Heinrich Wokalek

Measurement of chemiluminescence in freshly drawn human blood

SummaryThe present investigations were undertaken to find out whether chemiluminescence measurements of stimulated granulocytes can be carried out in freshly drawn blood and — because of the ease of the method — be introduced into routine diagnostics.Blood was drawn from the cubital vein of healthy volunteers at various times and under various conditions. Subsequently the zymosan induced and luminol amplified chemiluminescence was recorded and analyzed. It could be demonstrated that variations existed between individuals which can, however, be minimized when photon counts obtained under standard conditions were related to the number of granulocytes present in the blood samples. It could be further demonstrated that also platelets are activated by zymosan as well and that they, contribute to the total chemiluminescence by a share of about 5%. Platelet chemiluminescence can effectively be suppressed by aspirin. Opsonising factors in plasma (presumably antibodies and/or complement) play a decisive role in the intensity and kinetics of blood chemiluminescence. Measurements of zymosan induced chemiluminescence in freshly drawn unfractionated and fractionated blood seem to be especially suited to monitor and analyze deviations and defects of the cellular and humoral defence mechanisms.ZusammenfassungDie vorliegenden Untersuchungen bilden die Grundlage, um die im Prinzip sehr einfache Messung der Chemilumineszenz stimulierter Granulocyten in frisch gewonnenem Vollblut durchzuführen und damit für die einfache Routine-Diagnostik anwendbar zu machen.Gesunden Freiwilligen wurde zu verschiedenen Zeiten und unter verschiedenen Bedingungen kubital Venenblut entnommen und anschließend die durch Zymosan induzierte und durch Luminol verstärkte Chemilumineszenz analysiert. Die Chemilumineszenz-Kurven verschiedener Individuen zeigten Variationen, die jedoch verringert werden, wenn die unter Standardbedingungen gemessenen Photonen auf die Zahl der im Blut vorhandenen Granulocyten bezogen werden. Es zeigte sich ferner, daß Blutplättchen ebenfalls durch Zymosan aktiviert werden und zu etwa 5% an der gesamten Chemilumineszenz beteiligt sind. Die Plättchen-Chemilumineszenz läßt sich indes durch Zusatz von Aspirin nahezu vollständig ausschalten. Von entscheidender Bedeutung für die Intensität und Kinetik der im Vollblut gemessenen Chemilumineszenz ist die Anwesenheit opsonierender Plasma-Faktoren (Antikörper und/oder Komplement).Die Messung der Chemilumineszenz im Vollblut erscheint uns geeignet, sowohl Abweichungen der zellulären wie der humoralen Abwehrfunktionen rasch und einfach zu erkennen.

Involvement of complement in psoriasis and atopic dermatitis, measurement of C3a and C5a, C3, C4 and C1 inactivator

SummaryNormal complement components and activation products were determined in the peripheral blood of 35 patients with atopic dermatitis (AD) and 24 patients with psoriasis at a mild to intermediate stage. None of the patients had received systemic or local steroid therapy 6 weeks prior to blood collection. Levels of C3, C4 and C1 inactivator (C1 INA) were determined in serum by radial immunodiffusion, whereas C3a and C5a levels were measured by radioimmunoassay. In comparison to healthy non-atopic controls, the levels of C3, C4 and C1 INA were found to be significantly increased in both diseases. No substantial differences were detected between patients with psoriasis vulgaris and psoriasis guttata, which suggests that the dissimilarities found were not due to preceding or concomitant infections. In AD, there was a tendency towards increased C3a levels, whereas in psoriasis, C3a levels were significantly increased. In both diseases, no measurable amounts of C5a could be detected. The results indicate that, in both AD and psoriasis, the complement participates in the inflammatory process. Elevated levels of C3a suggest that there is a continuous activation of the complement system leading to the generation of inflammatory mediators.

Time course of wound healing.

Wound healing is a special kind of inflammation. Undisturbed wound healing is subject to a fixed time schedule of biochemical and cellular events. It is virtually impossible to deal with the time course of wound healing without describing the cellular and non-cellular events involved. The activity and mode of cell action after injury are coordinated by spatial and chronological factors, as well as by different mediators and cell-cell interacting signals. During wound healing the sequence of different signals and message substances, such as mediators of inflammation, fulfill a key function in wound repair. The report describes the time course of healing and the control of cellular events by different mediators and cell interactions. Emphasis is placed on temporal aspects, including the various signals leading to typical cellular events in wound healing.

