this effect, in vitro H2O2 treatment of RAW 264.7 macrophages similarly caused an increase in surface TLR4. The H2O2-induced increase in surface TLR4 was prevented by depleting intracellular calcium or disrupting the cytoskeleton, suggesting the involvement of receptor exocytosis. Further, fluorescent resonance energy transfer between TLR4 and the raft marker GM1 as well as biochemical analysis of the raft components demonstrated that oxidative stress redistributes TLR4 to lipid rafts in the plasma membrane. Preventing the oxidant-induced movement of TLR4 to lipid rafts using methyl-beta-cyclodextrin precluded the increased responsiveness of cells to LPS after H2O2 treatment. Collectively, these studies suggest a novel mechanism whereby oxidative stress might prime the responsiveness of cells of the innate immune system.
    Full-text · Article · Sep 2006 · Journal of Experimental Medicine
    0Comments 155Citations
  • No preview · Article · Feb 2005
    0Comments 577Citations
  • [Show abstract] [Hide abstract] ABSTRACT: The optimal duration of antimicrobial treatment for ventilator-associated pneumonia (VAP) is unknown. Shortening the length of treatment may help to contain the emergence of multiresistant bacteria in the intensive care unit (ICU). To determine whether 8 days is as effective as 15 days of antibiotic treatment of patients with microbiologically proven VAP. Prospective, randomized, double-blind (until day 8) clinical trial conducted in 51 French ICUs. A total of 401 patients diagnosed as having developed VAP by quantitative culture results of bronchoscopic specimens and who had received initial appropriate empirical antimicrobial therapy were enrolled between May 1999 and June 2002. A total of 197 patients were randomly assigned to receive 8 days and 204 to receive 15 days of therapy with an antibiotic regimen selected by the treating physician. Primary outcome measures-death from any cause, microbiologically documented pulmonary infection recurrence, and antibiotic-free days-were assessed 28 days after VAP onset and analyzed on an intent-to-treat basis. Compared with patients treated for 15 days, those treated for 8 days had neither excess mortality (18.8% vs 17.2%; difference, 1.6%; 90% confidence interval [CI], -3.7% to 6.9%) nor more recurrent infections (28.9% vs 26.0%; difference, 2.9%; 90% CI, -3.2% to 9.1%), but they had more mean (SD) antibiotic-free days (13.1 [7.4] vs 8.7 [5.2] days, P<.001). The number of mechanical ventilation-free days, the number of organ failure-free days, the length of ICU stay, and mortality rates on day 60 for the 2 groups did not differ. Although patients with VAP caused by nonfermenting gram-negative bacilli, including Pseudomonas aeruginosa, did not have more unfavorable outcomes when antimicrobial therapy lasted only 8 days, they did have a higher pulmonary infection-recurrence rate compared with those receiving 15 days of treatment (40.6% vs 25.4%; difference, 15.2%, 90% CI, 3.9%-26.6%). Among patients who developed recurrent infections, multiresistant pathogens emerged less frequently in those who had received 8 days of antibiotics (42.1% vs 62.0% of pulmonary recurrences, P =.04). Among patients who had received appropriate initial empirical therapy, with the possible exception of those developing nonfermenting gram-negative bacillus infections, comparable clinical effectiveness against VAP was obtained with the 8- and 15-day treatment regimens. The 8-day group had less antibiotic use.
    Preview · Article · Dec 2003 · JAMA The Journal of the American Medical Association
    0Comments 726Citations
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