[Platelet activation in relapse idiopathic nephrotic syndrome in children]. / Aktywacja plytek krwi w zaostrzeniu idiopatycznego zespolu nerczycowego u dzieci.

The role that platelets play in pathogenesis and thromboembolic complications of the idiopathic nephrotic syndrome (INS) in children still remains unclear. The aim of the study was to analyse of platelet activation in whole blood during first 8 weeks of ins. Study group comprised 24 children with 34 relapses of INS by ISKDC (group A). Obtained results were compared to 16 healthy children (group B). We assessed activation by the count of platelet aggregates, microparticles and surface expression of selected markers--CD62P (P-selectin), CD42b (part of von Willebrand factor receptor) at the onset INS, after 2 weeks of therapy. We found the increased counts of platelet aggregates and microparticles at the onset of INS with a systematic decrease in following 2 weeks. Furthermore, expression of CD42b was significantly lower at the beginning of therapy. There were no clear correlation between markers of activation and biochemical parameters in the study group. According to these findings we conclude that increased activation of blood platelets is an independent risk factor of thromboembolic complication in the early stages of relapse of INS. The role of platelets in pathogenesis or induction of ins relapse remains the matter for further investigation.

Changes in the extrinsic and intrinsic coagulation pathways in humans after decompression following saturation diving.

We have investigated the effect of simulated saturation diving on the activation of intrinsic and extrinsic coagulation pathways. Thirty-one male divers divided into two groups were tested in decompression habitat LSH-200. The first group of 16 divers was subjected to hyperbaric exposure at pressure of 180 kPa with air as a breathing mixture, and the second group of 15 divers, exposed to a pressure of 400 kPa with a heliox breathing mixture (helium-oxygen mixture: pO2, 40 kPa; pN2, 40 kPa; pHe, 420 kPa). The concentrations of tissue factor, tissue factor pathway inhibitor, factors XII, X, VII, and I, prothrombin fragment F1 + 2, and thrombin-antithrombin complex as well as platelet count, prothrombin time, activated partial thromboplastin time, plasmin-antiplasmin complex (PAP) and D-dimers were measured. We did not detect activation of the extrinsic coagulation pathway after decompression. There was a statistically significant decrease in platelet counts and factor I, XII and X concentrations after air-diving, and a potent and statistically significant increase of PAP concentration in both groups of divers. We suggest that saturated air or heliox diving followed by decompression have little if any effect on thrombin generation. Saturated air diving, however, may induce a decrease in platelet count and factor XII concentration. The observed elevation of PAP concentrations in both groups of divers suggests possible activation of fibrinolysis. The exact effect of diving and decompression on fibrinolytic system has to be further investigated.

[Generation of reactive oxygen species by peripheral blood neutrophils in patients with pulmonary tuberculosis]. / Wytwarzanie reaktywnych form tlenu przez neutrofile krwi obwodowej chorych na gruzlice pluc.

Oxygen metabolism of neutrophils was measured using chemiluminescence method in patient with pulmonary tuberculosis. Patients were included into three groups. Group I--20 patients (10 men, 10 women) aged 24-74 years (mean 48.3 years) with pulmonary tuberculosis BK(+). Group II--20 patients (15 men, 5 women) aged 19-67 years (mean 45.1 years) with pulmonary tuberculosis BK(0). The control group consisted 16 clinically healthy persons (12 men, 4 women) aged 28-59 years (mean 42.5 years). Blood samples (5 ml) were collected for examination from cubital vein early morning before breakfast. A luminometer 1251 coupled with an IBM PC AT compatible computer was used for the measurement of chemiluminescence. The chemiluminescence of non-stimulated neutrophils and those stimulated by a receptor stimulus formyl-Met-Leu-Phe (fMLP) as well as an extrareceptor stimulus phorbol myristate acete (PMA) was measured. The results of our study demonstrated that chemiluminescence of non-stimulated as well as stimulated by fMLP and PMA neutrophils was lower in comparison to the values of chemiluminescence in the control group.

Spontaneous apoptosis of neutrophils in whole blood and its relation to apoptosis gene proteins.

