Endotoxin hypersensitivity in chronic heart failure.

BACKGROUND: Raised concentrations of endotoxin (lipopolysaccharides, LPS) have been demonstrated in patients with chronic heart failure (CHF). Tolerance of monocytes to LPS can be induced by negative feedback mechanism through LPS itself, resulting in a downregulation of cytokine response to LPS challenge. As endotoxin desensitization has also been suggested for CHF, we investigated the response to LPS challenge in CHF patients. METHODS: We prospectively studied 100 patients with CHF (62 +/- 13 years) and 21 controls (58 +/- 10 years, LVEF 60 +/- 3%). HLA-DR expression and TNFalpha generation of monocytes after ex vivo stimulation by LPS (stimulation with LPS 50 and 500 pg/ml) were determined. 46 CHF patients were in NYHA class II (LVEF 29 +/- 8%) and 54 in NYHA class III (LVEF 27 +/- 7%). RESULTS: HLA-DR expression in controls (25,837 +/- 7915 ABS/cell) was comparable to CHF NYHA II patients (23,720 +/- 8488 ABS/cell, n.s.), but lower in patients classified NYHA III (20,327 +/- 5073 ABS/cell, p < 0.01). Stimulated TNFalpha production ex vivo was higher in CHF NYHA III (LPS 50: 437 +/- 284; LPS 500: 946 +/- 500 pg/ml, each p < 0.05) and CHF NYHA II (LPS 50: 397 +/- 277; LPS 500: 933 +/- 483 pg/ml, each p < 0.05) compared to controls (LPS 50: 315 +/- 134; LPS 500: 715 +/- 339 pg/ml). CONCLUSIONS: In chronic heart failure TNFalpha generation capacity increases while HLA-DR expression decreases compared to controls. Thus patients with CHF display enhanced susceptibility to inflammatory stimuli.

The value of cardiopulmonary exercise testing and brain natriuretic peptide plasma levels in predicting the prognosis of patients with chronic heart failure.

BACKGROUND: A peak VO2 above 14 ml/min/kg at cardiopulmonary exercise testing and brain natriuretic peptide (BNP) levels is used to estimate survival in patients with chronic heart failure (CHF). Limited data, however, exist comparing the prognostic value of both markers simultaneously in patients with mild to moderate CHF. METHODS: We prospectively studied 85 consecutive patients (59+/-13 years, 63 men) with CHF (mean LVEF 26+/-6%). All patients underwent cardiopulmonary exercise testing with determination of peak VO2 and measurement of plasma BNP at rest. The incidence of cardiac decompensation and cardiac death was recorded in the follow-up. RESULTS: During a mean follow-up of 427+/-150 days, four deaths and ten cardiac decompensations occurred. Kaplan-Meier estimates of freedom from clinical events differed significantly for patients above and below the median BNP of 292 pg/ml and also for patients above and below a peak VO2 of 14 ml/min/kg (p<0.05 each). BNP and peak VO2 (area under the ROC 0.75 vs. 0.72) showed a comparable discrimination of CHF patients with adverse cardiac events. The prognostic information of BNP was at least as powerful as that derived from peak VO2. A BNP above 324 pg/ml was associated with a risk ratio of 8.8 for adverse cardiac events. CONCLUSIONS: In patients with mild to moderate CHF, BNP measurements appear to be an alternative to peak VO2 determined by cardiopulmonary exercise testing for the assessment of prognosis in CHF. BNP may facilitate the ambulatory management of patients with mild to moderate CHF since it is less expensive, less time-consuming, and free of procedural risk compared to exercise testing.

Human endotoxemia induces down-regulation of monocyte CC chemokine receptor 2.

Upon injection of Escherichia coli lipopolysaccharide into human volunteers, the monocyte density of CC chemokine receptor 2 (CCR2) decreased. Minimal CCR2 density was observed 4 h after injection. Peak plasma concentrations of the CCR2 ligand monocyte chemotactic protein 1 and of tumor necrosis factor alpha were reached after 4 h and 2 h, respectively.

Monitoring temporary immunodepression by flow cytometric measurement of monocytic HLA-DR expression: a multicenter standardized study.

