Correlation between flagellin A (flaA) genotypes and antimicrobial susceptibility patterns of Campylobacter jejuni strains isolated from children with gastroenteritis in Athens, Greece.

BACKGROUND AND OBJECTIVE: Campylobacter jejuni is one of the most common enteric pathogens worldwide. The bacterium is transmitted to humans via contaminated food and water. In the majority of cases the disease is self-limiting, but treatment is indicated in immunocompromised patents, in severe cases with septicemia, and in children. The subtyping of clinical, animal, and food C. jejuni isolates is very important for epidemiological studies. In the present study, 192 Campylobacter jejuni isolates characterized by pulsed-field gel electrophoresis (PFGE) of SmaI digested genomic DNA were further examined with respect to their antimicrobial resistance and their flagellin A (flaA) genotypes in order to disclose any correlation between a certain flaA type and a specific antimicrobial susceptibility pattern. METHODS: C. jejuni clinical isolates were collected from infected children up to 14 years of age from five general hospitals in the area of Attica, Greece, during the period 2004-7. C. jejuni strain isolation and identification from stool samples were performed by conventional bacteriological methods. SmaI restriction fragments were prepared as described previously for the PFGE analysis. Antimicrobial susceptibility was tested and interpreted by determination of the minimal inhibitory concentration (MIC) by use of the agar dilution method as described by the Clinical Laboratory Standards Institute. FlaA typing was performed by PCR amplification of the corresponding gene, and the product was digested with DdeI and visualized by agarose gel electrophoresis. Data were analyzed using the software Gene Profiler 1-D Gel Analysis and Data Basing for Windows((R)). RESULTS: A statistically significant correlation between certain flaA genotypes (flaA 17 Greece [GR], flaA 19 GR and flaA 39 GR) and resistance to some antimicrobial agents (ampicillin, amoxicillin/clavulinic acid [co-amoxiclav], erythromycin, nalidixic acid, and ciprofloxacin) was detected in C. jejuni strains isolated from infected children. CONCLUSIONS: Further investigations on a molecular basis are warranted in order to clarify whether certain C. jejuni flaA types are associated with specific antimicrobial resistance attributes.

Low cholesterol along with inflammation predicts morbidity and mortality in hemodialysis patients.

Low and not high cholesterol seems to predict high mortality in hemodialysis (HD) patients. The confirmation of this reverse epidemiology as well as its possible interconnection with the increased inflammatory activity observed in this population is being explored in the present study. A group of 136 HD patients was prospectively studied for 2 years, and cardiovascular disease (CVD) as well as all-cause mortality and morbidity were recorded. Baseline lipid profile, inflammatory status, and patients' characteristics were studied as potential survival and hospitalization predictors. During the 24-month follow-up, 21 deaths (52.4% due to CVD) and 38 hospitalizations (55.3% due to CVD) were recorded. In multivariate Cox regression analysis, decreased interleukin-10 (IL-10) and decreased total serum cholesterol (TChol) were the only independent predictors of CVD mortality while C-reactive protein and decreased TChol predicted all-cause mortality. Interleukin-10 at baseline was 11.29 +/- 21.49 vs. 5.51 +/- 4.57 pg/mL (P<0.018) and TChol 167.37 +/- 47.84 vs.122.04 +/- 26.48 mg/dL (P<0.000) in survivors vs. nonsurvivors from CVD, while C-reactive protein at baseline was 9.37 +/- 11.54 vs. 23.15 +/- 18.76 mg/L (P<0.000) and TChol 169.26 +/- 46.42 vs. 133.26 +/- 46.33 mg/dL (P<0.003) in survivors vs. nonsurvivors from any cause of death. Using the same method of statistical analysis, IL-6 and decreased soluble gp130 (sgp130)--an antagonist of IL-6 action--were found to be the only independent prognostic factors for hospitalization due to CVD while decreased soluble gp130 remained the sole predictor of hospitalization due to any cause. In conclusion, reverse epidemiology regarding cholesterol is confirmed in the present study. Furthermore, inflammatory activity also predicts, independently of or in conjunction with low-cholesterol, CVD and all-cause morbidity and mortality in HD patients.

Telomerase activity is decreased in peripheral blood mononuclear cells of hemodialysis patients.

