Robert A. Stolarek

Breath analysis of hydrogen peroxide as a diagnostic tool.

The potential diagnostic significance of exhaled hydrogen peroxide (H(2)O(2)) in pulmonary and systemic disorders has received considerable interest over the last few decades. Despite large physiologic variability and low specificity, airway H(2)O(2) generation has been found to be consistently increased by inflammatory conditions. Furthermore, the level of exhaled H(2)O(2) has been associated with efficacy of treatment in various pulmonary diseases. To evaluate this potential biomarker, detection methods including standardization protocols have been developed. Despite these advances, more comprehensive and controlled studies are required. In this manuscript we review progress to date in the analytical measurement of exhaled H(2)O(2) and speculate on its potential clinical significance as a diagnostic tool.

Increased Levels of Soluble TNF-α Receptors and Cellular Adhesion Molecules in Patients Undergoing Bioincompatible Hemodialysis

Background: The study aimed to differentiate the effects of hemodialysis (HD) and chronic renal failure (CRF) on the levels of circulating tumor necrosis factor-α (TNF-α) and TNF-α receptors p55 and p75, soluble vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), soluble endothelial-leukocyte adhesion molecule-1 (sE-selectin) and sP-selectin in 18 patients on regular HD treatment with cuprophane membrane in relation to 15 non-dialyzed CRF patients and 15 healthy controls. Methods: The serum concentrations were determined with standard ELISA assays. Results: Blood serum p75 and p55 were approximately tenfold increased in CRF (36.7 ± 6.2 and 27.1 ± 5.6 ng/ml) and HD patients (45.6 ± 18.4 and 28.7 ± 5.9 ng/ml) before the HD session (HD 0), during (HD 20) the session (45.7 ± 18.4 and 28.5 ± 7.3 ng/ml) and after (HD 240) the HD session (52.1 ± 17.4 and 30.9 ± 8.2 ng/ml) in comparison to control values (5.6 ± 1.3 and 2.4 ± 0.8 ng/ml, respectively) (p < 0.01). The highest increment of p75 at the end of HD session (HD 240) was also significantly higher than at preceding time points (HD 0 and 20) (p < 0.05). However, the remaining study parameters did not change during an HD session. Also, there were no relevant changes in TNF-α levels if (HD 0) 22.7 ± 21.5 ng/ml and (HD 240) 21.1 ± 18.9 ng/ml were compared. Chronic HD status was related to the increase of sVCAM-1 and sICAM-1 levels. Prior to HD, T0 sVCAM-1 and sICAM-1 concentrations were 2,180.4 ± 761.8 and 567.3 ± 218.8 ng/ml, during HD (T20): 2,172.7 ± 759.2 and 602.3 ± 379.9 ng/ml, and after HD (T240): 2,401.6 ± 756.4 and 648.3 ± 183.5 ng/ml, respectively (p < 0.05 vs. controls and CRF patients). sVCAM-1 and sICAM-1 serum levels (1,262.2 ± 472.9 and 165.6 ± 50.4 ng/ml) were similar in CRF patients and healthy controls (854.4 ± 241.5 and 217.6 ± 74.2 ng/ml, respectively). Even though serum sE- and sP-selectin in CRF patients did not differ from the control (39.8 ± 21.3 vs. 42.1 ± 18.9 ng/ml and 187.9 ± 66.9 vs. 198.8 ± 62.2 ng/ml, respectively), their levels were increased in HD patients up to 111.9 ± 54.6 and 453.2 ± 231.1 ng/ml in patients prior to HD, 118.7 ± 66.2 and 350.8 ± 114.8 ng/ml during the HD session and then 132.3 ± 61.1 and 368.3 ± 126.6 ng/ml, respectively, after its completion (p < 0.05 in comparison with CRF patients and controls). Conclusions: The increased circulating TNF-α receptors appear more associated with the uremic milieu than HD-related systemic inflammation, whereas increased soluble cellular adhesion molecules in patients undergoing bioincompatible HD may be related to the enhanced systemic inflammation specifically due to maintenance HD.