BACKGROUND: Single-center trials have shown that monocytic HLA-DR is a good marker for monitoring the severity of temporary immunodepression after trauma, major surgery, or sepsis. A new test for measuring monocytic HLA-DR is now available. METHODS: We evaluated a new test reagent set for monocytic HLA-DR expression (BD Quantibritetrade mark HLA-DR/Monocyte reagent; Becton Dickinson) in single-laboratory and interlaboratory experiments, assessing preanalytical handling, lyse-no-wash (LNW) vs lyse-wash (LW) values, reference values, and the effect of use of different flow cytometers and different instrument settings on test variance. RESULTS: For preanalytical handling, EDTA anticoagulation, storage on ice as soon as possible, and staining within 4 h after blood collection gave results comparable to values obtained for samples analyzed immediately after collection (mean increase of approximately 4% in monocytic HLA-DR). Comparison of LNW and LW revealed slightly higher results for LNW ( approximately 18% higher for LNW compared with LW; r = 0.982). Comparison of different flow cytometers and instrument settings gave CVs <4%, demonstrating the independence of the test from these variables and suggesting that this method qualifies as a standardized test. CV values from the interlaboratory comparison ranged from 15% (blood sample unprocessed before transport) to 25% (stained and fixed before transport). CONCLUSIONS: For the BD Quantibrite HLA-DR/Monocyte test, preanalytical handling is standardized. Single-laboratory results demonstrated the independence of this test from flow cytometer and instrument settings. Interlaboratory results showed greater variance than single-laboratory values. This interlaboratory variance was partly attributable to the influence of transport and can be reduced by optimization of transport conditions.

CD14 gene -260 C/T polymorphism is associated with chronic heart failure.

BACKGROUND: Patients with chronic heart failure (CHF) show inflammatory changes and elevated plasma levels of TNFalpha and endotoxins. However, the role of the CD14 C(-260)T polymorphism in patients with CHF is unclear. Therefore, we sought to determine whether the C=>T promoter polymorphism (position -260) of the CD 14 gene is associated with a higher risk for the development of CHF. METHODS: We studied 100 patients with CHF (mean age 62+/-3 years, LVEF 28+/-8%) and 100 healthy controls (59+/-10 years, p=NS; LVEF 60+/-4%, p<0.05). CD14 genotyping was performed using a PCR-RFLP technique. RESULTS: Among CHF patients, the frequency of the T allele was lower (38% vs. 48%, p<0.05) and the frequency of the C allele higher (62 % vs. 52 %, p<0.05) than among controls. The distribution of CD14 genotypes in healthy controls was as follows: CC 32%, CT 40%, and TT 28%. Among CHF patients, the TT genotype was significantly underrepresented compared to controls: CC 38%, CT 48%, and TT 14% (p<0.05). CONCLUSIONS: The C -260T polymorphism of CD14 seems to influence the susceptibility for the development of CHF. The T allele is less frequent among CHF patients than among controls. The TT genotype could be a new genetic protective factor against the development of CHF.

Urotensin II in patients with chronic heart failure.

BACKGROUND: Human Urotensin II (hU-II) is the most potent vasoconstrictor known to date. HU-II receptors are predominant in the human heart and arterial vessels, suggesting hU-II to be of importance as a cardiovascular mediator. METHODS: We studied 32 consecutive patients (60+/-12 years) with chronic heart failure (CHF) and 10 control subjects (54+/-12 years, n.s.) with cardiopulmonary exercise testing. Blood samples for the measurement of plasma hU-II and big-endothelin-1 (big-ET1) were obtained at rest and at peak exercise. RESULTS: Peak VO(2) was significantly higher in controls than in CHF patients (19.8+/-3.8 vs. 14.7+/-3.6 ml min(-1) kg(-1), P<0.001). Big-ET1 levels were increased in CHF compared to controls at rest (2.8+/-1.8 vs. 1.7+/-0.1 fmol/ml, P<0.01) and at peak exercise (2.7+/-1.7 vs. 1.6+/-0.2 fmol/ml, P<0.005). HU-II concentrations were comparable in patients with CHF and controls at rest (2990+/-1104 vs. 3290+/-508 pg/ml, n.s.) and peak exercise (3063+/-1185 vs. 3213+/-1188 pg/ml, n.s.). Resting hU-II levels demonstrated no correlation with peak VO(2) in controls or CHF patients. CONCLUSIONS: The measurement of circulating plasma levels of hU-II does not seem to be very helpful in studying the effects of hU-II in human cardiovascular regulation. A local paracrine or autocrine mediator effect of hU-II in CHF is possible.

