Elisabeth M Kukovetz

Possible involvement of myeloperoxidase in lipid peroxidation

1. Exposure of liposomes to the MPO-H2O2-Cl- system results in oxidation of lipids. Malondialdehyde and 4-hydroxynonenal are formed. 2. Oxidation of liposomes by stimulated rat neutrophils, assessed by malondialdehyde formation, is inhibited by KCN. This indicates involvement of MPO in the process. 3. The MPO-H2O2 system oxidizes mildly LDL but in the presence of chloride a propagation phase, with a rapid increase of conjugated diene formation, was observed.

Influence of Age on the Release of Reactive Oxygen Species by Phagocytes as Measured by a Whole Blood Chemiluminescence Assay

Polymorphonuclear and mononuclear phagocytes play an important role in host defense, but may also cause tissue injury through excessive inflammation. Reactive oxygen species (ROS) are not only directly ore indirectly involved in a wide variety of clinical disorders, such as atherosclerosis, reperfusion injury, pulmonary toxicity and cancer, but they are also important in the aging process. This process is associated with increasing susceptibility to infection. In this study we investigated the influence of age and sex on phagocyte activation by means of a whole blood chemiluminescence (CL) assay. Circulating phagocyte activity was measured in 55 healthy volunteers (24 females, 31 males) aged from 6 to 92 years. Using an automated luminescence system, phagocytes were stimulated by polystyrene beads and Luminol-enhanced CL was determined in terms of peak height and peak time in freshly withdrawn, peripheral venous whole blood. An extremely significant positive correlation (p < 0.0001) between the maximum of light emission after stimulation and increasing age was found. This finding is true for the total population of blood phagocytes as well as for a single cell. In contrast the time of the appearance of the maximum of light emission showed an extremely significant inverse correlation (p < 0.0003) with increasing age. The influence of sex on the CL-parameters showed no significant difference between women and men. It is concluded that the increased susceptibility of circulating phagocytes to oxidative burst in elderly subjects may be the consequence of several biological events. Senescent cells express more and also have new antigens on their surfaces that trigger an autoimmune response. Cellular senescence appears earlier in old organisms. Therefore phagocytes in aging individuals may be increasingly involved in their scavenger tasks that grow with the catabolic bias in cell turnover. Moreover, atherosclerotic alterations in the intima and endothelial lesions are physiologic concomitants of age and may lead to a stimulation of circulating phagocytes.

PMN-related parameters for the monitoring of wound healing in traumatology.