Influence of 13-cis retinoic acid on zymosan-induced chemiluminescence of granulocytes

SummaryThe effect of 13-cis retinoic acid (Ro-4-3780) on zymosan-induced chemiluminescence (CL) of freshly drawn human blood and isolated granulocytes has been examined in vitro. A marked enhancement was observed; optimal concentrations were 100 μg/ml in whole blood CL and 10 μg/ml in isolated granulocyte CL. It was found that already 5 μg/ml of 13-cis retinoic acid in isolated granulocytes showed a significant enhancement in zymosan-induced CL. The enhancement by 13-cis retinoic acid was completely inhibited by 10-5 M sodium azide, which is an inhibitor of myeloperoxidase activity. CL response was not enhanced by retinoic acid in mouse bone marrow originated cultured macrophages, which do not have myeloperoxidase activity. These findings strongly suggest that 13-cis retinoic acid might stimulate myeloperoxidase-dependent CL.ZusammenfassungDer Effekt von 13-cis-Retinsäure (RO-4-3780) auf die Zymosan-induzierte Chemilumineszenz (CL) wurde in vitro im Vollblut und an isolierten Granulocyten untersucht. Dabei wurde eine deutliche Verstärkung der CL im Vollblut (optimale Konzentration: 100 μg/ml) und bei isolierten Granulocyten (optimale Konzentration: 10 μg/ml) beobachtet. Bereits eine 13-cis-Retinsäure-Konzentration von 5 μg/ml führte zur einer signifikanten Verstärkung der Zymosan-induzierten CL an isolierten Granulocyten. Die durch 13-cis-Retinsäure bewirkte Verstärkung der CL ließ sich durch den Myeloperoxidasehemmer Natriumazid (NaN3) in einer Konzentration von 10-5 M komplett hemmen. Aus dem Knochenmark kultivierte Makrophagen der Maus, welche keine Myeloperoxidaseaktivität besitzen, zeigten keine Steigerung der CL nach 13-cis-Retinsäure-Behandlung. Diese Befunde sind ein deutlicher Hinweis dafür, daß 13-cis-Retinsäure die Myeloperoxidase-abhängige CL von Granulocyten stimuliert.

Modulation of granulocyte oxidative response by recombinant interferon α2 and γ

SummaryInterferon (IFN) has been described to influence various cellular functions. In this study we investigated whether the oxidative response of polymorphonuclear leukocytes (PMN) is also affected by IFN. In order to exclude the possible influence of impurities in IFN preparations, only recombinant human IFN α2 or γ were used. Lucigenin-dependent chemiluminescence (CL) of PMN was measured to assess the production of oxygen radicals. IFN γ at a concentration of more than 10 ng/ml elicited a minimal CL response in PMN. When PMN were incubated with IFN γ for 1 h and then stimulated with chemotactic peptide f-met-phe (FMP), zymosan-activated serum (ZAS), zymosan particles, or phorbol-myristate acetate (PMA), the CL response was increased as consequence of the generally enhanced oxidative metabolism. IFN α2 showed no such effect at any concentration tested. A 5-min pretreatment with IFN γ decreased the ZAS response but did not affect the reaction to the other stimuli. The possibility of a generation of IFN by PMN during the assay could be excluded as no IFN activity could be detected in an antiviral assay after stimulation of PMN for 6 h with PolyIxPolyC, LPS, ConA, C. parvum, PMA, zymosan, or FMP. The modulation of granulocyte activity by IFN γ may be important in the regulation of the anti-inflammatory response of PMN.

Stimulierbarkeit eingeschränkter Spermienmotilität und mucociliärer Clearance der Lunge durch Theophyllin-Ethylendiamin

SummaryThe influence of oral aminophylline (Phyllotemp ret., 2×225 mg) on the mucociliary clearance of the lung and on sperm motility was examined in 13 patients with asthenospermia vs. placebo because the anatomic structure of the cilia of the sperm cells and of the respiratory epithelium cells is similar. The mucociliary clearance was measured by means of radioactive tagged (99 mTc) monodisperse particles (erythrocytes from the patients). To attain a reproducible deposition pattern, the particles were inhaled during a slow inspiratory vital capacitiy maneuvre. The i.v. application of aminophylline (6 mg/kg body weight) to four normal subjects increased the mucociliary clearance in three of the four volunteers vs. placebo.The elimination rate of the particles increased under treatment with aminophylline from 29%±12% to 46%±19%, but only in seven of the 13 patients with asthenospermia. Sperm motility increased in all of the patients from 33%±12% to 54%±14%. No correlation was found between the increase in the mucociliary clearance and the sperm motility. The reasons for this difference will be discussed. Theophylline can be recommended for the treatment of male infertility in cases of asthenospermia.The influence of oral aminophylline (Phyllotemp ret., 2 X 225 mg) on the mucociliary clearance of the lung and on sperm motility was examined in 13 patients with asthenospermia vs. placebo because the anatomic structure of the cilia of the sperm cells and of the respiratory epithelium cells is similar. The mucociliary clearance was measured by means of radioactive tagged (99 mTc) monodisperse particles (erythrocytes from the patients). To attain a reproducible deposition pattern, the particles were inhaled during a slow inspiratory vital capacity maneuvre. The i.v. application of aminophylline (6 mg/kg body weight) to four normal subjects increased the mucociliary clearance in three of the four volunteers vs. placebo. The elimination rate of the particles increased under treatment with aminophylline from 29% +/- 12% to 46% +/- 19%, but only in seven of the 13 patients with asthenospermia. Sperm motility increased in all of the patients from 33% +/- 12% to 54% +/- 14%. No correlation was found between the increase in the mucociliary clearance and the sperm motility. The reasons for this difference will be discussed. Theophylline can be recommended for the treatment of male infertility in cases of asthenospermia.