Patients with acute on chronic liver failure display "sepsis-like" immune paralysis.

BACKGROUND/AIMS: Cellular immune depression is linked to high mortality in sepsis, but has yet to be systematically analysed in liver cirrhosis. The aim of the present study was to directly compare functional immune parameters in patients with acute on chronic liver failure (ACLF), severe sepsis, and non-decompensated cirrhosis. METHODS: Patients with ACLF (n=27) were investigated at admission to a medical ICU. Patients with stable liver cirrhosis (n=24) and severe sepsis (n=31) served as control groups. In all subjects, serum levels of IL-6 and IL-10, ex vivo production of TNF-alpha in a whole blood assay, and monocyte surface HLA-DR expression were determined. RESULTS: In patients with ACLF or sepsis, ex vivo TNF-alpha production and HLA-DR expression were severely decreased compared to subjects with stable cirrhosis (both P<0.001). Contrary, IL-6 levels were highest in septic patients, followed by subjects with ACLF and cirrhotic patients (both P<0.05). Immune dysfunction in ACLF was independent of aetiology of liver cirrhosis and associated with high mortality. CONCLUSIONS: Patients with ACLF and severe sepsis show a similar degree of cellular immune depression. The reduced cellular immune function in subjects with ACLF might contribute to the increased infectious morbidity of these patients and provide a rational basis for prevention strategies.

Gentamicin supplementation of polyvinylidenfluoride mesh materials for infection prophylaxis.

Hernia repair evolved from pure tissue repair to mesh repair due to decreased recurrence rates. However, concern exists about mesh-related infections occurring even several years after initial operation. Therefore, a polyvinylidenfluoride (PVDF) mesh material was constructed and surface modified by plasma-induced graft polymerization of acrylic acid (PVDF+PAAc). Antimicrobial treatment was sought by binding of gentamicin (PVDF+PAAc+Gentamicin). In vitro efficacy and cytotoxicity was measured by agar diffusion test, L929 cytotoxicity testing and by analyzing the amount of gentamicin release from the mesh surface. In vivo biocompatibility was evaluated in 45 Sprague-Dawley rats. 7, 21 and 90 days after mesh implantation the amount of inflammatory and connective tissue as well as the percentage of proliferating (Ki67) and apoptotic cells (TUNEL) were analyzed at the perifilamentary region. Agar diffusion tests showed sufficient local antimicrobiotic effects against the bacteria tested after 24h of incubation. No signs of cytotoxicity could be identified by L929 testing. Furthermore, surface modification did not affect the in vivo biocompatibility. At the end of the observation period, no significant differences were found for the perifilamentary amount of inflammatory cells and connective tissue and the percentage of Ki67 and TUNEL positive stained cells. The presented data confirm that an antibiotic surface modification of PVDF mesh samples is feasible. By analyzing cytotoxicity in vitro as well as biocompatibility in vivo no side effects were observed.

Hyperglycemia at admission to the intensive care unit is associated with elevated serum concentrations of interleukin-6 and reduced ex vivo secretion of tumor necrosis factor-alpha.

