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European Journal of Heart Failure 2004 6(4):483-491; doi:10.1016/j.ejheart.2003.12.004
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© 2004 European Society of Cardiology

Selective intestinal decontamination in advanced chronic heart failure: a pilot trial

Viviane M. Conraadsa,*, Philippe G. Jorensb, Luc S. De Clerckc, Hendrik K. Van Saened, Margaretha M. Ievene, Johan M. Bosmansa, Annemie Schuerweghc, Chris H. Bridtsc, Floris Wuytsf, Wim J. Stevensc, Stefan D. Ankerg, Mathias Rauchhaush and Christiaan J. Vrintsa

a Department of Cardiology, University Hospital Antwerp Wilrijkstraat 10, 2650 Edegem, Belgium
b Department of Intensive Care Medicine, University Hospital Antwerp Wilrijkstraat 10, 2650 Edegem, Belgium
c Department of Immunology, University Hospital Antwerp Wilrijkstraat 10, 2650 Edegem, Belgium
d Department of Medical Microbiology, University of Liverpool UK
e Department of Microbiology, University Hospital Antwerp Wilrijkstraat 10, 2650 Edegem, Belgium
f Department of Medical Statistics, University Hospital Antwerp Wilrijkstraat 10, 2650 Edegem, Belgium
g Department of Cardiology, Franz-Volhard-Klinik, Charité Medical School Campus Berlin-Buch, Berlin, Germany
h Department of Cardiology, Universitätsklinik und Poliklinik für Innere Medizin III Halle, Germany

* Corresponding author. Tel.: +32-3-821-46-72; fax: +32-3-825-08-48. E-mail address: Viviane.Conraads{at}uza.be


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Background and aims: Endotoxin, derived from intestinal aerobic Gram-negative bacilli (AGNB), could be an important monocyte activator in chronic heart failure (CHF). The effect of selective decontamination of the digestive tract (SDD) on intracellular monocyte cytokine production, monocyte CD14 expression, circulating endotoxin and cytokines, and flow-mediated dilation (FMD) was studied in patients with severe CHF.

Methods and results: Ten patients with CHF (NYHA class III–IV) were enrolled in a non-placebo controlled pilot trial involving the administration of SDD (polymyxin B, tobramycin) for 8 weeks. One patient was later excluded due to cardiac transplantation. Before treatment, after 4 and 8 weeks therapy, and 6 weeks post-treatment, monocyte CD14 expression, intracellular monocyte production of interleukin-1β [IL-1β], interleukin-6 [IL-6], tumour necrosis factor (TNF)-{alpha} with and without lipopolysaccharide (LPS) stimulation were measured. Concentrations of endotoxin and cytokines (IL-1β, IL-6, TNF-{alpha}) were also determined. AGNB in faeces, intestinal endotoxin and FMD were assessed at baseline, after 4 weeks of treatment and 6 weeks post-treatment. SDD eradicated intestinal AGNB (P<0.00001) and decreased faecal endotoxin concentrations (P<0.00001). There was a significant decline in monocyte CD14 expression (P=0.03) and in IL-1β (P=0.0001), IL-6 (P=0.02) and TNF-{alpha} (P=0.0002) production after 4 and 8 weeks of treatment in the basal state and for IL-1β (P=0.008) and IL-6 (P=0.005) after LPS stimulation. FMD significantly improved at 4 weeks and returned to baseline after treatment discontinuation (P=0.002). Circulating concentrations of endotoxin and cytokines remained unchanged.

Conclusion: Reduction of the intestinal endotoxin pool led to a decrease in monocyte CD14 expression and intracellular cytokine production in patients with severe CHF. The improvement of peripheral endothelial function could be a marker of the anti-inflammatory effect of SDD.

Key Words: Chronic heart failure • Inflammation • Endotoxin • Selective digestive decontamination • Endothelial dysfunction

Received June 30, 2003; Revised October 10, 2003; Accepted December 7, 2003


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Immunological dysbalance with systemic inflammation is increasingly recognised as a feature of the syndrome of chronic heart failure (CHF). Over expression of interleukin-6 (IL-6), tumour necrosis factor (TNF)-{alpha}, and soluble TNF receptors correlate with heart failure severity and have important prognostic significance [1–4].