SummaryIn the search for objective methods to monitor the course of wound healing, the proteinase PMN elastase (n=56 pat.), the lipid peroxidation product malondialdehyde (MDA) (n=18 pat.), and polymorphonuclear neutrophil granulocytes (PMN) migratory behaviour were measured [1, 6, 7, 11]. This “stimulated PMN-locomotion” was quantified by a new PMN migration filter assay (n=10 pat.) [2]. We determined the clinical course during “per primam (pp)” wound healing (group 1), “pp” wound healing with secondary inflammatory disease (group 2), manifestation of a bacterial wound infection during healing-“per secundam (ps)” (group 3) and manifest wound infection (“ps”) at the time of admission (group 4).In group 1 PMN elastase returned to normal values on the 10th postsurgical day. Median values in group 3 reflected a highly significant difference (p<0,01) on day 4 and 5 compared with group 1. In group 2 and 4 medians reflected consistent high values without reaching normal ranges throughout. MDA did not exceed the normal range in group 1, in group 3 low levels persisted, and in group 4 a recurring increase was noticed.The total migration index median (TMI) in Group I, which quantifies the percentage of stimulated PMN, reflected its highest value immediately post-surgically and dropped to the lowest on the 13th postsurgical day (decrease by 54%). The mean invasion depth (T/2), a parameter of PMN distribution, showed only slight variation with time. In a group 3-patient, T/2 reflected a maximal migratory stimulation on day 6, 4 days before clinical infection signs could be noticed; then it dropped to the lowest on day 10. This decrease probably reflects a PMN behavioural change from migration to phagocytosis [9].RésuméLa protéinase PMN élastase (n=56 malades), le produit de la peroxidation lipidique malon dialdehyde (MDA) (n=18) aussi bien que la migration des granulocytes neutrophiles polymorphonucléaires (PMN) ont été mesurées dans le cadre de la recherche de méthodes pour surveiller le cours de la cicatrisation. Cette “PMN Locomotion simulée” a été quantifiée par un nouvel essai pour la migration des PMN (n=10). Nous avons déterminé le cours clinique de la cicatrisation dans un groupe de cicatrisation “per primam” (pp) (groupe 1), “pp” cicatrisation et surinfection secondaire (groupe 2), surinfection bactérienne durant la cicatrisation “per secundam” (ps) (groupe 3) et infection initiale au moment de l’admission (groupe 4).Dans le groupe 1 l’élastase PMN est retournée à des valeurs normales au dixième jour après l’intervention. Les valeurs moyennes du groupe 3 ont montré une différence significative (p<0,01) le quatrième et cinquième jour par rapport au groupe 1. Dans les groupes 2 et 4 les moyennes ont montré des valeurs élevées permanentes sans atteindre des valeurs normales. MDA est resté dans les limites de la normale dans le groupe 1; dans le groupe 3 les valeurs sont restées basses et dans le groupe 4 on a pu constater un accroissement répété des valeurs.Dans la migration PMN (concernant le groupe 1), la moyenne de l’index total de migration (TMI), qui mesure le pourcentage de PMN stimulé, présente la valeur la plus élevée immédiatement après l’intervention pour tomber au plus bas le treizième jour après l’intervention (diminution de 54%). La profondeur moyenne d’invasion (T/2), un paramètre de distribution de PMN, a montré seulement une légère variation avec le temps. Avec un malade du groupe 3, T/2 a montré une stimulation migratoire maximale le sixième jour, 4 jours avant que des signes cliniques d’infection aient pu être remarqués; puis T/2 est tombé au plus bas le dixième jour. cette diminution de T/2 reflète probablement des conditions de PMN changeant de la migration à la phagocytose.

Polymorphonuclear leucocyte migration response in uneventful wound healing following trauma surgery

In a study of the clinical importance of polymorphonuclear granulocytes (PMN) for the monitoring of wound healing we investigated the postsurgical course of nine patients all of whom had undergone trauma surgery and had no wound complications. The “stimulated random PMN locomotion” was evaluated by a new migration filter device which preserves the cells in their genuine priming state, simulating in vivo conditions. The percentage of all activated PMN, expressed by the total migration index (TMI) reflected the highest median immediately after surgery (zmax = 30.1 %) and dropped to the lowest value on day 13 (zmax = 13.9%). The mean invasion depth (T/2) of the cells along the migration distance into the filter showed only slight variations over time. The neutrophil migration activity (NMA), described by T/2 and TMI, behaved in a similar way to TMI. In studying physiological healing, preliminary results indate that TMI, which expresses PMN activation, is an efficient tool in the postoperative monitoring of patients, and might in the future serve as a basis for an early warning system for wound healing complications.

The influence of trauma on changes in neutrophil granulocyte function assessed by an analysis of granulocyte migration

