Zbigniew Baj

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.

The Influence of Hyperlipidemia on Platelet Activity Markers in Patients after Ischemic Stroke

Background: To investigate the relationship between hyperlipidemia and platelet activation markers – platelet and soluble P-selectin (sP-selectin), and platelet-derived microparticles (PDMPs) – in patients after ischemic stroke. Methods: 41 patients after ischemic stroke (>3 months) confirmed by CT were divided into 2 groups: with hyperlipidemia (HL, n = 21) and normolipidemia (NL, n = 20). Twenty healthy subjects served as controls. CD62P-positive platelets and PDMPs in whole blood were analyzed by the use of a flow cytometer and anti-CD61 and anti-CD62P monoclonal antibodies. Platelets were activated by thrombin (0.08 units). The level of sP-selectin in serum was measured by ELISA. Results: We observed a significantly higher CD62P expression and percentage of CD62P-positive resting and thrombin-activated platelets in the HL as compared to the NL group. The sP-selectin concentration was also significantly higher in HL than NL subjects (p < 0.05). Moreover, we observed a significantly higher percentage of PDMPs in patients after stroke (NL: p < 0.05; HL: p = 0.005) in comparison with the control group. Conclusions: Patients after stroke present symptoms of platelet hyperreactivity. HL in the patients may be a risk factor for vascular events due to the increase in platelet activation.

Platelet reactivity and mean platelet volume as risk markers of thrombogenesis in atrial fibrillation

Atrial fibrillation (AF) is associated with increased risk of thromboembolic complications. One of the markers of the increased risk of hypercoagulable state is platelet hyperreactivity. The aim of the study was to assess impact of arrhythmia on platelet reactivity. METHODS The study included 36 (mean age 48,3; range 21-60) male patients with lone atrial fibrillation, with exclusion of concomitant diseases known to trigger hypercoagulable state. The AF patients underwent cardioversion to restore sinus rhythm and were subsequently under observation for 1month. Echocardiography, ECG and blood collection was performed before cardioversion (T0) and 4weeks after successful cardioversion (T1). During the study period patients have been contacted and examined every week and 24h ECG monitoring was performed. Platelet reactivity was assessed based on changes of CD62 and CD42b expression on platelet surface after stimulation with thrombin. Also changes in MPV were assessed. RESULTS In all patients sinus rhythm was maintained at the end of the study period, however in 14 patients recurrences of AF were observed, confirmed by 24h ECG monitoring (atrial fibrillation recurrence group - AFR) and 22 patients maintained sinus rhythm throughout the whole study period (SR group). Mean fluorescence intensity (MFI) of CD62 on thrombin stimulated platelets decreased significantly 4weeks after electrical cardioversion as compared to T0 (48.04±22.42 vs 41.47±16.03; p<0.01). Also MFI of CD42b on thrombin stimulated platelets decreased significantly 4weeks after electrical cardioversion as compared to T0 (22.16±10.82 vs 12.06±5.99; p<0.0001). Platelets reactivity estimated by CD 62 expression in SR group decreased significantly after 4weeks observation (58.01±15.26 vs 46.57±13.44; p<0.001) opposite to AFR group 35.66±21.87 vs 34.54±16.4; p-ns). Moreover there were significant differences between basal reactivity during AF between SR and AFR groups (58.01±15.26 vs 35.66±21.87; p-0.01). MFI of CD42b on thrombin stimulated platelets decreased significantly both in AFR and SR groups (22.05±11.36 vs 13.8±6.03; p<0.001 and 21.87±14.18 vs 10.04±5.09; p<0005). MPV decreased significantly 4weeks after electrical cardioversion as compared to T0 (8.81±0.19 vs 8.42±0.14; p<0.0001). CONCLUSION The changes of platelet reactivity to thrombin observed after restoration of sinus rhythm in patients prove that arrhythmia intrinsically leads to increased reactivity of platelets.

Effect of sinus rhythm restoration on platelet function in patients with lone atrial fibrillation.