Serum metalloproteinases MMP-2, MMP-9 and metalloproteinase tissue inhibitors TIMP-1 and TIMP-2 in patients on hemodialysis

BackgroundWe assessed the effect of hemodialysis (HD) and chronic kidney disease (CKD) on the serum levels of metalloproteinase-2 (MMP-2), MMP-9 and metalloproteinase tissue inhibitors (TIMP-1) and TIMP-2.Methods18 patients on regular HD treatment with low-flux, cuprophane membrane, 15 non-dialyzed patients with CKD and 15 healthy controls were sampled. The serum MMP and TIMP concentrations were determined by ELISA assays.ResultsMMP-9, TIMP-1, and TIMP-2 serum levels were significantly decreased in HD patients to 32.7xa0±xa020.1xa0ng/ml, 178.8xa0±xa073.0xa0ng/ml, and 103.4xa0±xa055.3xa0ng/ml compared with 482.3xa0±xa0139.5, 367.6xa0±xa075.5xa0ng/ml, and 299.7xa0±xa063.2xa0ng/ml in patients with CKD and 594.6xa0±xa0154.7xa0ng/ml, 354.5xa0±xa081.2xa0ng/ml, and 272.4xa0±xa091.8xa0ng/ml in healthy controls, respectively, (Pxa0<xa00.001 vs. HD patients). MMP-2 was lower in patients with CKD: 405.6xa0±xa0106.1xa0ng/ml compared with 516.9xa0±xa081.7xa0ng/ml in controls (Pxa0=xa00.02). The MMP-2/TIMP-2 ratio was increased in HD patients compared with both patients with CKD and controls. In the course of an HD session, MMP-2 and TIMP-1 serum levels were significantly decreased from pre-HD 570.0xa0±xa0256.5 and 178.8xa0±xa066.9xa0ng/ml to post-HD 492.6xa0±xa0212.5 and 144.6xa0±xa044.2xa0ng/ml (Pxa0=xa00.004 and 0.013, respectively). However, the MMP-9/TIMP-1 ratio increased from pre-HD 0.15 (2.19) (median, range) to 0.23 (0.33) after a HD session (Pxa0=xa00.03). CRP was positively correlated with MMP-9 and MMP-9/TIMP-1 ratio in HD patients and patients with CKD (rxa0=xa00.67; Pxa0=xa00.03).ConclusionsThe MMP-9/TIMP-1 ratio increased during HD sessions, although their absolute levels were lowered. This change may represent a chronic state of enhanced fibrosis in patients undergoing HD.

Increased H2O2 level in exhaled breath condensate in primary breast cancer patients

PurposeThis study was designed to assess exhaled hydrogen peroxide (H2O2), blood serum antioxidant capacity, and tumor necrosis factor-α (TNFα) in primary breast cancer (PBC).MethodsThe study included 34 consecutive, non-smoking PBC patients (aged 62.5xa0±xa013.5 at surgery) prior to the treatments, qualified for modified radical mastectomy and not undergoing any adjuvant systemic therapy, and 33 healthy controls. The post surgery pathological assessment included tissue expression of estrogen (ER) and progesterone (PR) receptors, and epidermal growth factor receptor type 2 (HER-2/neu). Exhaled H2O2 was determined fluorometrically in the exhaled breath condensate (EBC). Blood serum antioxidant capacity and TNFα levels were assessed with ferric reducing ability of plasma (FRAP) and ELISA immunoassay, respectively.ResultsIn PBC patients, 10 ER, 11 PR, and 9 HER-2/neu positive tumors were identified and HER-2/neu score was 2+ in 20% of all tumors. Median (Me) H2O2 was increased up to 0.44xa0μM (interquartile range IR: 0.20–1.25xa0μM) compared with healthy control of 0.36xa0μM (IR: 0.12–0.48xa0μM; pxa0<xa00.05). The H2O2 concentration in EBC was significantly correlated (τxa0=xa00.27; pxa0=xa00.03) and increased in cases with nodal metastases (nxa0=xa012; pxa0=xa00.04). Serum TNFα was increased up to 51.7xa0±xa021.0xa0pg/ml compared with controls 17.2xa0±xa03.65xa0pg/ml (pxa0<xa00.05). FRAP was increased to 1.41xa0±xa00.37xa0mM Fe2+ compared with control 1.19xa0±xa00.17xa0mM Fe2+; (pxa0=xa00.006).ConclusionsThis is the first study to demonstrate increased H2O2 in exhaled breath condensate in patients with localized breast malignancy and its relation with clinical severity.

Increased whole blood chemiluminescence in patients with chronic renal failure independent of hemodialysis treatment.

