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Methicillin-resistant Staphylococcus aureus-induced vascular leakage is associated with excessive production of nitric oxide and vascular endothelial growth factor
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- Kinga A Powers, Katalin Szászi, Rachel G Khadaroo, Patrick S Tawadros, John C Marshall, András Kapus, Ori D Rotstein[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.Journal of Experimental Medicine 09/2006; 203(8):1951-61. · 13.91 Impact FactorThis article is viewable in ResearchGate's enriched formatRG Format enables you to read in context with side-by-side figures, citations, and feedback from experts in your field. - Am J Respir Crit Care Med. 02/2005; 171:388-416.
- Recurrent Pseudomonas aeruginosa pneumonia in ventilated patients: relapse or reinfection? Crit Care Med. Combes A C Figliolini, Trouillet, Jl . 2003. 1102-1107.
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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
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).
The role of regulatory T cells in the resistance of CCR4
knockout mice during severe sepsis
Raphael Molinaro1, Alessandra Monteiro de França1,
Josi Alves-Filho2, Fernando de Queiroz Cunha2,
Marcelo Bozza3, Steven Kunkel4, Claudia Benjamim1
1Department of Pharmacology, UFRJ, Rio de Janeiro, RJ, Brazil;
2Department of Pharmacology, USP, Ribeirão Preto, SP, Brazil;
3Microbiology Institute, UFRJ, Rio de Janeiro, RJ, Brazil;
4Department of Pathology, UMMS, Ann Arbor, MI, USA
Critical Care 2007, 11(Suppl 4):P2 (doi: 10.1186/cc5981)
Background Studies reveal that regulatory T (Treg) 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 Tregcells 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, Treg
cells from CCR4–/–CLP mice did not inhibit proliferation of T
effector cells as observed for Tregcells from wild CLP mice, at a
proportional ratio of T effector:Tregcells. Interesting, Tregcells 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 Tregcells
from wild CLP mice inhibit 80%, much more than expected.
Conclusion These results suggest that Tregcells 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 Tregcells in immunosuppression following severe
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,
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.
The modified light chain of inter-alpha inhibitor/antibody
fusion protein, MR1007, improves survival in the rabbit sepsis
Masaki Nakamura, Takashi Takeuchi, Yukiko Yatagai,
Katsuki Naito, Kamon Shirakawa, Yoshitaka Hosaka,
Shoji Furusako
Discovery Research, Mochida Pharmaceutical Co., Ltd, Gotemba,
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.
Microbial metabolites in the blood of patients with sepsis
Anastasia Khodakova, Natalia Beloborodova
Bakulev Scientific Center for Cardiovascular Surgery, Moscow,
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.
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
with sepsis
(n = 12)
Patients before
(n = 36)
Patients with
smooth recovery
(n = 9)
Patients with
(n = 10)
(n = 16)Aromatic acid (ng/ml)
p-Hydroxyphenylacetic acid
3-Phenylpropionic acid
Phenyllactic acid
p-Hydroxyphenyllactic acid
1-Indolacetic acid
3-Indolacetic acid
2,140 (631–3,516)
367 (217–1,098)
1,543 (564–2,731)
246 (183–628)
72 (62–93)***
35 (20–54)**
47 (37–64)**
195 (159–371)***
262 (113–385)***
93 (56–141)*
114 (53–220)***
4 (0–29)
58 (37–93)**
254 (134–373)***
47 (0–218)*
181 (60–542)
263 (109–313)**
89 (68–126)*
288 (269–772)*
275 (228–499)***
456 (344–667)*
0 (0–3)
112 (89–177)*
465 (314–836)
0 (0–27)
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 4
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 =
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.
Impact of autologous centrifuged shed mediastinal blood on
procalcitonin, C-reactive protein levels and postoperative
complications during the early period following cardiac
Judita Andrejaitiene1, Audrone Veikutiene2,
Edmundas Širvinskas1, Rimantas Benetis3
1Institute for Biomedical Research, Kaunas Medical University,
Kaunas, Lithuania; 2Kaunas University of Medicine Hospital,
Department of Cardiothoracic and Vascular Surgery, Kaunas,
Lithuania; 3Kaunas 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.