Atrial fibrillation (AF) is associated with increased risk of thrombo- embolic complications. The aim of thestudywasto assessif arrhythmia, independent of other risk factors leads to increased platelet activation. The study involved 34 (mean age 50 +/� 9.03, range 21-59) male patients with lone persistent atrial fibrillation. The exclusion criteria were: age N60, coronary artery disease, left ventricular dysfunction (ejection fraction EF b 40%), congenital and acquired heart defects, artificial heart valve, diabetes, thyroid disease, inflammatory diseases, cancer, renal disease, and active smoking. The exclusion criteria precluded more than 95.4% of patients with AF hospitalized in our Department within the last 6 years. The AF patients underwent cardioversion to restore sinus rhythm and remained subsequently under observation for 1 month. Echocardi- ography, ECG and blood collection was performed before cardioversion (T0) and 4 weeks after successful cardioversion (T1). During the study period, patients were contacted and examined weekly along with 24-hour ECG monitoring. In all patients sinus rhythm was maintained at the end of the study period, however in 12 patients recurrence of AF was observed, confirmed by 24-hour ECG monitoring (atrial fibrillation recurrence group — AFR). In 10 patients the episodes of arrhythmia were asymp- tomatic, while only 2 patients complained of arrhythmia symptoms. In 22 patients no recurrence of AF in 24-hour ECG monitoring was observed (sinus rhythm group — SR). Parameters of resting platelets collected from peripheral blood activation was measured using flow cytometry. Platelet activation was assessed by expression of p-selectin (CD62) on platelets (CD61 positive cells). The platelet aggregate number was presented as a percentage of CD61+ blood elements bigger than platelets. The platelet derived microparticles (PDMPs) were assessed based on FSC histogram profile as CD61+ particles smaller than platelets. The leukocyte-platelet aggregates were detected based on coexpression of CD11b and CD62

Pentoxifylline decreases neutrophil respiratory bursts in patients with stable angina.

Abstract— We estimated the effect of pentoxifylline (PTX) on the respiratory burst (examined by chemiluminescence method) of unprimed and primed neutrophils with tumor necrosis factor‐α (TNF‐α) in patients with stable angina pectoris. Chemiluminescence of non‐stimulated as well as formyl‐methionyl‐leucyl‐phenylalanine (fMLP) and phorbol myristate acetate (PMA) stimulated neutrophils was measured. We studied 45 patients with stable angina subjected to percutaneous transluminal coronary angioplasty (PTCA) procedure, who were randomly divided into two groups. The study group consisted of 24 patients who were administered pentoxifylline orally, and the control group consisted of 21 patients without pentoxifylline administration. Blood samples for examination were collected from the coronary sinus and peripheral vein just before the PTCA procedure. Pentoxifylline decreased the respiratory burst of non‐stimulated and fMLP‐stimulated neutrophils without affecting the chemiluminescence of PMA stimulated neutrophils. Moreover, pentoxifylline diminished the chemiluminescence non‐stimulated and stimulated by fMLP but not by PMA of TNF‐α primed neutrophils. We presume that administration of PTX in stable angina patients may have a beneficial effect.

Impact of anemia treatment with methoxy polyethylene glycol-epoetin beta on polymorphonuclear cells apoptosis in predialysis patients with chronic kidney disease

BACKGROUND Some data in literature indicate increased apoptosis of polymorphonuclear cells (PMNs) in chronic kidney disease (CKD), what seems to be connected with anemia. Erythropoiesis-stimulating agents, used in anemia treatment in CKD may affect cells apoptosis. Aim of this study was to investigate impact of anemia treatment with methoxy polyethylene glycol-epoetin beta (CERA) on PMNs apoptosis in predialysis patients with CKD. METHODS Percentage of early and late apoptotic PMNs was measured by flow cytometry based on annexin V and propidium iodide binding. CD90 (Fas), CD95L (FasL), CD16 and CD11b expression on PMNs were evaluated by flow cytometry after incubation with respective monoclonal antibody. RESULTS Percentage of PMNs in early and late apoptosis in CKD patients before CERA treatment was significantly higher to control group, which was accompanied by significantly higher Fas and Fas-L expression and significantly lower expression of CD16. CERA treatment downregulated significantly percentage of early, apoptotic PMNs but percentage of late apoptotic cells did not change and was still significantly higher to control group. In all investigated groups we observed a significant negative correlation between hemoglobin concentration and percentage of apoptotic PMNs, as well as Fas and FasL expression and significant positive correlation between Hb and CD16 expression. CONCLUSIONS Our results indicate that PMNs apoptosis is increased in predialysis patients with CKD and anemia treatment with CERA may diminish readiness of PMNs to undergo apoptosis. This antiapoptotic impact of anemia treatment with CERA seems to concern early apoptotic PMNs before they undergo to late, irreversible stage of apoptosis.