Bacterial lipopolysaccharide (LPS) or endotoxin is a potent stimulus of pro-inflammatory cytokine production, acting via the CD14/Toll-like receptor-4 on monocytes and macrophages. Endotoxin concentrations are raised in patients with severe CHF [5]. In children with congenital heart disease, endotoxemia is associated with worse clinical outcome [6]. Oedematous CHF patients show a depressed carrier-mediated saccharide transport, suggestive of intestinal ischemia and strongly correlating with circulating cytokines [7]. These observations lend support to the hypothesis of translocation of aerobic gram-negative bacilli (AGNB) and/or absorption of endotoxin from the gut into the blood stream.

The aim of the present pilot study was to test the effects of selective eradication of intestinal AGNB, using an enteral non-absorbable polymyxin B/tobramycin regimen, on intracellular monocyte cytokine production and circulating cytokines in patients with severe CHF. In addition, the effect of this intervention on peripheral vascular function was studied.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Patients
We prospectively enrolled 10 patients with severe CHF in this non-randomised, non-placebo controlled pilot study (eight men, two women). Median age was 73 years (range 44–78 years). Patients were in New York Heart Association Class (NYHA) III or IV, with a median left ventricular ejection fraction of 19% (range 14–37%). All patients had stable symptoms and medical therapy for at least 1 month and were seen regularly in the outpatient clinic. Patients were on standard medical treatment consisting of ACE-inhibitors (n=5), other vasodilators or AT1 receptor blockers (n=5), diuretics (n=10, furosemide median dosage=90 mg, range 40–200 mg, four patients additionally on chlorthalidone 50 mg), spironolactone (n=7, median dosage 25 mg, range 0–50 mg), digoxin (n=5), beta-blockers (n=1), aspirin (n=2), and amiodarone (n=3).

Patients with acute/chronic infections, allergies, rheumatoid disease, cancer, or treatment with anti-inflammatory drugs were excluded. This study was approved by the Local Ethical Committee. All patients gave written informed consent. Investigators (sonographer, laboratory personnel) had no knowledge of the intervention sequence in the study (baseline, during treatment, after treatment).

2.2. Intervention
Patients with CHF received a total daily dose of 800 mg of polymyxin B and 320 mg of tobramycin (selective decontamination of the digestive tract [SDD]) [8]. Objections from the Local Ethical Committee precluded treatment of control subjects. The antibiotics were given as 10 ml solutions, every 6 h, for a period of 8 weeks. One patient was withdrawn because of heart transplantation. Another patient stopped the treatment after 1 month because of side effects (gastrointestinal upset, diarrhea).

The sampling procedure for both AGNB assessment and endotoxin measurement was standardised as follows. Faecal samples (minimum 2 g) were obtained prior to SDD (baseline), after 4 and 8 weeks of treatment and, finally, 6 weeks after discontinuation of treatment. Additionally, we collected faecal specimens 1 week after antibiotic treatment and 2 weeks after discontinuation of therapy for the determination of AGNB concentrations. Samples obtained for endotoxin measurements were frozen at –20 °C within 1 h after collection.

Fasting blood samples (30 ml) were collected between 08.00 and 09.00 h into heparinised tubes (Vacuette®, Greiner, Labortechnik, Kremsmünster, Austria) for intracellular cytokine measurements, at baseline, after 4 and 8 weeks of therapy, and 6 weeks after discontinuation of antibiotics. In addition, blood was sampled for circulating cytokines (20 ml) into ethylenediaminetetraacetic acid (EDTA) tubes (Vacutainer®, Becton and Dickinson, Meylan, France). Plasma was separated by centrifugation within 30 min of collection and aliquots were stored at –20 °C.