Apoptosis of neutrophils limits their pro-inflammatory potential. We tested the ability of fresh and cultured whole blood neutrophils to undergo spontaneous apoptosis and expression of p53, Fas/Apo-1, bcl-2 protein in the cells using flow cytometry. Neutrophil apoptosis was estimated using Annexin V and propidium iodide binding and verified under light microscopy. The percentage of early and late apoptotic neutrophils in the blood samples increased significantly after 20 h culture from 12.3 +/- 14.2% and 4.3 +/- 4.2% to 39.5 +/- 14% and 15.3 +/- 9.6%, respectively. The majority of late apoptotic neutrophils had altered morphology in FSC/SSC dot plot compared to alive or early apoptotic neutrophils. Cultured neutrophils presented markedly lower expression of bcl-2 protein compared to fresh blood cells: 211 +/- 321 median of fluorescence intensity (MFI) and 787 +/- 1152 MFI, respectively. The increased percentage of late apoptotic cells after culture paralleled the increase in the Fas/Apo-1 expression and negatively correlated with bcl-2 expression. We noted intracellular expression of p53 protein in neutrophils, although the expression did not correlate neither to the percentage of the apoptotic neutrophils, nor to the Fas/Apo-1 or bcl-2 expression. Our results suggested that neutrophil apoptosis is gene regulated, moreover, we present a possibility to assess the neutrophil apoptosis and cellular expression of the proteins of apoptosis related genes in whole blood samples.

Does potassium dichromate induce apoptosis in lymphocytes?

The aim of this work was to investigate the possibility of apoptosis in the human peripheral blood lymphocytes after treatment with potassium dichromate (K2Cr2O7), a potential occupational carcinogenic and mutagenic agent. Lymphocytes were stimulated by phytohemagglutinin, cultured for 72 h and incubated with either 0.2 mM or 0.4 mM K2Cr2O7 for the last 24 h or 48 h of culture. The condensation and margination of chromatin with emerging 'half-moon' structure, characteristic of apoptosis were observed. Phosphatidylserine displaced from the inner to outer side of the cellular membrane in 54% of cells after a 48-h incubation with 0.4 mM K2Cr2O7 (annexin-V+/PI-); 39% of these cells were of late apoptotic--secondary necrotic form (annexin-V+/PI+). Following the agarose gel electrophoresis of DNA, a 'ladder pattern' typical of apoptosis, was found. The results of the present study demonstrate that K2Cr2O7 can induce in the human peripheral blood lymphocytes changes similar to apoptotic ones.

[Effect of trimetazidine on biological activity of neutrophils in patients with transient myocardial ischemia induced by exercise testing]. / Wplyw trimetazydyny na aktywnosc biologiczna neutrofilów u chorych z przemijajacym niedokrwieniem miesnia sercowego prowokowanym testem wysilkowym.

We estimated the effect of trimetazidine on biological activity of neutrophils in patients with exercise test--induced transient myocardial ischaemia. The study group comprised 16 patients (10 men and 6 women) aged 40-56 years (mean 48.2 years) with stable angina. Exercise test was performed on cycloer-gometer (Medicor, Hungary). Trimetazidine in a dose 3 x 20 mg/day was applied 24 hours prior to the exercise test. The control group consisted of 14 patients (9 men and 5 women) aged 37-59 years (mean 47.9 years), with stable angina, in whom exercise test was performed but the drug was not administered. Blood samples for examination were taken from basilic vein before the exercise test and 10 min afterwards. To evaluate the ability of neutrophils to aggregate the leukergy test of Fleck modified by Berliner and Aronson was applied. Oxidative metabolism of nonstimulated and stimulated by fMLP and PMA was measured using chemiluminescence method. It was shown that in patients with stable angina trimetazidine caused decrease in neutrophil aggregation. The drug did not inhibit neutrophil biological activity af-ter exercise test--induced myocardial ischaemia.

The effect of air and nitrox divings on platelet activation tested by flow cytometry.

