Gerd Egger

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stimulated by polystyrene beads and Luminol-enhanced CL was determined in terms of peak height and peak time in freshly withdrawn, peripheral venous whole blood.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.

Basic fibroblast growth factor (BFGF) immunoreactivity as a possible link between head injury and impaired bone fracture healing

Healing of fractures of long bones or large joints is often accelerated in patients with severe traumatic brain injury (TBI). However, in these patients an early fracture healing is accompanied by hypertrophic callus formation or heterotopic ossifications which might even result in an ankylosis of the affected joints. It seems that enhanced osteogenesis in patients suffering from TBI could be caused by some humoral factors, since the sera of these patients strongly promote the growth of osteoblast cells in vitro. However, humoral growth promoting factors which could perhaps induce enhanced osteogenesis are not yet identified. Hence, the aim of this study was to analyse if basic fibroblast growth factor (bFGF) could be related to the phenomenon of enhanced osteogenesis, since bFGF stimulates the growth of osteoblasts in vitro and could be found both in the brain and the bone tissue. For that purpose the values of bFGF immunoreactivity were determined in the sera of patients with TBI and bone fractures (n = 8) as well as in the sera of patients with either TBI alone (n = 10) or bone fractures alone (n = 7), during a period of three months after injury. Quantification of the bFGF immunoreactivity was done using the ELISA based on monoclonal antibodies raised against the recombinant human bFGF. The bFGF immunoreactivity values obtained were also compared with the values determined in the sera of normal, healthy persons (n = 9). In the group of patients with bone fractures alone only a transient increase of bFGF immunoreactivity (threefold above the normal values) was observed in the second week after injury. A similar increase of the values of bFGF immunoreactivity was also determined in the sera of patients with TBI only, but it lasted longer (from the 1st until the 7th to 8th week after injury). In the case of patients with TBI and bone fractures a specific pattern of post-traumatic dynamic change of the values of serum bFGF immunoreactivity was observed. Namely, the increase of bFGF immunoreactivity (up to seven-fold above the normal values) was determined even during the first week after injury. Afterwards, periods of high values of bFGF immunoreactivity observed during the 2nd, 4th and the 7-10th weeks after injury were interrupted by sudden decreases even to the normal values (during the 3rd and the 5-6th week after injury).(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of the values of basic fibroblast growth factor determined by an immunoassay in the sera of patients with traumatic brain injury and enhanced osteogenesis and the effects of the same sera on the fibroblast growth in vitro.

In patients with severe traumatic brain injury, the early healing of fractures is accompanied by hypertrophic callus formation or heterotopic ossifications, which might even result in ankylosis of the affected joints. Analysis of the sera of patients with traumatic brain injury revealed post-traumatic dynamic changes of basic fibroblast growth factor immunoreactivity, similar to those observed during fracture healing associated with enhanced osteogenesis. The aim of this study was to determine whether such changes in basic fibroblast growth factor concentrations could be related to the phenomenon of enhanced osteogenesis. Basic fibroblast growth factor immunoreactivity was determined (using an IEMA kit) in the sera of patients with traumatic brain injury and bone fractures (n = 8) and in the sera of patients with either traumatic brain injury alone (n = 10) or bone fractures alone (n = 7), and the effects of these sera on L929 fibroblast growth were analysed in vitro. The results did not prove a causative relationship between the changes of basic fibroblast growth factor immunoreactivity and in vitro growth promoting effects of the sera. However, it is apparent that, in addition to changes in the growth-promoting activity and basic fibroblast growth factor concentration of serum, other as yet unknown post-traumatic changes can cause enhanced osteogenesis.

Migratory activity of blood polymorphnuclear leukocytes during juvenile rheumatoid arthritis, demonstrated with a new wholeblood membrane filter assay

Polymorphonuclear leukocyte (PMN) migration is measured in whole blood in a migration chamber consisting of a membrane filter (3-μm pores, 140μm thick) with an integrated chemoattractant depot (FMLP in solid form) attached to a plastic container. Control chambers lack FMLP (blanks). One test unit requires 300μl blood. Numbers and distribution of the PMN immigrants into the filters are determined microscopically. Altogether 26 measurements of PMN migration in five juvenile rheumatoid arthritis (JRA) patients with varying disease activity were compared with the reactions of a healthy control group (N=32). Correlations were calculated with conventional laboratory parameters (WBC, PLT, BSR, CRP, Hgb, serum Fe) and disease activity. In comparison with healthy controls, PMNs of JRA patients generally show a markedly increased penetration depth into the filters irrespective the presence of the chemoattractant or the disease activity. Increased migratory reactions to FMLP in comparison to blanks were found during high disease activity only. The PMN penetration depth correlates positively with the CRP, and reciprocally with the Hgb blood levels. The migration assay combines fast and simple processing with good preservation of the genuine PMN activation state.