BACKGROUND: Telomerase preserves telomere length and structure, preventing cellular senescence, which is associated with alteration of the chromosomal ends. We hypothesized that telomerase activity is altered in peripheral blood mononuclear cells (PBMCs) of hemodialysis (HD) patients. To investigate this hypothesis as well as the relationship between telomerase and inflammation, we measured the activity of this reverse transcriptase as well as the level of several inflammatory markers in PBMCs and serum of an end-stage renal failure (ESRF) population and a non-renal-failure group of subjects. METHODS: In PBMCs isolated from 42 HD and 39 non-renal-failure subjects of the same age (51.0 +/- 12.4 and 51.4 +/- 12.1 years, respectively) telomerase activity was measured using PCR-ELISA; the method was based on the telomeric repeat amplification protocol. RESULTS: Telomerase activity in PBMCs was detected in 18 (42.9%) HD and 28 (71.8%) non-renal-failure subjects (p = 0.013). Among positive subjects, percent telomerase activity in PBMCs was significantly higher in non-renal- failure (117 +/- 112 %) than in HD (47.6 +/- 57.1 %) subjects (p = 0.008). Detectable telomerase activity was lower in long-term than in short-term HD patients (13.3 +/- 8.9 vs. 75.0 +/- 64.8%, respectively, p = 0.015). Although higher in HD group, inflammatory indexes (C-reactive protein, interleukin-6, IL-6, soluble IL-6 and soluble gp130) were not correlated to telomerase activity in PBMCs. CONCLUSION: Telomerase activity in PBMCs is reduced in HD patients. It seems that, at least in this type of cell in this population, defense from senescence, as assessed by telomerase activity, is altered and associated with the chronicity of uremia/HD procedure.

Serum oxidized low-density lipoprotein is inversely correlated to telomerase activity in peripheral blood mononuclear cells of haemodialysis patients.

BACKGROUND: Telomerase preserves telomeres' function and structure preventing cellular senescence. Its activity is reduced in peripheral blood mononuclear cells (PBMC) of haemodialysis (HD) patients. The purpose of this study is to investigate the potential correlation between increased oxidative stress/inflammation and telomerase activity in PBMC of HD patients. METHODS: Telomerase activity was measured by PCR-ELISA in PBMC isolated from a group of 42 HD patients and 39 subjects with estimated glomerular filtration rate >or=80 mL/min (control group). Serum oxidized low-density lipoprotein (ox-LDL), tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) were also measured in both groups by ELISA. RESULTS: Ox-LDL was negatively correlated to percentage telomerase activity in PBMC (r = -0.506, P = 0.000 in the whole group of 81 HD and normal subjects and r = -0.559, P < 0.001 in HD patients). TNF was also inversely associated with percentage telomerase activity in the whole group studied (r = -0.492, P = 0.000) while IL-10 was not. In stepwise multiple linear regression, taking into consideration the most important characteristics of the HD patients and control group, the only significant predictors for percentage telomerase activity in PBMC were ox-LDL and TNF (beta = -0.421, t = -4.083, P = 0.000 and beta = -0.381, t = -3.691, P = 0.000, respectively) while examining separately HD patients, the predictors for the same parameter were ox-LDL and HD duration (beta = -0.671, t = -4.709, P = 0.000 and beta = -0.349, t = -2.447, P = 0.023, respectively). CONCLUSION: Ox-LDL serum level is inversely correlated to telomerase activity in PBMC of HD patients. Our study proposes a new consequence of increased oxidative stress in HD patients: the premature cellular senescence potentially related to atherosclerosis through LDL oxidation.

Release of interleukin-6 and its soluble receptors by activated peripheral blood monocytes is elevated in hypocholesterolemic hemodialysis patients.