BACKGROUND: Nitrogen (N2) microbubbles activate the blood platelets and coagulaltion system. HYPOTHESIS: Breathing nitrox rather than air may reduce the level of platelet activation associated with decompression. METHODS: We tested platelet counts and the expression of functional membrane molecules on platelets in 10 divers subjected to saturated compression in nitrox at 4 ATA and in 9 divers subjected to compression in air at 2.8 ATA. Blood samples were taken before and immediately after the test. We measured the percentages of microplatelets, platelet aggregates and platelets bearing the activation marker C-D62P, and bearing molecules forming receptors for fibrinogen (CD61) and for von Willebrand factor (CD42b) using flow cytometry and specific monoclonal antibodies. Symptoms for DCS were also evaluated. RESULTS: DCS symptoms were not noted in either the nitrox or air group. In both groups we observed a marked increase in the percentage of activated platelets bearing CD62P molecules and an enhanced number of microplatelets and a marked drop in the platelets count in the blood of (divers in the air group. CONCLUSION: In all divers we observed certain changes in the platelet system, nevertheless decompression in nitrox resulted in a lesser degree of platelet activation. Though this study cannot exclude platelet activation as an etiological factor in DCS, the findings suggest platelet activation can occur in the absence of observable sign of DCS. Thus, platelet activation may be too sensitive a marker to serve as a predictor of DCS.

[Comparison of platelet activation and procoagulant activity of blood cells in healthy pregnant women and EPH-gestosis patients]. / Porównanie wybranych parametrów koagulologicznych oraz stanu pobudzenia krwinek plytkowych u ciezarnych kobiet z ciaza fizjologiczna i powiklana gestoza EPH.

OBJECTIVES: EPH-gestosis is one of the most frequent complications of pregnancy, labour and puerperium. Despite its unknown ethiology, many authors suggest a vital role played by platelets as an ethiopathogenic factor. The aim of this study was to observe the level of platelets activation in EPH-gestosis subjects. STUDY DESIGN: Platelets activation level has been observed in 16 EPH-gestosis third semester pregnant women and 14 normotensive third semester pregnant controls. Platelets double-labeled with monoclonal antibodies and a flow cytometer have been used in assessment of platelet activation level. Anti-P-selectin (GMP 140- Granule Membrane Protein) has been used as a marker of the platelet released reaction. Fibrinogen, D-dimers, antithrombin and thrombin-antithrombin complexes levels have been analyzed. RESULTS: An increased platelet activation (p < 0.005) as well as the increased level of thrombin-antithrombin (p < 0.005) in pregnant EPH-gestosis women comparing to control group, has been observed in the study. CONCLUSIONS: A state of hypercoaguability and enhanced platelet activation in pregnant EPH-gestosis women have been observed.

Tumor necrosis factor-alpha binding by peripheral blood lymphocytes and polymorphonuclear neutrophils in patients with multiple sclerosis.

Tumor necrosis factor-alpha (TNF-alpha) in multiple sclerosis (MS) is responsible for peripheral blood leukocyte priming. The aim of this study was to evaluate fluorescein isothiocyanate (FITC)-labelled TNF binding ability by peripheral blood lymphocytes and polymorphonuclear neutrophils (PMNs) of MS patients, measured using flow cytometry (FACScan). Three groups of MS patients (total 34) were examined. Higher serum levels of TNF soluble receptors sp55 and sp75 were found in the MS patients during MS acute exacerbation (n = 10) and in chronic progressive forms (n = 11) as compared to MS remission (n = 13) and other neurological diseases (n = 14). Peripheral blood lymphocytes and PMNs of patients with acute exacerbation of MS bound TNF-FITC more effectively (p <0.01) as compared with the chronic progressive forms of MS, MS remission and other neurological diseases. The obtained results suggest a greater enhancement of TNF activity during MS acute exacerbation.

Expression of aminopeptidase N (APN) on peripheral blood mononuclear cells' surface as a marker of these cells' transendothelial migration properties in the course of multiple sclerosis.