System approach to the diagnosis and treatment of septic
arthritis in newborns
Gennadiy Khanes1, Svitlana Bidnenko2,
Volodymyr Grygorovskyy3, Iryna Maksakova1,
Valeriy Romashko4
1Neonatal Surgery Department, Ukrainian Specialized Pediatric
Hospital OkhMatDyt, Kyiv, Ukraine; 2Bacteriologic Laboratory of
Scientific Research Institute of Traumatology and Orthopedics of
the Academy of Medical Sciences of Ukraine, Kyiv, Ukraine;
3Morphologic Laboratory of Scientific Research Institute of
Traumatology and Orthopedics of the Academy of Medical
Sciences of Ukraine, Kyiv, Ukraine; 4Immunologic 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
Page 5
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.
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
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
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
streptococcal superantigens, and resistin was assessed in the
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.
with lipopolysaccharide or
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
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Page 6
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
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
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.
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
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 4Sepsis 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
(%) Proceedings
P value
Cultures obtained before antibiotics
Antibiotics in a due time (2-hour interval)
Activated protein C
Page 7
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.
The role of CC-chemokine receptor 4 in murine polymicrobial
Stefan Maier1, Tobias Traeger1, Wolfram Keßler1, Pia Körner1,
Hendrik Mehmcke1, Yolande Chvatchko2, Marlene Mikulczak1,
Tobias Ebker1, Claus-Dieter Heidecke1
1Department of Surgery, Universität Greifswald, Germany; 2Serono
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.
LL-37 at the local site of streptococcal skin and soft-tissue
Pontus Thulin1, Linda Johansson1, Parham Sendi1,
Erika Hertzén1, Adam Linder2, Per Åkesson2,
Bertil Christensson2, Malak Kotb3, Donald Low4,
Birgitta Agerberth1, Anna Norrby-Teglund1
1Karolinska Institutet, Stockholm, Sweden; 2Lund University, Lund,
Sweden; 3Veterans Affairs Medical Center, Memphis, TN, USA;
4University 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
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.
HMGB1 expression in streptococcal soft-tissue infections
correlates with disease severity
Parham Sendi1, Linda Johansson1, Pontus Thulin1,
Adam Linder2,Per Åkesson2, Bertil Christensson2,
Donald Low3, Anna Norrby-Teglund1, Carl-Johan Treutiger1
1Karolinska Institutet, Stockholm, Sweden; 2Lund University, Lund,
Sweden; 3University 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,
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Page 8
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.
Can procalcitonin reflect the etiology of the bacteremia?
Ekaterina Chernevskaya, Natalia Beloborodova,
Tatyana Vostrikova
Bakoulev Scientific Center for Cardiovascular Surgery, Moscow,
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%),
Conclusion A high PCT level in patients with suspected infection
may be indicative of Gram-negative infection before obtaining the
culture results.
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
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
Number of cases
Procalcitonin (ng/ml)
White blood cell count (× 109/l)
Temperature (°C)
Multiple organ failure (number of patients (%))
101 49
5.4 (1.78–12.21)
15.4 (11.1–23.8)
37.5 (37–38)
16/80 (20%)
0.86 (0.28–2.19)
14.2 (11–22)
37 (37–38)
7/32 (21.8%)
Not significant
Not significant
Not significant
Page 9
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.
Effects of vasopressin and terlipressin in ovine septic shock
on mesenteric blood flow and survival
Sebastian Rehberg1, Christian Ertmer1, Matthias Lange1,
Andrea Morelli2, Hugo Van Aken1Martin Westphal1
1Department of Anaesthesiology and Intensive Care, University of
Münster, Münster, Germany; 2Department 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.
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
Bruce Davis1, Karen Becker1, Henry Rinder2, Kathleen Davis1
1Trillium Diagnostics, Brewer, ME, USA; 2Department 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
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 10
(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.
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.
Physiological parameters, location of infection and organ
failure are significant predictors of misdiagnosing severe
Diane Chamberlain1, Christine Wilson1, Tamara Hunt2
1Flinders University, Faculty of Health Sciences, Adelaide,
Australia; 2Flinders 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 4Sepsis 2007
Table 1 (abstract P21)
Genic expression of apoptosis in tolerance to lipopolysaccharide
TNF receptor-associated factor 4 (TRAF4)
Fas antigen ligand (FASL)
TNF-related apoptosis-inducing ligand (TRAIL)
Fas I receptor
Inhibitor of apoptosis protein 3
Caspase 2 precursor
Caspase 7, apoptosis-related cysteine protease
Caspase 11
Caspase 2, apoptosis-related cysteine protease
Apoptotic protease activating factor 1
Fas-associated factor 1
Programmed cell death 1 protein precursor
Bid, apoptotic protease
Caspase 8, apoptosis-related cysteine protease
aRatio = controls/tolerants: down, down to 0.05; up, up to 5.0.