BACKGROUND: A reverse association between cholesterol level and cardiovascular disease mortality is observed in hemodialysis (HD) patients; this paradoxical relationship may be explained by the coexistence of inflammation. Interleukin-6 (IL-6) is a central regulator of inflammation; its action is augmented by the soluble IL-6 receptor (sIL-6R) and inhibited by the soluble gp130 (sgp130). In order to investigate the potential association of inflammation with cholesterol levels in the HD population, release of soluble IL-6 components by peripheral blood mononuclear cells (PBMCs) was measured in two groups of HD patients with distinctly different lipid profile and in a control group. METHODS: Twenty-two HD patients with low serum cholesterol (range 85-171 mg/dl), 23 HD patients with high cholesterol (189-342 mg/dl) and 21 normolipidemic non-renal failure subjects were enrolled in the study. IL-6, sIL-6R and sgp130 were measured by ELISA in the serum and in the supernatant collected from cell cultures of activated or resting PBMCs isolated from all three groups. RESULTS: Serum IL-6 and sgp130 level was higher while sIL-6R was lower in both groups of HD patients compared to the control group. The ex-vivo release of the IL-6 and sgp130 by unstimulated PBMCs did not differ significantly between the three groups but that of the sIL-6R was higher in non-renal failure than in hypercholesterolemic HD subjects. Production of sIL-6R by stimulated PBMCs was higher in low-cholesterol HD patients (p < 0.001) and the same was valid for the sgp130 release (p = 0.034). Release of IL-6 by activated PBMCs was higher in the low-cholesterol compared to the high-cholesterol HD patients group (p = 0.011 for post hoc test). Major serum lipid fractions were inversely correlated to IL-6 and sIL-6R production from stimulated PBMCs in HD but not in non-renal failure subjects. Finally, release of the sgp130 by PBMCs was significantly reduced in 13 hypertriglyceridemic--and hypercholesterolemic--HD patients. CONCLUSION: Production of soluble components of a crucial pro-inflammatory and potentially atherogenic cytokine, namely the IL-6, by stimulated PBMCs appears to be inversely correlated with the serum cholesterol levels in HD patients.

Serum and peripheral blood mononuclear cells infectious burden: correlation to inflammation and atherosclerosis in haemodialysis patients.

BACKGROUND: Infectious agents may be implicated in the inflammatory atherosclerotic process. Not only specific microorganisms but also the infectious burden, defined as the number of pathogens to which a patient is exposed, has been associated with atherosclerosis. In the present study, the infectious burden, determined directly (by identification of viable pathogens in peripheral blood mononuclear cells (PBMC)) and indirectly (by serum antibodies detection) is correlated to the inflammatory and atherosclerotic status in haemodialysis (HD) patients, a population at high risk for cardiovascular disease. METHODS: The viable forms of four microorganisms (Chlamydia pneumoniae, herpes virus 1 and 2 and cytomegalovirus) were identified in patients PBMC by cell cultures and subsequent polymerase chain reaction. Serum IgG against the above pathogens and Helicobacter pylori were also determined. Inflammation was assessed by measurement of C-reactive protein (CRP), serum amyloid A (SAA), three pro- and one anti-inflammatory cytokines and four adhesion molecules. Atherosclerosis was defined by a scoring system using medical history data. RESULTS: The number of viable pathogens identified in PBMC in the 122 HD patients included in the study were zero in 22.1% of them, one in 33.6%, two in 43.4% and three in one patient. The number of IgG antibodies determined was one in 6.6% of patients, two in 32%, three in 48.4% and four in 13.1%. Seropositivity was not significantly different between patients with or without the respective viable pathogen identified in PBMC. Atherosclerosis was present in 40.2% of patients, and CRP, SAA and interleukin-6 were all increased in these patients. Neither inflammatory indexes nor atherosclerosis were significantly different in patients with a higher number of viable pathogens detected in PBMC or in those with a higher antibodies number. CONCLUSIONS: The direct infectious burden determination (the number of viable pathogens in PBMC) does not coincide with the serum (by IgG detection) infectious burden. Although inflammation correlates to atherosclerosis, neither PBMC nor the serum infectious burden is associated with these two entities in the inflamed and atherosclerotic HD patients.

Treatment with fluvastatin rapidly modulates, via different pathways, and in dependence on the baseline level, inflammation in hemodialysis patients.