We examined the effects of trauma on polymorphonuclear leucocyte (PMN) migratory parameters and PMN elastase release, with the aim of tracing an acute inflammatory reaction from its very beginning to the phase of recovery. Fifteen patients who underwent monotrauma surgery, followed by uneventful healing, served as inflammation model. PMN activation was studied by measuring their readiness to migrate (TMI) and their penetration potency (DC) in a whole blood membrane filter device, in which a chemoattractant depot (FMLP) was integrated. Control chambers lacking FMLP provided parameters of the spontaneous migration. In healthy controls (n = 64), the numbers of invading PMNs decreased continuously from the outermost layer towards the interior of the filter device. FMLP did not influence the mobilization rate of PMNs immigrant from the blood into the filter, but those cells that did migrate penetrated deeper (P < 0.05). After trauma, the spontaneous and FMLP-stimulated DC was increased (P<0.05). Trauma also tended to inhibit PMN migratory activity episodically; depression of the unspecific immune function (low TMI values) was found on the 3rd (P<0.0001) and 12th (P<0.01) postsurgical days. There was no correlation between the migratory parameters and the inflammation parameter, PMN elastase release. Preliminary results indicate that analyses of PMN migratory parameters by a whole blood membrane filter assay could provide a valuable adjunct in monitoring trauma-associated immunologic changes.ZusammenfassungEs wurde die Trauma-bedingte Auswirkung auf Migrationsparameter neutrophiler Granulozyten (PMN) und auf die PMN-Elastase-Freisetzung untersucht, mit dem Ziel, eine akute Entzündungsreaktion von Anbeginn an bis zur Erholungsphase im Verlauf zu kontrollieren. 15 Patienten, die sich einem unfallchirurgischen Eingriff, gefolgt von blander Wundheilung, unterzogen, dienten als Entzündungsmodell. Die Erfassung des PMN-Aktivierungsgrads erfolgte im Vollblut durch Messung ihrer Migrationsbereitschaft (TMI) und ihres Eindringvermögens (DC) in ein Membranfiltersystem, welches ein Chemotaxindepot (FMLP) beinhaltete. Kontrollkammern waren FMLP-frei und charakterisierten die Spontanmigration. Bei der gesunden Kontrollgruppe (n=64) nahm die Zahl der ins Filtersystem eingedrungenen PMN kontinuierlich von der oberflächlichsten bis zur tiefsten Schicht ab. FMLP beeinflußte die Mobilisierungsrßte der vom Blut ins Filtersystem eingewanderten PMN nicht, aber jene Zellen, die wanderten, drangen tiefer vor (p < 0.05). Nach dem Tauma war die spontane und FMLP-stimulierte DC erhöht (p<0.05). Es war eine Tendenz, die PMN-Migrationsaktivität episodisch zu hemmen, bemerkbar. Eine Depression der unspezifischen Immunfunktion (niedrige TMI-Werte) fanden sich am 3. (p < 0.0001) und 12. postoperativen Tag (p < 0.01). Es bestand keine Korrelation zwischen den Migrationsparametern und dem Entzündungsmarker PMN-Elastase. Vorläufige Ergebnisse deuten darauf hin, daß die Analyse von PMN-Migrationsparametern im Vollblut-membranfilterassay ein wertvolles Attribut in der Verlaufskontrolle Trauma-assoziierter immunologischer Veränderungen repräsentieren könnte.

Neutrophil Migration Activity (NMA) — A new migration test for the monitoring of wound healing in traumatology?

SummaryA new PMN migration test was introduced in traumatology to measure the acute, inflammatory response after surgery in a patient with uncomplicated wound healing and in a case of acute, early postoperative infection. Polymorphonuclear neutrophil granulocyte (PMN) — behaviour was studied by quantifying the neutrophil migration activity (NMA), which combines PMN-stimulation and PMN-distribution in a migration filter device simulating “in vivo”-conditions. While in uncomplicated wound healing the NMA was within the normal range, in an acute, early infection migratory stimulation reached a maximum on the 5th postsurgical day followed by a decrease during the following days. This decrease is probably an expression of an increased PMN-turnover and excessive phagocytosis. PMN-behaviour changing from migration to phagocytosis is regarded as an indicator of wound healing complications before clinical symptoms occur.RésuméUn nouveau test de migration PMN a été introduit en traumatologie afin de mesurer la réaction inflammmatoire aiguë après une intervention chirurgicale chez un patient présentant une cicatrisation normale et chez un patient présentant une infection post opératoire aiguë immédiate. L’état des granulocytes neutrophiles polymorphonucléaires (PMN) ont été étudiés, en quantifiant l’activité neutrophile de la migration (NMA), qui décrit la stimulation PMN aussi bien que la distribution, dans un filtre à migration simulant les conditions in vivo. Tandis que lors de la cicatrisation normale la NMA est dans les limites de la normale, elle présente un maximum de stimulation migratoire au cinquième jour d’une infection aiguë avec décroissance progressive ultérieure. Cette diminution est probablement l’expression d’un PMN turn over augmenté et d’une phagocytose excessive. On considère que la condition PMN qui change de la migration à la phagocytose est un indicateur des complications associèes à la cicatrisation avant l’apparition des symptomes cliniques.