Blood Polymorphonuclear Leukocyte Activation in Atherosclerosis: Effects of Aspirin

The aim of the study was to demonstrate an activation of polymorpho-nuclear leukocytes (PMNs) in chronic progressive atherosclerosis (ATH). A group of patients with ATH, and a group of ATH patients under aspirin (ASA) therapy were compared with control persons without atherosclerotic alterations (healthy controls). Each group comprised 15 male age-matched subjects. The following inflammatory parameters related to PMN activities were measured: the polymorphonuclear leukocyte (PMN) blood count; blood PMN migration and reactive oxygen species release in vitro; the blood levels of PMN elastase, malondialdehyde, antibodies to oxidized LDL and soluble ICAM-1. In ATH patients, the PMN blood counts and the share of blood PMNs migrating upon platelet activating factor and leukotriene B4 stimulation were significnatly above the values of the healthy controls, while the other parameters were not significantly altered. ASA treatment attenuated the inflammatory response and reduced the differences between ATH and the healthy controls. It can be concluded that, in patients with chronic progressive atherosclerosis, PMNs are involved in the inflammatory process underlying the disease.

Impaired blood polymorphonuclear leukocyte migration and infection risk in severe trauma

OBJECTIVES We investigated the association of impaired blood polymorphonuclear leukocyte (PMN) migration with the incidence of bacterial infections in patients with severe trauma. METHOD Twenty-six intensive-care patients with different injury severity scores were enrolled in a prospective study. PMN migration was measured daily using 300 microl fresh whole blood in a membrane filter assay. Migration was evaluated in an automated image analyzer that recorded numbers and distribution of the immigrant PMNs within a filter. The relevant parameter was the percentage of PMNs that migrated from the blood samples into the filters upon f-Met-Leu-Phe stimulation. RESULTS Nine patients developed posttraumatic infections verified microbiologically. These patients showed a reduced PMN migratory capacity in comparison with the 17 patients without infections. A migrating portion of six per cent or less at least three days in succession preceded infections by one to 19 days and indicated infection in eight true positive versus three false positive cases, and 14 true negative versus one false negative case, i.e. specificity was 82.3% and sensitivity 88.8%, p=0.0008. Trauma severity had no influence on PMN migration. CONCLUSIONS Trauma patients with impaired PMN migration are at risk for bacterial infections. Whole-blood migration tests can define the infection risk and thus may be useful predictive markers for infections.

Impaired Whole-Blood Polymorphonuclear Leukocyte Migration as a Possible Predictive Marker for Infections in Preterm Premature Rupture of Membranes

Objectives: Steroids, used in pretermpremature rupture of membranes (pPROM), to reduce the risk of morbidity and mortality of the preterm neonate, impair the maternal polymorphonuclear leukocyte (PMN)-based immune system. In spite of combination with antibiotics, prenatal and postnatal bacterial infections of mother and child are frequent. This pilot study focuses on the influence of steroids in pPROM on maternal PMN functional capacity and subsequent infections. Methods: After opting for expectant management, eight women with pPROM and no signs of infection were treated by steroids (betamethasone 5.7 mg, i.m. every 24 hours, for three days) and antibiotic therapy with either amoxicillin and clavulanic acid, piperacillin or ampicillin i.v. up to delivery. The conventional inflammation parameters of PMN blood count and C-reactive protein (CRP) were measured daily in parallel with PMN migratory capacity towards N-formyl-methionyl-leucyI-phenylalanine stimulation and under blank conditions, estimated by a whole blood membrane filter assay. Results: In all patients PMN migration decreased during the application of steroids. Three patients showed a decrease in PMN migration below critical values and in spite of antibiotic prophylaxis acute pyelonephritis developed 2–6 days later. PMN count and CRP were not predictive of maternal infection. Conclusion: Reduced PMN function, caused by steroid treatment in pPROM, is suggested to be a reason for serious bacterial infections in spite of antibiotic prophylaxis. PMN migration reflects individual PMN defensive capacity.

Blood polymorphonuclear leukocyte migratory activities during rheumatoid arthritis.

Blood polymorphonuclear leukocyte (PMN) migratory activity was investigated in adult rheumatoid arthritis (RA) patients and in healthy control subjects using fresh whole blood in a novel membrane filter assay. The PMNs migrated under FMLP stimulation and under blank control conditions (spontaneous migration). Essential evaluation criteria were the percentage of PMNs that migrated from the entire blood sample into the filters (TMI) and the penetration depth of the migrating cell bulk into the filters (DC). PMNs from healthy subjects penetrate deeper under FMLP stimulation than under blank control conditions. Migration depends on age and sex: the TMI decreases, while the DC and the reactivity towards FMLP increase with age. FMLP triggers a stronger DC reaction in females than in males. Compared with healthy subjects, patients with RA develop an increased PMN reaction, whereas FMLP inhibits migration in comparison with the blank controls. There is no correlation between disease activity estimated by joint functions and PMN migratory activity, while there are strong correlations between disease activity and the classical RA laboratory parameters WBC, platelets, BSR, CRP, hemoglobin and rheumatoid factor. PMNs therefore probably do not play a major role in joint injury. Gold therapy inhibits DC reactivity. PMN migration in RA differs markedly from the reactions in juvenile rheumatoid arthritis, where high disease activity is associated with high PMN migratory activity, and the correlations between classical laboratory parameters and disease activity follow other patterns than in RA.

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.