BACKGROUND: Hemodialysis (HD) patients are frequently in an elevated inflammatory state which is correlated to the atherosclerosis-related and overall morbidity and mortality in this population. Statins, beyond their antilipidemic effects, are also considered to have anti-inflammatory, immunomodulating and antioxidant properties. The individual response of HD patients to a short course of fluvastatin, the mechanisms involved in the immunomodulating and anti-inflammatory effects of this drug and the time interval to the appearance of these effects are investigated in this longitudinal study. METHODS: In a group of 51 HD patients, fluvastatin 40 mg/day was administered for 4 weeks. Serial measurements of the lipid profile, C-reactive protein (CRP), interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), interleukin-10 (IL-10), and serum oxidized LDL (ox-LDL), were performed before, during, and after the treatment period. RESULTS: Total cholesterol was significantly reduced after 14 days of treatment with fluvastatin (from mean +/- SD 216.7 +/- 34.3 to 179.2 +/- 42.3 mg/dl, p < 0.001). IL-6 and ox-LDL were reduced on day 28 (p < 0.001 and p < 0.01, respectively) and IL-10 was increased on day 14 (p = 0.05); CRP did not change significantly during the treatment period while sIL-6R was increased on day 28 of fluvastatin administration (p < 0.05). In a subgroup of patients with CRP, IL-6, sIL-6R, and ox-LDL baseline serum values > or = the median and IL-10 < or = the median, CRP was reduced on day 28 of fluvastatin treatment (p < 0.01), IL-6 and ox-LDL were reduced earlier, on day 14 (p = 0.05 and p < 0.05, respectively) while sIL-6R did not change significantly during the treatment period. CONCLUSIONS: Treatment with fluvastatin rapidly modulates inflammation in HD patients. Enhancement of anti-inflammatory mechanisms and attenuation of the inflammatory and oxidative state contribute to this modulation. Patients in an elevated baseline inflammatory state respond more rapidly and effectively to the treatment. This immediate and multi-potent action of the statins could be clinically useful in acute atherosclerosis complications or in the treatment of chronic inflammation in HD patients.

Exploring inflammation in hemodialysis patients: persistent and superimposed inflammation. A longitudinal study.

BACKGROUND: Inflammation is frequently elevated, and seems to be episodic in hemodialysis (HD) patients. Whether, its episodic character is due to the temporal variability, in periods free of clinical events, of the inflammatory indices or due, to the acute phase response induced by common inflammatory stimuli, has not been investigated yet in a longitudinal study. This study explores inflammation forms, characteristics and causes which are probably related to the high cardiovascular disease (CVD) morbidity in HD patients. METHODS: In 37 HD patients, high-sensitivity C-reactive protein (hs-CRP), serum amyloid A (SAA) and interleukin-6 (IL-6) were weekly measured for 16 consecutive weeks. Inflammatory clinical events, in the week before every measurement, were recorded. Repeated measures ANOVA were applied for statistical analysis. RESULTS: Fifty-one of 533 patient-weeks were positive for a clinical event. Mean +/- SD (range) hs-CRP was 7.01 +/- 16.06 (0.2-169) mg/l for all the weeks of the study, 38.25 +/- 39.35 (2.1-169) mg/l for the weeks with clinical events and 3.70 +/- 3.86 (0.2-26.1) mg/l for the weeks free of events. Variations for SAA and IL-6 were similar. 'Clinical events' strongly influenced acute-phase proteins and IL-6 levels. The effect of the factor 'time' (as assessed by inflammatory indices variation in weekly repeated measurements) was significant for all the 3 indices measured, independently of the factor 'clinical events'. CONCLUSIONS: In periods free of clinical events, microinflammation characterizes HD patients and fluctuates in time. Inflammation due to common clinical events is added, periodically, to this microinflammation. The high level persistent microinflammation as well as the superimposed--due to clinical events--inflammation could be related to the CVD in these patients.

The variability and accurate assessment of microinflammation in haemodialysis patients.

BACKGROUND: Systemic microinflammation is correlated with atherosclerosis. It needs a reliable assessment. This study explores the temporal variations of three inflammatory indexes [C-reactive protein (CRP), serum amyloid A (SAA) and interleukin-6 (IL-6)] in a period free of clinical events and tests the reliability of their multiple measurements for the assessment of microinflammation in haemodialysis (HD) patients, a population at high risk of atherosclerotic cardiovascular disease. METHODS: For 4 months, serum CRP, SAA and IL-6 were measured in 29 HD patients during the weeks they were free of inflammatory clinical events (> or =12 measurements for each index in every patient). The components of the variance as well as the reliability of two to five measurements for each index, aimed at assessing microinflammation precisely, were computed. RESULTS: The median (interquartile range) of CRP was 2.3 (0.9-4.9) mg/l, of SAA 3.7 (2.1-9.3) mg/l and of IL-6 4.4 (2.2-7.7) pg/ml. Patients were approximately equally distributed between three groups of low, intermediate and high variability for each index. The contribution of intraindividual (biological) variation to the total of variance was 71.3%, 69.3% and 86.7% for CRP, SAA and IL-6, respectively (higher than in all other similar studies in healthy populations). Using two measurements, the estimated reliability was 57-68% for CRP in two-thirds of the patients (comparable with that found in healthy subjects) and 57% for SAA and IL-6 in only one-third of the patients. Increasing the number of measurements up to five did not change the reliability. CONCLUSIONS: Individual factors significantly influence the levels of inflammatory indexes in HD patients in periods free of inflammatory clinical events. The mean of two weekly CRP measurements, but not of SAA or IL-6, seems to assess microinflammation in most patients with a sufficient reliability.