CD13 Ag and CD11a, CD11b, CD18 molecule expression on peripheral blood mononuclear cells (PBMC) were studied as these cells' adherent or transendothelial migration properties in three different multiple sclerosis (MS) patients groups (total 38): with clinically active MS (acute exacerbation of MS and primary chronic progressive MS (CP-MS)) and with MS remission. The control group consisted of patients, suffering from other non-inflammatory neurological diseases (OND). The results of our study suggest that CD11a/CD18 molecules expression on PB lymphocytes, although higher on these cells' surface in the course of MS as compared to OND, does not differentiate clinical forms of MS. CD11a molecule expression on monocytes did not differ significantly in all tested MS patient groups in comparison to OND. Although the expression of CD11b/CD18 molecules on monocytes' surface shows their activation in the course of MS, it does not differentiate them either. However, CD13 Ag of APN expression on PBMC surface may be an immunological marker of MS clinical form. CD13 Ag expression may also be a sensitive marker of these cells' transendothelial migration properties.

Increased expression of neutral endopeptidase (NEP) and aminopeptidase N (APN) on peripheral blood mononuclear cells in patients with multiple sclerosis.

We studied CD10 Ag of neutral endopeptidase (NEP) and CD13 Ag of aminopeptidase N (APN) expression on peripheral blood mononuclear cells (PBMC) as cells activation markers and for their transendothelial migration properties in three groups of MS patients (total 58); with acute exacerbation of MS (n = 18), primary progressive MS (n = 17) and with MS remission (n = 23). The control group (OND) consisted of 24 patients, suffering from other noninflammatory neurological diseases. CD10 Ag and CD13 Ag expression on PBMC was higher in clinically active MS (acute exacerbation and progressive MS) compared to MS remission and OND groups. Our study suggests that CD10 Ag and CD13 Ag can be useful mononuclear cell activation markers in the course of MS. CD13 Ag expression on PBMC may be also the sensitive marker of these cells transendothelial migration properties.

[Increased percentage of "double phenotype" form T lymphocytes in blood of patients with multiple sclerosis]. / Zwiekszona wartosc odsetkowa "podwójnie dodatniej" formy fenotypowej limfocytów T we krwi chorych na SM.

We studied the percentage of double positive (CD4+CD8+) form of T-cells in a group (total 77) multiple sclerosis (MS) patients, measured by means of monoclonal antibodies anti-CD3, CD4/FITC, CD8/RPE and flow cytometry FACScan (Becton Dickinson). In our study we have shown that the percentage of the double positive T cells is higher (p < 0.05) in the peripheral blood of patients with acute exacerbation of MS (n = 21), and in the course of chronic progressive MS (n = 27) comparing to MS remission (n = 29) and other neurological diseases (n = 25) groups. In the study we have shown that the percentage of double positive T-cells in peripheral blood depends mainly on disease activity.

[TNF-alpha binding ability as a sign of peripheral blood neutrophils preactivation in the course of multiple sclerosis]. / Zdolnosc do wiazania czynnika martwicy nowotworu--alpha przez neutrofile krwi obwodowej chorych na stwardnienie rozsiane.

Usually neglected is the role of neutrophils in causing of immunological disturbances in patients with multiple sclerosis (MS). Nevertheless, it has been indicated over the recent years that these cells possess a sufficient potential to affect both immune response and inflammation. This potential may result in MS through the process of priming of these cells by proinflammatory cytokines like TNF. We studied TNF and its soluble receptors sp55 and sp75 serum levels and binding of fluorescein isothiocyanate (FITC)-stained TNF by neutrophils. We studied three different groups of MS patients: 10 patients in relapse of the disease, 13 in its remission, and 11 in its chronic progressive form (CP-MS). The control was provided by 14 neurological patients (OND) with non-inflammatory diseases. The performed studies showed higher TNF sp55 and sp75 soluble TNF receptors serum levels in the patients with relapse, comparing with other MS patients and OND. TNF binding by neutrophils of MS patients during relapse was also higher, than other MS patients and OND. These result suggest the preactivation of neutrophils in the relapse of MS.

Priming effect of met-enkephalin and beta-endorphin on chemiluminescence, chemotaxis and CD11b molecule expression on human neutrophils in vitro.

