G. Piasecka

Increased serum concentrations of conjugated diens and malondialdehyde in patients with pulmonary tuberculosis.

During pulmonary inflammation increased amounts of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) are produced as a consequence of phagocyte respiratory burst. One of the manifestation of these free radical-mediated processes is lipid peroxidation (LP). The aim of our study was to assess the concentration of lipid peroxidation products (LPPs), conjugated diens (CD) and malondialdehyde (MDA), in patients with active TB. Forty-two patients were enrolled into the study. Half (group I) had advanced TB and were sputum smear-positive. The remainder (group II) had only small radiographical changes and were sputum smear-negative. Serum concentrations of CD and MDA were measured at days 0, 7, 14 and 28 in group I and day 0 in group II. We found that in all patients with active TB CCD (1.0 +/- 0.05A233) and CMDA (2.01 +/- 0.16 nmol dl-1) were significantly elevated compared to healthy controls (0.67 +/- 0.03A233 and 1.36 +/- 0.08 nmol dl-1, respectively) (P < 0.001). The highest levels of LPPs were in patients with advanced TB. These concentrations were stable during the first month of anti-tuberculous therapy. Our data indicated that, as in bacterial pneumonia, LPPs were enhanced in active TB. The levels of LPPs depended on the form of the disease as they were higher in subjects with advanced disease than in those with only small radiographical changes. Further studies are needed to assess the role of antioxidants as adjuvant therapy in patients with pulmonary TB.

Effect of bacterial lipopolysaccharide on the content of lipid peroxidation products in lungs and other organs of mice

The influence of lipopolysaccharide fromEscherichia coli (LPS, 17 mg/kg body weight) on the lipid peroxidation process in organs of mice was studied. The content of conjugated dienes (CD), lipid peroxides (LP), malondialdehyde (MDA) (all three lipid peroxidation by-products), peroxidase (PO) activity and wet-to-dry weight ratio in lungs, heart, spleen, kidneys and liver were determined 1.5 h after intravenous injection of LPS. Animals observed at this time-point had reduced activity and decreased body temperature by about 2°C, however, all analysed organs did not reveal any changes of wet-to-dry weight ratio comparing to organs from mice injected with sterile, pyrogen free 0,9% NaCl. Only extracts from heart and lungs showed significant increase in the tissue level of at least two lipid peroxidation products. The heart content of CD, MDA, and LP was about 1.5-, 1.3-, and 2.4-fold higher than in control group. In lungs CD and MDA increased 3.3- and 1.3-times but in spleen only content of LP was elevated. In these organs the suppression of PO activity was also observed. Liver and kidneys did not reveal any convincing enhancement of lipid peroxidation process and alterations of PO activity. Since free radical reactions are involved in lipid peroxidation process and inactivation of PO these results suggest that heart, lungs and spleen are the organs mostly exposed to oxidative stress during the first 1.5 h after single injection of LPS in mice.

Comparison of PAF- and fMLP-induced [Ca2+]i transients in human polymorphonuclear leukocytes

Changes of [Ca2+]i in human polymorphonuclear leukocytes (PMNL) were studied. PMNL suspension was activated three times every 5 min with 10(-7) M PAF and fMLP. Both PAF and fMLP, induced three consecutive [Ca2+]i transients in PMNL suspended in medium with 1 mM Ca2+. The first Ca2+ response was a result of Ca2+ release from internal stores and the extracellular Ca2+ influx, while the second and third responses were completely dependent on Ca2+ influx from extracellular space. The contribution of Ca2+ from intracellular stores to the first PAF-induced Ca2+ response was about 1.4-fold lower in comparison with the first fMLP induced Ca2+ response (27 +/- 1 vs 37 +/- 6% (p < 0.05). Previous addition of PAF enhanced 3-fold (p < 0.001) the PMNL response to fMLP while cells pretreated with fMLP failed to increase their [Ca2+]i after challenge with PAF. PMNL from 40% of donors did not respond to PAF in the presence of 100 nM Ca2+. However, the cells responding to PAF as the cells treated with fMLP or cyclopiazonic acid released almost the entire Ca2+ from intracellular stores after challenge. Subtraction of mean [Ca2+]i transients in the presence of 100 nM Ca2+ from that obtained in medium with 1 mM Ca2+ showed that, in PMNL stimulated with PAF in contrast to the cells treated with fMLP, the onset of Ca2+ influx from extracellular space precedes Ca2+ release from intracellular stores. These results suggest that PAF-induced Ca2+ influx from extracellular space is at least partly independent of Ca2+ release from intracellular stores.

Changes of intracellular free calcium concentration in human polymorphonuclear leukocytes after repeated stimulations with N-formyl-methionyl-leucyl-phenylalanine.

