Binding of Mucin by E. coli from Human Gut.

Cells of E. coli isolates from the gut of healthy volunteers (N=5) and patients with Crohn's disease (N=5) and laboratory E. coli strain DH5α bound mucin in vitro in similar amounts ranging from 0.02 to 0.12 mg/mg of bacterial dry weight. Binding was evaluated by the decrease in optical absorption of mucin solution at 214 nm after incubation with bacteria. Detailed analysis of mucin binding by one of isolates showed that during incubation of 0.09 mg/ml bacteria in 0.15 M NaCl containing 0.1 mg/ml mucin at 25oC, maximum binding was reached in 30 min, while in the presence of 14 mM α-methyl mannoside, mucin binding decreased by 46% (p<0.05). Confocal microscopy revealed intensive binding of FITC-labeled mucin to the surface of a small number of bacterial cells. Mucin binding did not significantly affect zeta potential of bacteria and their energetic status assessed by ATP content; at the same time, ATP content in the extracellular environment slightly increased.

Evaluation of ATP content in hair bulbs in human scalp.

The content of ATP in scalp hair bulbs in humans was measured in the hair roots from 15 healthy volunteers. Light and electron microscopy confirmed the presence of outer and an inner root sheaths in the root of pulled out anagen hair. Incubation of samples in buffer solution led to extraction of ATP, which was measured by the chemiluminescent method. Mechanic disintegration of hair bulbs and their freezing-defrosting did not increase ATP output. The results of microscopy indicated that ATP extraction procedure was associated with separation of the outer radical sheath from the inner one without impairing the structure of the inner sheath. The mean content of ATP was 12 ± 2 pmol per bulb. The use of pulled out hair bulbs for ATP measurements simplified the procedure as involved no surgical removal of follicles.

[Human serum albumin modified under oxidative/halogenative stress enhances luminol-dependent chemiluminescence of human neutrophils].

It is shown that human serum albumin, previously treated with HOCl (HSA-Cl), enhances luminol-dependent chemiluminescence of neutrophils activated by phorbol-12-myristate-13-acetate (PMA). The enzyme-linked immunosorbent assay revealed that addition of HSA-Cl to neutrophils promotes exocytosis of myeloperoxidase. Inhibitor of myeloperoxidase--4-aminobenzoic acid hydrazide, without any effect on lucigenin-dependent chemiluminescence of neutrophils stimulated with PMA, effectively suppressed luminol-dependent chemiluminescence (IC50 = 20 microM) under the same conditions. The transfer of the cells from medium with HSA-Cl and myeloperoxidase to fresh medium abolished an increase in PMA-induced luminol-dependent chemiluminescence, but not the ability of neutrophils to respond to re-addition of HSA-Cl. A direct and significant (r = 0.75, p) correlation was observed between the intensity of PMA stimulated neutrophil chemiluminescence response and myeloperoxidase activity in the cell-free media after chemiluminescence measurements. These results suggest the involvement of myeloperoxidase in the increase of neutrophil PMA-stimulated chemiluminescence response in the presence of HSA-Cl. A significant positive correlation was found between myeloperoxidase activity in blood plasma of children with severe burns and the enhancing effects of albumin fraction of the same plasma on luminol-dependent chemiluminescence of PMA-stimulated donor neutrophils. These results support a hypothesis that proteins modified in reactions involving myeloperoxidase under oxidative/halogenative stress, stimulate neutrophils, leading to exocytosis of myeloperoxidase, a key element of halogenative stress, and to closing a "vicious circle" of neutrophil activation at the inflammatory site.

Activity of antioxidant enzymes in the wound in patients with deep burns.

Analysis of wound discharge in children with deep burns over 3 weeks after the injury revealed gradual increase in catalase activity. The increase in activities of myeloperoxidase, glutathione-S-transferase, and catalase was maximum in patients with the most severe burns. Local complications were observed during the period of maximum myeloperoxidase activity, while the beginning of epithelialization was associated with its reduction. Analysis of wound impressions confirms long-term persistence of neutrophils in the wound.

Comparative study of cytokine content in the plasma and wound exudate from children with severe burns.

The content of 27 cytokines was measured in blood plasma from 19 children with severe uncomplicated burns (group 1) and complicated burns (septic toxemia, toxemia, and pneumonia; group 2). Before surgical treatment (day 4 (+/-2) after burn), significant differences were found in the concentrations of interleukin-1 receptor antagonist, interleukin-6, interleukin-8, interleukin-10, tumor necrosis factor-alpha, interferon-gamma, MCP-1, and granulocyte colony-stimulating factor. Cytokine concentration in group 2 patients was much higher than in group 1 patients and healthy children. The concentrations of interleukin-6, interleukin-8, and MCP-1 in group 1 patients significantly surpassed the normal level. Cytokine concentration in the plasma and wound exudates and myeloperoxidase activity in wound exudates from 4 patients of group 2 were measured over 18 days after burn. The inflammatory response was characterized by an increase in the content of interleukin-1beta, interleukin-8, MCP-1, tumor necrosis factor-alpha, MIP-1alpha, and granulocyte-macrophage colony-stimulating factor in the wound (as compared to that in the plasma). Activity of myeloperoxidase in all patients was shown to correlate with the amount of MIP-1alpha (r=0.47), tumor necrosis factor-alpha (r=0.47), and granulocyte-macrophage colony-stimulating factor (r=0.55, p<0.05). Interleukin-8 concentration was beyond the limits of calibration. No correlation was found between the concentration of any of 27 cytokines in blood plasma and exudate. Our results indicate that during active surgical treatment, the wound serves as the source of inflammatory cytokines. Cytokines play a role in the systemic response and increase the degree of local inflammation, which modulates the number and activity of wound neutrophils.