The opioid peptides are widely distributed throughout the body, and they are generated during stress and inflammatory reaction. Opioids are involved in the communication between the immune and neuroendocrine systems. In the present study we have investigated the ability of both met-enkephalin and beta-endorphin to stimulate and prime the human neutrophils for enhanced chemiluminescence (CL) and chemotaxis induced with fMLP, OZ or PMA. We have also tested the effect of beta-endorphin and met-enkephalin on CD11a, CD11b, CD18 and CD16 molecule expression on PMN in vitro. PMN from ten healthy donors were incubated in vitro with different concentrations of beta-endorphin or met-enkephalin, and the CL response was evaluated with luminometer. To assess the effect of opioid peptides on CD11a, CD11b, CD18 and CD16 molecule expression the whole blood samples were incubated with different concentrations of the opioids, then the white cells were labelled with respective PE-conjugated MoAb and evaluated by flow cytometry. We have shown that: (1) met-enkephalin and beta-endorphin at physiological concentrations relevant to that of in vivo (10(-8) and 10(-6) M) enhanced fMLP, PMA or OZ stimulated chemiluminescence and induced chemotactic response, (2) High concentrations of beta-endorphin (10(-3) M) or met-enkephalin (10(-5) M) decreased the CL response of PMN in vitro, (3) The opioid peptides at lower concentrations resulted in CD11b and CD18 molecule up-regulation on neutrophils. We may conclude that opioid peptides in physiological concentration are involved in neutrophil priming whereas in higher concentration exert immunosuppressive potency. Opioid peptides like inflammatory cytokines may prime the neutrophils inflammatory response.

Pentoxifylline decreases neutrophil respiratory bursts in patients with stable angina.

We estimated the effect of pentoxifylline (PTX) on the respiratory burst (examined by chemiluminescence method) of unprimed and primed neutrophils with tumor necrosis factor-alpha (TNF-alpha) 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-alpha primed neutrophils. We presume that administration of PTX in stable angina patients may have a beneficial effect.

[Increased expression of aminopeptidase N on lymphocytes in multiple sclerosis]. / Zwiekszona ekspresja aminopeptydazy N na limfocytach w przebiegu stwardnienia rozsianego.

Aminopeptidase N is an ectoenzyme. Its expression on lymphocytes is the effect of cell activation. We studied APN expression on peripheral blood lymphocytes of multiple sclerosis (MS) patients and in the control group of patients with other neurological diseases (OND). We observed increased APN expression on lymphocytes of MS patients during acute exacerbation and in the course of chronic progressive MS compared to MS remission and OND groups. No such differences were found in CD11a molecule of LFA-1 integrin expression on lymphocytes. We suppose that APN expression on lymphocyte surface can be a sensitive marker of these cell activation in the course of MS.

[The effect of pentoxifylline on oxidative metabolism in neutrophils primed with tumor necrosis factor alpha in patients with stable angina pectoris]. / Wplyw pentoksyfiliny na metabolizm tlenowy neutrofilów preaktywowanych przez czynnik martwicy nowotworów alfa u chorych ze stabilna dusznica bolesna.

In 14 patients with stable angina pectoris we examined the effect of pentoxifyline (PTX) on oxidative metabolism of TNF-alpha-priming neutrophils. The control group consisted of 21 patients with stable angina pectoris without pentoxifylline administration. Blood samples for examination were taken from basilic vein (peripheral blood) and coronary sinus immediately before PTCA procedure. In PTX-group was found the significant decrease in spontaneous CL of TNF-alpha-priming neutrophils from coronary sinus blood (1231.0 +/- 119.4 mV x min), in comparison to the control group (1374 +/- 124.4 mV x min). In PTX-group was found the significant decrease in fMLP stimulated CL of TNF-alpha-priming neutrophils from peripheral blood (4219.0 +/- 707.2 mV x min) and coronary sinus blood (4322.0 +/- 664.4 mV x min), in comparison to the control group (5248.0 +/- 595.8 mV x min and 4973.0 +/- 536.5 mV x min; respectively). There were no differences between both groups in PMA stimulated CL of TNF-alpha-priming neutrophils.