The effect of viable Chlamydia pneumoniae on serum cytokines and adhesion molecules in hemodialysis patients.

BACKGROUND: Chlamydia pneumoniae (Cp) induces the production of cytokines and adhesion molecules in infected host eukaryotic cells. The causes for pro-inflammatory cytokine and adhesion molecule increase in hemodialysis (HD) patients have not been fully elucidated. The possibility that, in this particularly atherosclerotic population, Cp, a microorganism implicated in the infectious-based inflammatory hypothesis of atherosclerosis' is also responsible for these molecules' increase is assessed in this study. METHODS: In 130 stable HD patients, serum interleukin-1 beta (IL-1), interleukin-6, tumor necrosis factor alpha, interleukin-10, L-selectin, E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 (VCAM-1) levels were determined. Cp presence was identified by inoculation of the patient's peripheral blood mononuclear cells (PBMCs) in Hep-2 cell lines and subsequent polymerase chain reaction (PCR) in DNA extracted from cell cultures, as well as by determination of serum IgG antibodies against Cp (IgGCp). RESULTS: Patients, positive or negative for IgGCp, had no statistically significant differences in all molecules measured. Patients with viable Cp in PBMCs had higher serum levels of IL-1 and soluble VCAM-1 than negative ones for IgGCp (IL-1 6.87 +/- 7.35 vs. 2.34 +/- 1.47 pg/mL; P = 0.0009 and VCAM-1 1647.16 +/- 513.64 vs. 1162.14 +/- 546.83 ng/mL; P = 0.0115, respectively). Viable Cp in PBMCs remained a significant predictor factor for IL-1 and VCAM-1 in statistical analysis, when patients' characteristics and dialysis conditions were also evaluated. CONCLUSIONS: Our results showed that some serum cytokine and adhesion molecule increase in HD patients could be attributed to viable Cp presence in PBMCs. These findings support the Cp-based inflammatory atherogenous hypothesis and add a better understanding of these molecules' increase in HD patients.

Detection of Chlamydia pneumoniae in peripheral blood mononuclear cells: correlation with inflammation and atherosclerosis in haemodialysis patients.

BACKGROUND: Chlamydia pneumoniae has been implicated as an inflammatory agent in atherosclerosis. Clinical studies in this field have yielded conflicting results, which may have resulted from a lack of standardization for C.pneumoniae detection. We attempted to accurately estimate C.pneumoniae prevalence and to examine whether C.pneumoniae is associated with atherosclerosis and inflammation in haemodialysis (HD) patients. To do this, we assessed C.pneumoniae presence by a combination of methods and correlated its levels with inflammatory and atherosclerotic indexes in these patients. METHODS: Chlamydia pneumoniae was identified by polymerase chain reaction (PCR) in DNA extracted from cell cultures inoculated with patient buffy coats and by serum IgG antibodies against C.pneumoniae (IgGCp). Inflammation was assessed by C-reactive protein and serum amyloid A and atherosclerosis was evaluated from clinical and laboratory data. RESULTS: Of the 130 patients, only nine had viable C.pneumoniae in peripheral blood mononuclear cells (PBMCs) while 64 had serum IgGCp. Although patients with viable C.pneumoniae had higher atherosclerotic scores, seropositive and negative patients showed similar scores. Patients with atherosclerosis exhibited higher inflammatory indexes. Neither patients with detectable C.pneumoniae in PBMCs nor seropositive subjects had higher inflammation than negative patients. CONCLUSIONS: We found that viable C.pneumoniae in PBMCs, assessed by cell culture and PCR, was present in a small percentage of HD patients and was correlated with atherosclerosis. Seropositivity was much higher in HD patients but was not associated with viable C.pneumoniae or with atherosclerosis. Further studies in HD patients using high sensitivity and specificity methods in larger populations will be necessary to clarify the relationship between C.pneumoniae and atherosclerosis.