OBJECTIVES: The aim of the study was to investigate the association between admission blood glucose concentrations and immune function variables and its correlation to mortality rate in patients of a medical intensive care unit. DESIGN: Prospective, observational study. SETTING: Medical intensive care unit of a university hospital. PATIENTS: Patients were 189 consecutive critically ill patients in the medical intensive care unit. INTERVENTIONS: At admission to the intensive care unit, serum concentrations of interleukin-6, interleukin-8, interleukin-10, and tumor necrosis factor-alpha were measured with immunometric assays. Additionally, ex vivo secretion of tumor necrosis factor-alpha after stimulation with lipopolysaccharide in a whole blood assay and cytometric human leukocyte antigen-DR expression on monocytes were determined in all study subjects. Simplified Acute Physiology Score II and Therapeutic Intervention Scoring System-28 were calculated for the first day in the intensive care unit. MEASUREMENTS AND MAIN RESULTS: The relationships between blood glucose concentrations and immunologic variables were analyzed using univariate and multivariate statistical methods. Overall, 75 patients (39.7%) presented with hyperglycemia. An elevated blood glucose concentration at admission was related to an increased risk of mortality in the intensive care unit (odds ratio, 2.6; p = .009). At univariate and multivariate analysis, hyperglycemia was associated with increased serum concentrations of interleukin-6 (p < .05), a reduced ex vivo production of tumor necrosis factor-alpha (p < .01), and a history of diabetes mellitus (p < .05), whereas other clinical (including Simplified Acute Physiology Score II and Therapeutic Intervention Scoring System-28) and immunologic variables were not statistically related to blood glucose. CONCLUSIONS: Our main findings show that admission hyperglycemia is statistically related to distinct changes of humoral and cellular immune functions. Furthermore, elevated glucose concentrations at admission are associated with increased intensive care unit mortality rate in a medical intensive care unit. Although these data do not explain cause and effect, our results provide a strong rationale for studying the immunologic effects of strict glycemic control in the intensive care unit during the course of critical illness.

Brain natriuretic peptide kinetics during dynamic exercise in patients with chronic heart failure.

BACKGROUND: The kinetics of brain natriuretic peptide (BNP) secretion in chronic heart failure (CHF) during dynamic exercise have been the subject of controversial debate. The present study was therefore aimed to further clarify whether marked changes in BNP levels occur during and directly after vigorous exercise in CHF patients. METHODS: We prospectively studied 37 patients with CHF (60+/-10 years, LVEF 26+/-6%) and 20 healthy controls (58+/-11 years, LVEF 60+/-3%). Standardized exercise testing was performed in all CHF patients and controls. Venous blood samples for measurement of BNP were obtained prior to symptom-limited exercise, at peak exercise and at 1 and 5 min of recovery time. RESULTS: BNP concentrations were significantly higher in CHF compared to controls at rest, peak exercise and at 1 and 5 min of recovery. BNP levels did not change significantly during exercise in the control group. In CHF patients, BNP levels showed no marked difference at rest (428+/-421 pg/ml), peak exercise (507+/-450 pg/ml, n.s.), 1 min (560+/-460 pg/ml, n.s.) and 5 min recovery (526+/-424 pg/ml, n.s.). Strikingly, 6 of 37 CHF patients (16%) showed a decrease in BNP at exercise compared to rest but none of the controls. CONCLUSIONS: BNP levels in CHF patients and healthy controls are not significantly altered by vigorous exercise. In contrast to controls, 16% of CHF patients showed a decrease in BNP levels at exercise. In CHF, BNP levels were inversely correlated with peak VO(2), VO(2)-AT and LVEF.

Comparison of three commercial assays for the measurement of 17alpha-hydroxyprogesterone (17alpha-OHPR): limitations of the quality control system.

The measurement of 17alpha-hydroxyprogesterone (17alpha-OHPR) is of value for the diagnosis and management of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. In the central laboratory from 2000 to 2002, we observed, using the assay from the manufacturer DSL, an elevation of the moving average of 17alpha-OHPR concentrations and a number of adrenocorticotropic hormone (ACTH) stimulation tests despite the lack of any changes to the internal and external quality control, of which the criteria were continuously fulfilled. We studied a population of n=49 patients for the measurement of 17alpha-OHPR, with and without extraction, to evaluate the quality of different commercially available radioimmunoassays. The internal and external quality controls were successful in determining 17alpha-OHPR. An excellent measurement and correlation of 17alpha-OHPR was expressed with the assay from the manufacturer IBL without extraction and from the manufacturer DSL with extraction. The quantitative determination of 17alpha-OHPR requires adequate specificity and accuracy of the 17alpha-OHPR radioimmunoassays. The results show that internal and external quality control systems are not sufficient to resolve analytical problems described in this study.

Relaxin kinetics during dynamic exercise in patients with chronic heart failure.

Background: We investigated circulating relaxin (RLX) and its potential role in human congestive heart failure (CHF) at rest and after physical exercise. Methods: A total of 10 healthy controls and 35 patients with stable CHF were enrolled in the study. Results: RLX plasma concentrations at rest and after exercise were comparable in patients with CHF and in controls. Conclusion: These findings suggest that RLX does not play a major role as a circulating hormone in human CHF.