The immunoregulatory abilities of polymorphonuclear neutrophils in the course of multiple sclerosis.

The polymorphonuclear neutrophils (PMN) possess sufficient potential to affect both immune response and inflammation, however it has not been yet described in the course of multiple sclerosis (MS). We have studied binding of fluorescein isothiocyanate (FITC)- stained TNF-alpha by PMN, the expression of CD11a, CD11b, and CD18 molecules of beta2-integrines and the expression of CD10 (neutral endopeptidase-NEP) and of CD13 (aminopeptidase N; APN) antigens on PMN in three different groups of MS patients. The control group included neurological patients (OND) with noninflammatory diseases. The obtained results have proved that during MS exacerbation and in the course of chronic progressive MS, PMN reveal several forms of preactivation, including significantly higher stained-TNF-alpha binding, higher expression of CD11b and CD18, as well as CD10 and CD13 antigens, in comparison with MS remission or OND. We suggest that the increased expression of these molecules on PMN of MS patients in exacerbation of the disease and to a lower degree in the course of CP-MS is a result of PMN priming, and directly prove the PMN involvement in the disease pathogenesis.

Role of tyrosine kinase enzymes in TNF-alpha and IL-1 induced expression of ICAM-1 and VCAM-1 on human umbilical vein endothelial cells.

The aim of this study was to analyse the potential roles of protein kinase enzymes in tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) induced expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on human umbilical vein endothelial cells (HUVEC). The authors observed a marked increase in ICAM-1 and VCAM-1 expression on HUVEC stimulated for 24 h by TNF-alpha (10 ng/ml) or IL-1 (20 ng/ml). Pre-treatment of HUVEC for 30 min with protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A (10 micrograms/ml and 0.5 microgram/ml, respectively) before stimulation with IL-1 did not affect the expression of these molecules. Similar results were observed with respect to VCAM-1 expression on HUVEC stimulated by TNF-alpha. In contrast, pre-incubation of HUVEC with PTK inhibitors prior to the addition of TNF-alpha significantly enhanced subsequent expression of ICAM-1, although spontaneous expression of ICAM-1 on unstimulated HUVEC was unaffected. Western blot analysis demonstrated a significant increase in phosphorylated tyrosine protein levels in HUVEC stimulated by TNF-alpha, and significantly lower levels of these proteins in TNF-alpha stimulated HUVEC pre-treated with PTK inhibitors. These results demonstrate that IL-1 induced ICAM-1 and VCAM-1 expression does not result from activation of PTK-dependent pathways. In the case of TNF-alpha induced responses, the selective co-stimulatory effect of this cytokine in combination with PTK inhibitors on ICAM-1 expression suggests a complicated intracellular pathway of TNF-alpha induced ICAM-1 expression, possibly involving down-modulation of increases in ICAM-1 by PTK enzymes.

Myoinositol may be a factor in uremic immune deficiency.

In 10 chronic uremic patients on regular hemodialysis treatment and 10 healthy subjects in vitro PHA-induced peripheral blood mononuclear cell (PBMNC) and T-cell enriched lymphocyte proliferative responses were found to be impaired in the presence of myoinositol in the concentration generally observed in the blood serum of chronic uremic patients on regular hemodialysis treatment (600 mumol/l), while it remained unchanged in the presence of myoinositol in the concentration observed in normal blood serum (30 mumol/l). However, both myoinositol concentrations did not affect PMA-induced PBMNC and T-cell enriched lymphocyte proliferative responses, which suggests that inhibitory effect of the high myoinositol concentration on PHA-induced immune cell proliferation is cell membrane-related. In addition, myoinositol (600 mumol/l) significantly depressed CD3, CD4 and HLA-DR antigen expression on PHA-activated PBMNC surface in chronic uremic patients and healthy subjects, while CD8 antigen expression remained unaffected. The results seem to indicate that myoinositol, in the concentrations observed in uremic blood serum, may possibly share the responsibility for uremic immune deficiency.