Abstract.Introduction:The luminol-enhanced whole blood chemiluminescence (LBCL) assay is a rapid assay for the measurement of reactive oxygen species (ROS) generation by circulating phagocytes. This study’s aim was to determine if patients on maintenance hemodialysis (HD) and non-dialyzed patients with chronic renal failure (CRF) have altered LBCL and if dialysis itself affects ROS production in the blood.Materials and Methods:Twenty-six HD patients, 11 non-dialyzed patients with CRF, and 20 gender- and age-matched healthy controls were studied. Resting (rCl) and 2xa0×xa0 10−5 M n-formyl-methionyl-leucyl-phenylalanine-stimulated LBCL (peak chemiluminescence: pCl, total light emission after agonist addition: tCl) calculated per 104 phagocytes present in the 3-μl blood samples were measured with a Bio-Orbit® 1251 luminometer at 37°C for 11xa0min.Results:Prior to the HD session, median rCL, pCL, and tCL were 1.5, 3.0, and 2.8 times higher in HD patients than in healthy controls (p<0.01) and tended to increase at the end of the session. Significant increases in tCl were observed at 30xa0min and 240xa0min (end) of HD (1023.5 vs. 1810.6 vs. 2006.8 arbitrary unitsxa0×xa0 s/104 phagocytes, n=9, p<0.05). Median pCl and tCl were 5.0 and 4.3 times higher in non-dialyzed patients with CRF than in healthy controls (p<0.001). However, no significant differences were found between pre- and post-HD LBCL of HD patients and the LBCL of non-dialyzed patients with renal failure.Conclusions:Blood from patients with renal failure generates elevated amounts of oxidants independently of HD treatment. This may add to the understanding of the nature of oxidative stress and suggests the need of anti-oxidant treatment in these patients.

TNF-α priming effect on polymorphonuclear leukocyte reactive oxygen species generation and adhesion molecule expression in hemodialyzed patients

Abstract.IntroductionThe study aimed to assess reactive oxygen species generation and the expressions of some surface antigens on polymorphonuclear leukocytes (PMNs) in patients on regular hemodialysis (HD) treatment.Materials and MethodsThe respiratory burst of PMNs was determined with luminol-dependent chemiluminescence (CL) in resting cells and following N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol 12-myristate 13-acetate (PMA), or opsonized zymosan (OZ) stimulation and expressed in arbitrary CL units times assay-time (aUxa0xa0×xa0xa0min). The expressions of CD11b/CD18, CD10, and CD13 receptors were determined with flow cytometry.ResultsBasal PMN CL was increased in HD patients to up to 1285xa0±xa0129 aUxa0xa0×xa0xa0min compared with 895xa0±xa088 aUxa0xa0×xa0xa0min in healthy controls (pxa0<xa00.05). The CL of unprimed PMNs increased after fMLP stimulation from 3085xa0±xa0746 to 4529xa0±xa0808 aUxa0xa0×xa0xa0min, and after OZ stimulation from 12945xa0±xa01296 to 14678xa0±xa01355 aUxa0xa0×xa0xa0min. PMA-stimulated CL of PMNs was similar to control values. The oxidative burst in PMNs from HD patients and healthy controls was similar in response to TNF-α alone. The CL of TNF-α-primed PMNs in HD patients was significantly lower than CL measured in healthy controls (pxa0<xa00.05). The expressions of CD10 and CD13 metalloproteinase receptors were also increased (pxa0<xa00.05). Although CD11b expression was significantly increased at rest and after fMLP stimulation, the expression of another β-integrin heterodimer compound, CD18, was not increased.ConclusionsThese results provide evidence that TNF-α priming of PMNs is down-regulated in HD patients despite constitutive up-regulation of resting cytotoxicity and enhanced expression of adhesion and metalloproteinase receptors.

Serum antioxidant capacity is preserved in peritoneal dialysis contrary to its robust depletion after hemodialysis and hemodiafiltration sessions.