A rapid transient rise in the intracellular free calcium concentration ( Ca2+]i) is an important step in human polymorphonuclear leukocytes (PMNL) activation. This can be caused by many inflammatory mediators and has been implicated in the regulation of various cellular reactions. In this study we investigated the changes of [Ca2+]i in human PMNL activated three times with 10(-7)M n-formyl-methionyl-leucyl-phenylalanine (FMLP). PMNL in the presence of 1 mM Ca2+ were able to respond to three consecutive stimulations with FMLP. The first Ca2+ response was the highest one and was a result of Ca2+ release from internal stores (which was responsible for about 30% of maximal increment in [Ca2+]i) and the extracellular Ca2+ influx. Experiments with PMNL suspended in a medium containing 100 nM Ca2+ and pretreated with 1 nM Ni2+ (an inorganic calcium channel blocker) revealed that the second and third response is completely dependent on the extracellular Ca2+ influx. Changes of the time interval between stimulations had no influence on the occurrence of extracellular Ca2+ influx related to second addition of FMLP. Elongation of the time interval up to 30 min did not restore the release of Ca2+ from internal stores. It indicates the occurrence of dissociation of Ca2+ release from intracellular stores and extracellular Ca2+ influx during the second and third PMNL response to FMLP.

Effect of cytochalasin B on intracellular free calcium concentration in human polymorphnuclear leukocytes after repeated stimulation withn-formyl-methionyl-leucyl-phenylalanine

SummaryCytochalasin B can influence various functions of human polymorphonuclear leukocytes, including chemotaxis, lysosomal enzyme release, and reactive oxygen species generation. In this study we investigated the effect of cytochalasin B on the increase in intracellular free calcium concentration after three consecutive additions of 10−7 MN-formyl-methionyl-leucyl-phenylalanine. The interval between stimulations was 5 min. Intracellular free calcium concentration was monitored using the fluorencent calcium indicator FURA-2AM. Cytochalasin B (3.3 μg/ml) added 60 s before the cell stimulation enhanced all three polymorphonuclear leukocyte calcium responses by increasing theN-formyl-methionyl-leucylphenylalanine-induced calcium influx from the extracellular space. Cytochalasin B increased the peak intracellular free calcium concentration and elevated the plateau phase level, but had no influence on its shape. In addition, pretreatment with cytochalasin B of polymorphonuclear leukocytes suspended in low calcium medium restored their capacity to respond to a third stimulation withN-formyl-methiolnyl-leucyl-phenylalanini. Finally, in resting cells cytochalasin B caused a moderate increase in intracellular free calcium concentration which was independent of extracellular calcium.

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.

Effect of substance P and its precursor α-protachykinin on intracellular free calcium concentration in human polymorphonuclear leukocytes

The increase in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation. The undecapeptide substance P can influence various functions of human polymorphonuclear leukocytes, including chemotaxis, phagocytosis, and respiratory burst. In this study we investigated the ability of low-concentration (that can occur in vivo) substance P (10−7 M) and its precursor α-protachykinin (3×10−7 M) to increase the intracellular free calcium concentration in human polymorphonuclear leukocytes. Cells isolated from ten healthy donors were incubated with substance P or α-protachykinin in 1 mM calcium medium for 5 min and the intracellular free calcium concentration was monitored using the fluorescent calcium indicator Fura-2am. Polymorphonuclear leukocytes from 40% of donors responded to both agonists. The substance P- and α-protachykinin-induced increase in intracellular free calcium concentration was 59±13 nM and 58±12 nM and the extracellular calcium influx contributed to 87±8% and 54±8% of the calcium response, respectively. α-Protachykinin released almost all the calcium from intracellular stores, while substance P mobilized only 24±5% of this calcium pool. Finally, cells that responded to a single challenge with substance P and α-protachykinin were able to increase their intracellular free calcium concentration in response to each of three consecutive stimulations with these agonists. This may be an additional mechanism by which substance P and its precursor modify the function of human polymorphonuclear leukocytes.

Polymorphonuclear leukocytes from asthmatics release more calcium from intracellular stores and have enhanced calcium increase after stimulation withN-formyl-methionyl-leucyl-phenylalanine

Polymorphonuclear leukocytes isolated from peripheral blood of asthmatics appear to be primed to release more reactive oxygen species than cells of healthy subjects. The enhanced agonist-induced rise in the intracellular free calcium concentration may be responsible for this increased respiratory burst. To test this hypothesis we studied theN-formyl-methionyl-leucyl-phenylalanine- and cyclopiazonic acid-(an inhibitor of Ca2+-ATPase of intracellular calcium stores) induced calcium increase in the polymorphonuclear leukocytes of 28 subjects (16 with moderate asthma, 69.6% ±8.3% predicted normal peak expiratory flow and 12 normal controls) using a fluorescent probe Fura-2AM at 100 nM and 1 mM extracellular calcium concentrations. In 1 mM calcium, theN- formylmethionyl-leucyl-phenylalanine-induced calcium increase was 1.7-fold higher in asthmatics than in healthy subjects. Similarly, the contribution of calcium from intracellular stores to the calcium response toN-formyl-methionyl-leucyl-phenylalanine was higher in asthmatics (55% ±14% vs. 39%±14%, P<0.01). The pool of calcium released from intracellular stores byN-formyl-methionyl-leucyl-phenylalanine and cyclopiazonic acid was 2.3- and 2.2-fold larger than in control cells. There was a correlation between maximal intracellular calcium concentration related toN-formyl-methionyl-leucyl-phenylalanine-induced calcium release from intracellular stores and forced expiratory volume in 1 s expressed as percentage predicted and reversibility in asthmatics (r=0.63,r=−0.53,P<0.05). In conclusion, polymorphonuclear leukocytes of asthmatics exhibit an altered calcium response that is mainly dependent on increased calcium release from intracellular stores.

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.