2.3. Microbiological procedures
Quantitative counts of AGNB were carried out on the faecal specimens, using a suspension of 1 g of faeces well mixed in 9 ml of brain heart infusion broth (Becton Dickinson®). Ten-fold dilutions of the suspension were spread on the surface of MacConkey agar plates incubated aerobically for 18 h. The lower limit for the bacterial count was 100 colony forming units (CFU) per millilitre. All macroscopic different colonies of AGNB were isolates on pure culture and identified using standard techniques [9].

2.4. Faecal endotoxin
The endotoxin concentration was determined in the faecal supernatant by a micro technique of the Limulus amoebocyte lysate (LAL) test (Mallinckroth, St. Louis, MO, USA) as described elsewhere [9]. The endotoxin concentrations in the faeces were expressed as log 10 values in nanograms per gram of faeces.

2.5. Circulating endotoxin measurement
Endotoxin-free tubes (Endo Tube ET, Chromogenix AB, Sweden) were used to collect 8 ml blood for LPS assessment. Plasma levels of endotoxin were determined using the LAL assay (QCL-1000 test kit, Bio Whittaker Inc, Walkerswill, USA) as described elsewhere [5]. Normal endotoxin concentrations in healthy subjects are <0.5 EU/ml [10].

2.6. Intracellular cytokine measurement and CD14 expression
Intracellular cytokine production by monocytes was measured without stimulation (basal) and after 8 h of incubation with LPS (after LPS), as previously described [11]. Whole blood (1ml) stimulation during 8 h with 1 µg/ml Lipopolysaccharide E. Coli (LPS, Serotype 026:B6, Sigma, St. Louis, USA) together with 1.4 µmol/l brefeldin A (Sigma St. Louis, Mi, USA) was followed by incubation in sterile 15 ml polypropyleen tubes (Sarstedt, Nümbrecht, Germany) in order to prevent adherence of cells. After incubation, 100 µl of stimulated whole blood was incubated with CD14-FITC (BD Biosciences, Erembodegem, Belgium) for 15 min at 4 °C to identify the monocytes. Subsequently, red blood cells were lyzed and the remaining white blood cells were fixed at room temperature with Facslysis (BD). Cell membranes were made permeable with a 0.3% saponin (Sigma) solution in PBS, followed by incubation with PE-labeled anti-cytokine antibodies against IL-1β, IL-6, and TNF-{alpha} (BD) for 30 min at room temperature. Thereafter, cells were washed with 0.3% saponin in PBS and centrifuged for 10 min at 1000xg. Finally, the cell pellet was resuspended in 0.3 ml PBS and measured.

Twenty five thousand events were measured on a FACscan flow cytometer (BD) and analysed with WinMDI software (kindly provided by Trotter through http://facs.scripps.edu/software.html). Measurements were standardised using microspheres (DAKO FluoroSpheres, Code No K 0110 Glostrup, Denmark), according to the manufacturer's instructions. Analysis gates, containing approximately 2000 cells, were set on CD14-positive cells according to FITC emission and side scatter. Within this gate, intracellular cytokine production was evaluated. Results were expressed as the mean molecules of equivalent soluble fluorescein (MESF) units. Isotype matched irrelevant antibodies (mouse IgG1 RPE, rat IgG2a RPE, Serotec) were used as controls. CD14 expression on monocytes was acquired by flowcytometry. The mean fluorescence intensity (MFI) in 2000 monocytes, labeled with anti-CD14-FITC, was measured. After standardisation with DAKO microspheres, CD14 density on monocytes was expressed as MESF.

For the reason that of a viral respiratory infection, monocyte cytokine production after 4 weeks for one patient was excluded from analysis. Due to a technical problem, stimulated cytokine production at 4 weeks is missing for one patient.

The control group consisted out of five healthy age- and sex matched persons who had a negative anamnesis for acute/chronic infections, allergies, rheumatoid disease and cancer and were not taking chronic or short-term medication at the time of investigation.

2.7. Circulating cytokine measurement
Concentrations of IL-1β, IL-6, and TNF-{alpha} were measured using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's specifications (Quantikine, R and D Systems, High sensitivity kits: sensitivity of <0.1 pg/ml for IL-1β, <0.094 pg/ml for IL-6, 0.12 pg/ml for TNF-{alpha}). In order to minimise variation, ELISA testing in the present study was performed on the same day, using one badge. Freeze and thaw cycles were avoided. Samples were run in duplicate and the average of these two measurements was calculated.