Endotoxin sensitivity and immune competence in chronic heart failure.

BACKGROUND: Raised concentrations of endotoxin (lipopolysaccharide, LPS) are found in patients with chronic heart failure (CHF). Tolerance of monocytes to LPS can be induced by LPS itself resulting in a downregulation of cytokine response to LPS challenge. This phenomenon of LPS desensitization has also been suggested for CHF. METHODS: We investigated whether CHF patients really show a desensitization to LPS stimuli at rest or after physical exercise, which was used as a model of limited inflammatory reaction. Thirty-five patients with CHF (59+/-12 years, 8 women) and 30 healthy control subjects were prospectively studied with cardiopulmonary exercise testing. At rest and directly after exercise blood samples were taken for the quantitative determination of HLA-DR expression of monocytes as a measure for immune competence and for the measurement of TNFalpha generation after ex vivo stimulation by LPS. RESULTS: HLA-DR expression was comparable in CHF patients and controls at rest as well as after exercise. TNFalpha production by LPS-stimulated monocytes ex vivo was higher in CHF patients compared to controls at rest and after exercise. CONCLUSIONS: Thus, our data are the first to show that patients with stable CHF show a cellular hypersensitivity to LPS with a higher TNFalpha generation capacity at rest and after exercise compared to controls. CHF patients seem to have a marked susceptibility to low inflammatory stimuli and no desensitization to LPS.

Brain natriuretic peptide predicts right heart failure in patients with acute pulmonary embolism.

BACKGROUND: Plasma levels of brain natriuretic peptide (BNP) are increased in patients with left heart failure. In patients with severe pulmonary embolism (PE), primary right ventricular (RV) dysfunction is frequent. Little is known about BNP secretion in acute RV failure. METHODS: We prospectively studied 50 consecutive patients with confirmed PE (age range, 57 +/- 19 years; 36 men). PE was confirmed with pulmonary angiography, spiral computed tomography, or echocardiography and subsidiary analyses. On admission, echocardiography and BNP measurements were performed in all patients. RESULTS: Patients without RV dysfunction had significantly lower BNP levels than patients with RV dysfunction (55 +/- 69 pg/mL vs 340 +/- 362 pg/mL, P <.001). There was a significant correlation between RV end-diastolic diameter and BNP (r = 0.43, P <.05). BNP discriminated patients with or without RV dysfunction (area under the receiver operating characteristic curve, 0.78; 95% CI, 0.64-0.92). A BNP >90 pg/mL was associated with a risk ratio of 28.4 (95% CI, 3.22-251.12) for the diagnosis of RV dysfunction. All patients without LV systolic dysfunction who had syncope necessitating cardiopulmonary resuscitation had normal BNP levels. Patients with RV dysfunction had significantly more in-hospital complications (cardiogenic shock, inotropic therapy, mechanical ventilation). However, BNP levels were not predictive of mortality or in-hospital complications. CONCLUSIONS: BNP levels are frequently increased in patients with PE who have RV dysfunction, whereas patients without RV dysfunction show reference range BNP levels in the absence of left ventricular dysfunction. In acute PE, BNP elevation is highly predictive of RV dysfunction, but not of in-hospital complications and mortality.

Procalcitonin serum levels after out-of-hospital cardiac arrest.

The time course of Procalcitonin (PCT) serum levels was assessed in cardiac arrest survivors and compared with S-100 serum levels concerning their predictive values for neurological outcome. PCT and S-100 serum levels were analyzed serially on admission and during the following 3 days after hospitalization in 23 patients successfully resuscitated from out-of-hospital cardiac arrest. At day 14 patients were divided into groups according to the Glasgow-Outcome-Scale (GOS): one group with bad neurological outcome (GOS 1-3) and one group with good neurological outcome (GOS 4-5). Group comparisons were performed with the Mann-Whitney U-Test. The diagnostic performance of PCT and S-100 levels was analyzed using receiver operating characteristics (ROC). Patients with a bad neurological outcome had significantly higher S-100 levels than those with a good neurological outcome at all investigated time points and significantly elevated PCT levels at days 1-3. Highest levels for S-100 were found immediately after hospitalization (3.4 +/- 3.8 vs. 0.7 +/- 0.3 microg/l, P=0.003), and for PCT at day 1 (37 +/- 103 vs. 0.2 +/- 0.2 microg/l, P=0.0002). The results show that PCT serum levels are possibly elevated in patients with bad neurological outcome after cardiac arrest, without signs of severe infection or concomitant sepsis. Based on this observation, studies on larger numbers of patients should prove the predictive value of PCT in those patients.