Activity of antioxidant enzymes in the skin during surgical wounds.

Full-thickness skin wounds (460 mm(2)) in rats were associated with increased blood chemiluminescence and neutrophil infiltration of the wound tissue and surrounding skin (recorded by myeloperoxidase activity). Activities of glutathione peroxidase and glutathione S-transferase in the skin and wound tissue increased on days 4 and 8. A correlation was revealed between activities of these enzymes and myeloperoxidase activity. Activities of myeloperoxidase and catalase increased in patient's skin excised during plastic surgeries of more than 2.5 h duration.

Protective effect of complex antioxidant preparation containing vitamins and amino acids in rats with burn trauma complicated by endotoxemia.

Burn trauma increased blood chemiluminescence, while lipopolysaccharide in a dose of 1 mg/kg potentiated this effect, activated LPO, and decreased plasma antioxidant activity. In erythrocytes, superoxide dismutase activity increased, while activity of peroxide-utilizing enzymes decreased. Myeloperoxidase content increased in the lungs and epidermis. The preparation of alpha-tocopherol, selenium aspartate, and ubiquinone abolished the effect of lipopolysaccharide, but did not modulate the increase in chemiluminescence under the influence of this agent.

Effect of endotoxemia on skin antioxidant enzymes under experimental conditions.

Intraperitoneal injection of bacterial lipopolysaccharide in a dose of 1 mg/kg was followed by prestimulation of whole blood leukocytes in rats. Activities of peroxide- and lipoperoxide-utilizing antioxidant enzymes glutathione peroxidase, glutathione S-transferase, and catalase increased 1 day after lipopolysaccharide administration, while the content of malonic dialdehyde in the skin remained unchanged.

[Examination of the local antioxidative system of the eye in experimental corneal burn injury and the prospects for pharmacological correction of its parameters].

The present paper deals with the study of the efficiency of oral use of the antioxidative drug Immugen (a complex of alpha-tocopherol, oubichinone, selenium aspartate, methionine, and soyabean phospholipids) on a rabbit model of severe alkaline-induced corneal burn. The investigations have indicated that addition of Immugen to the rabbit feed exerts a significant positive effect on the parameters of the local antioxidative system of the eye and causes an increase in the activity of superoxide dismutase and catalase, and, on day 14, in antioxidative activity. The early experimental periods were marked by a slight rise in the frequency of deep corneal ulcerations. Moreover, the long-term clinical effect of use of Immugen appears as a significant increase in the area of the transparency-preserving affected cornea. The findings suggest that the antioxidants can show their optimal effect in the complex therapy for burn processes, including the use of proteinase inhibitors.

Wound-healing effect of papaya-based preparation in experimental thermal trauma.

Treatment with the phytopreparation from papaya accelerated wound healing and reduced the severity of local inflammation in rats with burn wounds. The effect of this phytopreparation can be related to an increase in the effectiveness of intracellular bacterial killing by tissue phagocytes due to the inhibition of bacterial catalase. Antioxidant activity of the preparation decreases the risk of oxidative damage to tissues.

Preventive and therapeutic effect of complex antioxidant preparation in rats with burn trauma.

The production of blood radicals and activity of superoxide dismutase in erythrocytes increased in rats with contact burn trauma (20%). In animals with burn trauma antioxidant activity of the plasma was much lower, while myeloperoxidase content in the lung tissue and epidermis was higher than in control rats. The complex of antioxidants (Immudzhen) inhibited radical generation at the peak of inflammation (day 4), increased antioxidant activity of the plasma, and normalized myeloperoxidase content in the lung tissue.

Direct enzymatic reduction of lucigenin decreases lucigenin-amplified chemiluminescence produced by superoxide ion.

The interaction of superoxide ion with lucigenin produces chemiluminescence (CL), which is widely used for the detection of this radical anion. However, in many biological systems lucigenin may be directly reduced to its semiquinone by some enzymes. We found that if the direct reduction of lucigenin takes place, it decreases superoxide production due to the competition with one-electron reduction of dioxygen to superoxide ion. Comparison of two methods of superoxide detection (lucigenin-amplified CL and cytochrome c reduction) showed that there are excellent correlations between the results obtained by the two methods. Hence, lucigenin-amplified CL remains a sensitive and reliable assay of superoxide detection.

Enhancement of antioxidant and anti-inflammatory activities of bioflavonoid rutin by complexation with transition metals.

The antioxidant and anti-inflammatory activities of two transition metal complexes of bioflavonoid rutin, Fe(rut)Cl(3) and Cu(rut)Cl(2), were studied. It was found that Cu(rut)Cl(2) was a highly efficient in vitro and ex vivo free radical scavenger that sharply decreased (by 2-30 times compared to the parent rutin): oxygen radical production by xanthine oxidase, rat liver microsomes, and rat peritoneal macrophages; the formation of thiobarbituric acid-reactive products in microsomal lipid peroxidation; and the generation of oxygen radicals by broncho-alveolar cells from bleomycin-treated rats. The copper-rutin complex was also a superior inhibitor of inflammatory and fibrotic processes (characterized by such parameters as macrophage/neutrophil ratio, wet lung weight, total protein content, and hydroxyproline concentration) in the bleomycin-treated rats. The antioxidant activity of Fe(rut)Cl(3) was much lower and in some cases approached that of rutin. Fe(rut)Cl(3) also stimulated to some degree spontaneous oxygen radical production by macrophages. We suggested that the superior antioxidant and anti-inflammatory activity of the copper-rutin complex is a consequence of its acquiring the additional superoxide-dismuting copper center. The inhibitory activity of Fe(rut)Cl(3) was lower, probably due to the partial reduction into Fe(rut)Cl(2) in the presence of biological reductants; however, similarly to the copper-rutin complex, this complex efficiently suppressed lung edema.