2.8. Endothelial function
The internal diameter of the brachial artery was measured using a vessel wall movement detector system (Wall Track System, Pie Medical, Maastricht, The Netherlands) consisting of a high-resolution ultrasound device (10 MHz linear-array transducer) connected to a data acquisition and processing unit [12]. Patients were examined after overnight fasting between 8 and 9 AM. Measurements were performed at baseline, after 4 weeks of therapy and 6 weeks after discontinuation of antibiotics. The brachial artery was occluded by inflation of a blood pressure cuff to suprasystolic pressure for 4 min. Thereafter (60 s), measurement of the internal diameter was repeated. According to current guidelines, flow-mediated dilation (FMD) is expressed as percentage, 100x(internal diameter after hyperemia–internal diameter before hyperaemia/internal diameter before hyperaemia) [13,14]. Measurements were repeated three times and the results were averaged. Inter-observer variability of diameter measurements was 1.4%. Five age- and sex-matched healthy subjects served as controls.

2.9. Statistical analyses
Data are given as median and range, unless otherwise stated. The Mann–Whitney U-test was used for comparisons of numerical data between groups. Repeated measures ANOVA were assessed using a quadratic model for the time factor. P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Bacteriological surveillance
Within 1 week, all but one patient were successfully decontaminated. The 4 log reduction in faecal concentrations was highly significant (P<0.00001). Two weeks after discontinuation of treatment, the intestinal AGNB had returned to pre-treatment concentrations. Susceptibility of Gram-negative aerobic bacilli to tobramycin and polymyxin was maintained. No cases of methicillin-resistant S. aureus were reported.

3.2. Faecal endotoxin concentration
The median log value of intestinal endotoxin at baseline (ng/g faeces) was 6 (range 5–6). This is equivalent to approximately 1mg of endotoxin/g faeces. Seven subjects showed a significant endotoxin reduction of 4 log during treatment (P<0.00001). The reduction is equivalent to a decrease in faecal endotoxin from 1 mg to 100 ng of endotoxin/g faeces. Six weeks after discontinuation of SDD, endotoxin concentrations had returned to baseline.

3.3. Circulating endotoxin concentrations
Plasma concentrations of endotoxin were increased at baseline (0.77 EU/ml [range 0.05–0.91 EU/ml]) and comparable with levels found in both oedematously decompensated patients [5] and in those with stable, but severe CHF in NYHA class IV without clinical signs of peripheral fluid retention [10]. SDD had no effect on circulating endotoxin concentrations (P=0.9).

3.4. Monocyte intracellular cytokine production and CD14 expression
Peripheral blood cell count showed no changes in absolute values nor in percentage of circulating monocytes during the study period (P>0.5).

Prior to SDD, basal intracellular cytokine concentrations were markedly higher in patients compared with controls (Table 1). After LPS stimulation, there was no difference (Table 2).


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  		Table 1 Pre-treatment intracellular monocyte cytokine production in the absence of LPS-stimulation

 


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  		Table 2 Pre-treatment intracellular monocyte cytokine production following LPS-stimulation

 
After 4 and 8 weeks of antibiotic treatment, basal intracellular concentrations of IL-1β (P=0.0001), IL-6 (P=0.02) and TNF-{alpha} (P=0.0002) showed a significant decline in patients with CHF (Figs. 1–3). Concentrations measured after 8 h of LPS stimulation were also significantly lower for IL-1β (P=0.008), IL-6 (P=0.005), but not for TNF-{alpha} (P=0.1) (Figs. 4–6). Six weeks after discontinuation of therapy, basal intracellular production of IL-1β, IL-6 and TNF-{alpha} (Figs. 1–3) and LPS stimulated production of IL-1β and IL-6 (Figs. 4–6) had increased again.


Figure 1
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  		Fig. 1 Intracellular monocyte production of IL-1β in the absence of LPS-stimulation. MESF=mean molecules of equivalent soluble fluorescein units.