Page 11
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.
In vitro and in vivo determination of anti-TNFα α activity in canine
plasma from donors subject to preconditioning with endotoxin
Michael Kotiw1, Michael Morgan2, Jesus Lopez1,
Steven Taylor2, Ian Shiels2
1Centre for Systems Biology, University of Southern Queensland
Toowoomba, Queensland, Australia; 2School 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
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
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 12
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.
Human protein C concentrate in the treatment of hemolytic
uremic syndrome
Burkhard Wermter1, Harald Koeditz1, Kathrin Seidemann1,
Thomas Jack1, Bernadette Brent1
Armin Wessel1, Michael Sasse1
1PICU Interdisciplinary Paediatric Intensive Care Unit and
2Paediatric Nephrology, MHH Medizinische Hochschule Hannover,
Critical Care 2007, 11(Suppl 4):P25 (doi: 10.1186/cc6004)
, Lars Pape2
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.
Biofilm forming P. aeruginosa induces an enhanced
inflammatory response in human monocytes
Cristina Ciornei1, Christophe Beloin2, Alexey Novikov3,
Martine Caroff3, Catherine Fitting1, Jean-Marc Ghigo2,
Jean-Marc Cavaillon1, Minou Adib-Conquy1
1Pasteur Institut, Cytokines & Inflammation, Paris, France; 2Pasteur
Institut, Genetics of Biofilms, Paris, France; 3Equipe '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 13
Laboratory markers to determine clinical significance of
coagulase-negative staphylococci in blood cultures
Piret Mitt1, Siiri Kõljalg2, Krista Lõivukene2, Epp Sepp2,
Irja Lutsar2, Matti Maimets1, Paul Naaber2
1Tartu University Hospital, Department of Infection Control, Tartu,
Estonia; 2University of Tartu, Departmentt of Microbiology, Tartu,
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.
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
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
11 ± 6
9 ± 4
2846 ± 369
1279 ± 285a
41 ± 3
28 ± 2
1637 ± 254
1020 ± 240b
1 ± 4
0 ± 0
40 ± 10
24 ± 5c
aP < 0.005, significantly different from mice treated with LPS alone.
bP < 0.001, significantly different from mice treated with LPS alone.
cP < 0.05, significantly different from mice treated with LPS alone.
Page 14
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.
Lung-derived macrophage migration inhibitory factor in adult
patients with septic shock, and its role in cardiocirculatory
Hans Lee1, Kaie Ojamaa1, Greg Ruskin1, Erfan Hussain1,
Maowen Hu1, Xinchun Lin1, Christine Metz1, Yousef Al-Abed1,
Edmund Miller1,2
1Laboratory of Cardiopulmonary Research, The Feinstein Institute,
Manhasset, NY, USA; 2Albert Einstein College of Medicine Bronx,
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.
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.
Real-time, low-cost detection of individual fungal cells in blood
using Fountain Flow™ cytometry
Paul E Johnson1,2, Christopher M Havens1,2,
Joseph F Johnson1,2
1Department of Physics & Astronomy, University of Wyoming,
Laramie, WY, USA; 2SoftRay, 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 15
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
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.
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,
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
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.
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What is your favorite kompot recipe?
What is your favorite kompot recipe?
April 19, 2014 1:53 PM Subscribe
I am looking for some old-fashion kompot recipes (preferably ones which include dried cherry but I am happy to try making different kinds). The arrival of warmer weather has brought back memories of drinking it on warm summer nights.
So, I haven't actually tried these recipes, which is why I didn't post earlier, but it looks like nobody else is jumping in, so here is a collection of kompot recipes from thekitchn.com, which I've had good luck with in the past. There are some with dried fruit, and some with fresh, and they look pretty straightforward. Good luck! They sound yum.
posted by instamatic at 3:37 PM on April 20, 2014
posted by instamatic at 3:37 PM on April 20, 2014
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