Renal replacement therapy (RRT) may differentially affect systemic generation of reactive oxygen species and depletion of antioxidant pools of low molecular weight molecules and proteins. This study was designed to assess the magnitude of the impairment of serum total antioxidant capacity (TAC) in relation to different RRT modalities. The study included patients on continuous ambulatory peritoneal dialysis (CAPD, Nu2003=u200321), hemodialysis (HD, Nu2003=u200321), hemodiafiltration (HDF, Nu2003=u200320), and healthy controls (Nu2003=u200333). TAC was assessed by the ferric reducing ability of plasma (FRAP) and with the 2,2‐diphenyl‐1‐picryl‐hydrazyl (DPPH) assay. In CAPD patients, predialysis FRAP and DPPH were increased: 1.46u2003mM and 10.5% vs. control 1.19u2003mM and 7.2%, respectively (Pu2003<u20030.001 in each). In HD and HDF patients, the FRAP and DPPH were significantly increased before and lowered after the RRT session (Pu2003<u20030.05) if compared with healthy controls. During an HD session, FRAP was decreased from pre‐HD 1.71u2003±u20030.29u2003mM to post‐HD 0.85u2003±u20030.20u2003mM (Pu2003=u20030.0001). The decrease of FRAP was lower during HDF (Pu2003<u20030.05 vs. HD), it decreased from pre‐HDF 1.41u2003±u20030.43u2003mM to post‐HDF 0.87u2003±u20030.23u2003mM (Pu2003=u20030.0001 vs. pre‐HDF). The HD session decreased DPPH from the pre‐HD median 10.3%, interquartile range (IR) 9.3–12.0% to post‐HD 2.6% IR 2.3–3.1% (Pu2003<u20030.0001). The adjustment of either urate or bilirubin up to pre‐HD levels did not restore lowered post‐HD levels of TAC. TAC remains preserved in CAPD, whereas the robust depletion of TAC, lower after HDF than HD sessions, cannot be attributed solely to the washout of dialyzable compounds.

Cefodizime enhances phagocytosis and intracellular killing of Staphylococcus aureus but does not influence polymorphonuclear leukocytes response to fMLP stimulation.

The influence of Cefodizime (CDZ) on in vitro activity of polymorphonuclear leukocytes (PMNL) from healthy subjects was assessed. Preincubation with CDZ enhanced phagocytosis and intracellular killing of Staphylococcus aureus by PMNL. Contrary to numerous clinical reports, no significant effect of CDZ preincubation on PMNL response to n-formyl-methionyl-leucyl-phenylalanine was found with respect to intracellular calcium changes, degranulation, hydrogen peroxide production, and chemiluminescence. These results suggest that augmented microbicidal activity of PMNL is not related to the enhanced production of reactive oxygen species in healthy subjects.

Coronary artery atherosclerosis in patients with the initial and the early stage of chronic renal failure

The recent clinical data indicate that the initial and the early stages of chronic renal failure (CRF) may lead to increased incidence of cardiovascular complications and increased extent of coronary artery disease (CAD). This retrospective study was aimed to determine the effects of coexisting diabetes mellitus type 2 (DM-2) and the extent of atherosclerosis in coronary vessels in patients with mildly reduced kidney function (glomerular filtration rate GFR = 89–60 ml/min) and moderately reduced kidney function (GFR = 59–30 ml/min). The study patients included 53 subjects with creatinine concentration above 120 μmol/l as a cut-off level for the initial stage and compensated CRF. The distributions of coronary artery stenosis were also analysed with respect to DM-2 coexistence and levels of haemoglobin glikolised (HbA1c). The odds ratio of pathological changes in coronary arteries in patients with GFR = 44–30 ml/min, with respect to the number of affected vessels — only one, more than one or more than two — were 7.22, 4.90 and 3.55, respectively. In CRF patients with GFR = 60–89 ml/min the odds ratio of one, more than one and more than two vessels with stenosis and CAD was 1.93, 1.70 and 1.53, respectively. DM-2 was not related to the risk of significant coronary artery stenosis and did not enhance the pre-existing changes in the study setting. Our results demonstrate that the initial and the early stages of CRF were significant risk factors for coronary stenoses and for enhancing the pre-existing changes.

IL-2, IL-6 and IL-8 levels remain unaltered in the course of immunosuppressive therapy after renal transplantation

Cytokines regulate the immune reactions elicited by renal transplantation (RT). This study was designed to investigate the blood serum levels of IL-2, IL-6, IL-8 in 25 RT patients (10 female and 15 male, mean 5.4±2.7 yrs after RT) three times over a six-month period during standard immunosuppressive therapy with cyclosporine A, azathioprine and prednisolone. The levels of IL-2, IL-6 and IL-8 were tested with ELISA Quantikine Human Interleukin Immunoassay (R&D Systems, detection level 7,0.7 and 10 pg/cm3, respectively). There was no significant alternation of blood serum levels of IL-2, IL-6 and IL-8 in the study patients.