Rapid and reliable genotyping for the Toll-like receptor 4 A896G polymorphism using fluorescence-labeled hybridization probes in a real-time polymerase chain reaction assay.

BACKGROUND: The Toll-like receptor 4 (TLR4) is involved in immune response to endotoxin as well as the pathogenesis of atherosclerosis. The TLR4 gene was shown to carry a single-nucleotide polymorphism (A896G). We developed a rapid-cycle polymerase chain reaction (PCR) which allows for rapid genotyping and, therefore, may be useful in clinical risk assessment. METHODS: Fluorescently labeled oligonucleotide hybridization probes were designed for the LightCycler instrument. A PCR product was generated in a single reaction followed by melting point analysis. Ninety-three German Caucasians were genotyped. The interleukin-1beta (IL-1beta) response to endotoxin was assessed after whole blood stimulation with endotoxin according to TLR4 genotypes. RESULTS: The method suggested by us is a time-saving technique requiring no additional manual steps. Frequencies of the A and G allele were 0.95 and 0.05, respectively. The study population was in Hardy-Weinberg equilibrium. The specificity of the method was confirmed by sequencing. The IL-1beta levels were lower in carriers of the G allele. CONCLUSIONS: This PCR assay is a rapid and reliable technique for TLR4 genotyping.

Cytokine response to endotoxin in individuals heterozygous for the Delta32 mutation of chemokine receptor CCR5.

Studies of mice with a targeted disruption of the CCR5 gene suggest that the CC chemokine receptor 5 (CCR5) is a determinant of the cytokine response to endotoxin. In humans, a naturally occurring mutation of the CCR5 gene is a 32-basepair (bp) deletion which precludes the translation of the gene into a functional transmembrane protein. To evaluate the cytokine phenotype of heterozygosity for the 32 deletion, we studied the endotoxin-stimulated release of tumor necrosis factor-alpha, Interleukin (IL)-6, IL-8, IL-10, and IL-12 in whole blood ex-vivo of healthy volunteers and patients undergoing elective cardiac bypass surgery. This operation represents a major surgical trauma associated with ischemia-reperfusion-injury and triggers a profound inflammatory response. In these patients, cytokine plasma concentrations were measured during and after cardiac surgery. No difference was found between the frequencies of the observed and expected CCR5 genotypes in the groups of individuals studied. Furthermore, no significant difference in ex-vivo or peri- and postoperative cytokine plasma concentrations was detected between CCR5 wild-type homozygotes and individuals carrying one defective CCR5 allele. Our results indicate that heterozygosity for the 32bp deletion of CCR5 is not associated with an altered cytokine response to endotoxin or to a major surgical trauma when compared with individuals homozygous for the wild-type allele.

Terminal renal failure in mice lacking transcription factor AP-2 beta.

Inactivation of the transcription factor AP-2 beta in a genetically mixed C57BL/6/129S1 mouse strain resulted in perinatal lethality as a consequence of massively enhanced apoptotic death of renal epithelial cells (Genes Dev 1997;11:1938-1948). Recently, we observed that the phenotype is modulated by genetic background because AP-2 beta mutant mice, backcrossed onto 129P2 background, survive approximately 2 weeks after birth, allowing for a detailed analysis of kidney function. Here we show that kidneys reveal varying amounts of cysts derived from all tubular structures (proximal and distal tubuli, collecting ducts). However, all mice died irrespective of the degree of cyst formation. Serum analysis of AP-2 beta mutant animals revealed defective tubular secretory function and ion homeostasis including severe hypocalcemia, hyperphosphatemia, and hyperuremia. Because hormonal calcium regulation was not impaired, the mice developed secondary renal hyperparathyroidism as typically observed in patients with terminal renal failure. We further demonstrate that molecular defects in the collecting duct system lead to insufficient water retention and urinary concentration. In summary, our studies reveal essential, nonredundant roles of AP-2 beta in renal tubular functions.