 


Figure 2
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  		Fig. 2 Intracellular monocyte production of IL-6 in the absence of LPS-stimulation. MESF=mean molecules of equivalent soluble fluorescein units.

 


Figure 3
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  		Fig. 3 Intracellular monocyte production of TNF-{alpha} in the absence of LPS-stimulation. MESF=mean molecules of equivalent soluble fluorescein units.

 


Figure 4
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  		Fig. 4 Intracellular monocyte production of IL-1β after LPS-stimulation. MESF=mean molecules of equivalent soluble fluorescein units.

 


Figure 5
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  		Fig. 5 Intracellular monocyte production of IL-6 after LPS-stimulation. MESF=mean molecules of equivalent soluble fluorescein units.

 


Figure 6
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  		Fig. 6 Intracellular monocyte production of TNF-{alpha} after LPS-stimulation. MESF=mean molecules of equivalent soluble fluorescein units.

 
Compared to baseline, monocyte CD14 expression was lower at 4 (P=0.0003) and 8 weeks of therapy (P=0.006). Six weeks after discontinuation, CD14 expression was still downregulated (P=0.0001 vs. baseline, Fig. 7).


Figure 7
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  		Fig. 7 Monocyte CD14 expression. MESF=mean molecules of equivalent soluble fluorescein units.

 
3.5. Circulating cytokine concentrations
Circulating concentrations of IL-1β, IL-6 and TNF-{alpha} remained unaltered during the study period: baseline concentration of IL-1β=0.79 pg/ml (range 0.4–2.09 pg/ml), P=0.6; IL-6=4.63 pg/ml (range 2.14–11.03 pg/ml), P=0.2; TNF-{alpha}=2.41 (range 1.54–6.63 pg/ml), P=0.8.

3.6. Peripheral vascular function
FMD prior to SDD was depressed in patients with CHF when compared to five healthy untreated controls (1.8% [range –0.2–3.3%] vs. 8.6%, [range 3.4–10.3%], P=0.003). For the treated CHF patients, the internal brachial artery diameter, measured before hyperaemia, remained unchanged throughout the study period (P=0.2). After 4 weeks of antibiotics, all but one out of nine treated patients showed an increase in FMD (Fig. 8). Mean FMD after 4 weeks was 58% higher than at baseline (mean absolute change: 1.03±1.09%, P=0.03). After discontinuation of SDD, FMD decreased to values seen at baseline (P=0.8 vs. baseline, P=0.02 vs. after 4 weeks).


Figure 8
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  		Fig. 8 Flow-mediated dilation of the brachial artery. FMD=flow-mediated dilation.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
This non-randomised, non-placebo controlled pilot study shows an anti-inflammatory effect of antibiotic eradication of the Gram-negative intestinal flora in patients with stable severe CHF. The attenuation of monocyte CD14 expression and intracellular monocyte cytokine production was accompanied by an improvement in vascular endothelial function as evidenced by flow-mediated dilation (FMD).

Anker et al. [15] were the first to propose that endotoxins, derived from the cell wall of intestinal aerobic Gram-negative bacilli (AGNB), could contribute to immune activation in CHF. Rauchhaus et al. [10] found a rise in endotoxin plasma concentrations from patients in NYHA class I to those in class IV. Monocytes play a pivotal role in the production of pro-inflammatory cytokines, including TNF-{alpha}, IL-1β and IL-6. By virtue of their CD14 and Toll-like receptor proteins, monocytes are highly susceptible to stimulation by endotoxin.

To test the hypothesis of endotoxin-derived immune activation in CHF, the present pilot study evaluated the effect of selective eradication of intestinal AGNB, using an enteral non-absorbable polymyxin B/tobramycin regimen. Besides the antimicrobial effect, this SDD protocol can also neutralise free endotoxin in the gut [8]. SDD significantly reduced AGNB carriage and elicited a marked decrease in faecal endotoxin load. The notable decline in intracellular monocyte cytokine production was accompanied by a downregulated monocyte CD14 expression, supporting the hypothesis of reduced endotoxin exposure. Indeed, the dynamic regulation of CD14 synthesis and membranous expression depends on endotoxin concentration [16,17]. Although not necessarily a consequence of reduced intracellular cytokine production, FMD after 4 weeks of SDD improved, followed by a return to baseline values, 6 weeks after discontinuation of therapy.