Platelet Reactivity in Patients With Stroke and Hyperlipidemia, GPIbα Assessment

The aim of this study was to assess platelet reactivity in patients after ischemic stroke and to investigate the influence of hyperlipidemia (HL) on platelet activity markers. A total of 41 patients after ischemic stroke were divided into the following 2 groups: patients with HL and patients with normolipidemia. Expression of CD42b on resting, thrombin-activated blood platelets, and fibrinogen level was assessed. The CD42b-positive platelets were analyzed using the flow cytometer, anti-CD61, and anti-CD42b monoclonal antibodies. The results confirmed increased platelet reactivity to thrombin in all patients after ischemic stroke manifested by significantly lower CD42b expression and percentage of CD42b(+) platelets after activation by thrombin. The influence of HL on the expression of CD42b on resting and thrombin-activated platelets was not found. However, increased level of fibrinogen but no influence of HL on fibrinogen concentration was observed in patients after ischemic stroke. Increased susceptibility to platelet agonists was found in patients after ischemic stroke in the convalescent phase.

Does the Selenium (SE) Level and Se-Dependent Enzyme Activity in Blood Plasma Correlate with Human Lymphocyte Subpopulations and Function?

Recent literature data on the effects of Se on subpopulations of T lymphocytes, on autologous mixed lymphocyte reaction (AMLR) and on natural killer (NK) cell cytotoxicity are limited or poorly defined. In healthy volunteers we have estimated se levels, glutathione peroxidase (GSH-Px) activity and lipid peroxide levels in human plasma and simultaneously, the subpopulations of T lymphocytes, proliferation in AMLR, and activity of NK cells. We found a significantly positive correlation between the selenium level and GSH-Px activity. The proliferative response in AMLR significantly correlated with plasma selenium levels but not with GSH-Px activity. NK cytotoxicity, subpopulations of T lymphocytes, and lipid peroxide levels did not correlate with both selenium concentration and GSH-Px activity. We suppose that the effect of Se on the proliferation of suppressor T lymphocytes (Ts) in AMLR is not mediated through GSH-Px activity and fluctuations of Se concentration within a physiological range in healthy persons do not affect NK cytotoxicity.

sP- and sE-selectin in stroke patients with metabolic disorders

BACKGROUND Activation of platelets and endothelial cells plays an important role in the pathogenesis of atherosclerosis and thrombotic disorders. The aim of our study was to assess the relationship between the metabolic disorders and markers of platelet activity and vascular injury in patient with acute ischemic stroke. MATERIAL AND METHODS The study group consisted of 84 patients with acute non-lacunar ischemic stroke divided into four subgroups with: (1) normolipidemia and normoglycemia, (2) normolipidemia and hyperglycemia, (3) hyperlipidemia and normoglycemia, (4) hyperlipidemia and hyperglycemia. 21 healthy subjects served as controls. We analyzed the concentration of adhesion molecules sP-selectin and sE-selectin in serum collected from all studied groups using ELISA method. RESULTS We observed significantly higher sE- and sP-selectin concentration in patients with hyperglycemia and hyperlipidemia compared to both control subjects and patients with normolipidemia and normoglycemia. We did not observe additional effect of comorbid hyperlipidemia and hyperglycemia on studied markers. Soluble E- and P-selectin concentration correlated positively with LDL, TC and HbA1c level in all stroke patients. CONCLUSION Soluble E- and P-selectin, blood markers of vascular injury and platelet activation, could be useful in the assessment of atherothrombotic properties of hyperglycemia and hyperlipidemia in stroke patients.

Platelet reactivity and mean platelet volume as new biomarkers in risk stratification in patient with atrial fibrillation

Thank you for very valuable comments to our study. We completely agree that there is still lack of methods of precise estimation of thromboembolic complications (TE) risk, especially in the group of CHA2DS2-VASc score 1. It is striking that these patients according to studies on Asian population have higher risk over 2% as compared to European studies [1,2]. According to our studies [3,4] even patients with lone atrial fibrillation have increased platelet reactivity which reflects procoagulable state. Thuswe agreewith Authors thatwe should search for more precise, reliable, easy and useful scores which include laboratory test to minimalize thromboembolic complications. Platelet activation and reactivity measured by flow cytometry could be additional marker, but in our opinion, in everyday clinical practice may be problematic due to several issues: short time to examination (optimal