SDD did not alter plasma cytokine concentrations. However, it should be recalled that circulating monocytes are not the only contributors to immune activation in CHF. Spillover of cytokines into the circulation from other sources, such as cardiac myocytes [18], cardiac non-myocyte cells [19], and hypoxic and underperfused tissues have also been mentioned [20]. In two separate studies [5,21], diuretic recompensation, despite endotoxin reduction in the study by Niebauer and colleagues [5], did not reduce pro-inflammatory cytokine concentrations. Moreover, Dutka et al. [22] and other investigators [23] have previously demonstrated that circulating TNF-{alpha} and IL-6 levels in patients with CHF show considerable variation.

Schwarz et al. [24] recently suggested that bacterial products, upon disturbance of the intestinal barrier, are transferred to the lymphatic vessels, where they can ‘prime’ monocytes. In the present study, reduction of intestinal endotoxin load could have decreased endotoxin transfer to the mesentheric lymph system, resulting in less activated monocytes without changes in circulating endotoxin levels. Alternatively, cyclic variations in the translocation of endotoxin or Gram-negative bacilli, due to changes in filling pressure and secondary congestion and/or ischemia, might have precluded the detection of a systematic decrease in circulating endotoxin levels. Moreover, the bioactivity of endotoxin, rather than the absolute levels of this substance could be the more relevant issue. It has been amply shown that the net pro-inflammatory effect of endotoxin depends on the balance between endotoxin itself and ‘anti-endotoxin properties’ such as LPS-binding protein (LBP), bactericidal/permeability-increasing protein (BPI), soluble CD14, lipoprotein concentrations and anti-endotoxin antibodies [25].

Impaired peripheral endothelium-dependent vasodilation is a major determinant of exercise intolerance in patients with CHF [26]. Serum of CHF patients downregulates endothelial NO synthase (eNOS) and increases endothelial apoptosis [27]. TNF-{alpha} augments nitric oxide (NO) breakdown at the endothelium, thereby enhancing superoxide radical formation [28]. Fichtschlerer and colleagues [29] demonstrated that etanercept, by virtue of its TNF-neutralizing effect, significantly improved FMD in patients with CHF.

Chemokine concentrations and gene expression in mononuclear blood cells are elevated in CHF [30]. Cytokines and endotoxin elicit increased endothelial expression of adhesion molecules and chemokines. It seems, therefore plausible that ‘de-activation’ of circulating monocytes, attracted to the activated vessel wall, could improve FMD. Interestingly, Verma et al. [31] identified neopterin, a specific marker of monocyte activation, as a novel indicator of endothelial dysfunction. In addition, interference of antibiotic killing with other CD14-dependent pathways, such as Tlr-4 activation by HSP70 (Heat Shock Protein 70) could be relevant [32].

4.1. Limitations
Due to the lack of a placebo group and the non-randomised design of the present study, a causal link between the observed anti-inflammatory changes and the partial recovery of endothelial dysfunction remains to be further explored. Despite the absence of a significant relation between changes in cytokine production or CD14 expression and FMD, we cannot exclude a net anti-inflammatory effect, since anti-inflammatory cytokines (i.e. IL-10, IL-1Ra) were not studied.

The considerable cost of the antimicrobial solution and the necessity to monitor both its efficacy and the impact on the intestinal microbiological flora will probably preclude the widespread continuous use of SDD. However, the application of SDD on an intermittent basis, preferably in a situation of cardiac decompensation leading to bowel oedema and/or ischemia and consequent bacterial or endotoxin translocation, might prove useful.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
We thank Ramon de Nooijer and Inge Goovaerts for their technical assistance. The constructive criticism of Benedicte de Winter is appreciated. Antibiotic therapy for this trial was provided by the pharmaceutical companies Glaxo Wellcome, Belgium and Eli Lilly Benelux.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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