Apmis

Mrowietz, Ulrich; Ternowitz, Thomas

doi: 10.1111/j.1699-0463.1987.tb00001.xpmid: 3577766

Using the cytoplasmic enzyme lactic dehydrogenase (LDH) as a marker, a method for routine measurements of highly purified monocyte chemotaxis is described. Standardization of this assay was carried out in order to achieve objective and reproducible results. The LDH‐method showed a good correlation with both the 51Cr‐chemotactic assay and the leading‐front technique. The sensititivity of the LDH‐method could be compared with the 51Cr‐assay and was significantly higher compared to the leading‐front technique. Being objective, inexpensive and requiring only a photometer as equipment, the LDH‐method was proved to be suitable for routine investigations of monocyte chemotaxis as a part of monocyte functional activity in laboratories engaged in leukocyte research.

Fomsgaard, Anders; Dinesen, Bo; Bæk, Leif

doi: 10.1111/j.1699-0463.1987.tb00002.xpmid: 3577769

To explore whether the intravenous administration of naturally occurring high‐titer human anti‐lipopolysaccharide IgG (anti‐LPS) is beneficial in the treatment of human septic shock, it is necessary to select blood donors according to anti‐LPS concentrations. The present paper describes an enzyme‐linked immuno‐adsorbent assay (ELISA) for the screening of blood donors to obtain such antibodies. Human specific IgG antibodies directed against smooth LPS from eleven different bacterial strains and species were quantitated. The ELISA is simple and uses only commercially available reagents. By optimizing assay conditions, a low unspecific background activity (OD below 0.010) was obtained, together with a large specific reading interval. 2021 human plasma samples from volunteer blood donors at six different blood‐banks in Denmark were screened by the ELISA. The median anti‐LPS antibody concentration was 9 mg/l. Five per cent of the donors had anti‐LPS concentrations above 40 mg/l (expected to be therapeutically useful). The age‐ and sex‐related distributions indicated that the highest prevalence of high anti‐LPS concentration was in males aged 40–49 years. It is concluded that specific anti‐LPS gammaglobulin based on naturally occurring anti‐LPS antibodies can be obtained in Denmark following the screening of blood‐donors, using the assay developed.

Hetland, G.; Eskeland, T.

doi: 10.1111/j.1699-0463.1987.tb00003.xpmid: 3554893

We conclude that the macrophages during cultivation produce the complement components of the classical pathway of complement, deposit complement components on EIgM and then phagocytose these cells via complement receptors. The conclusion is based on the following: EIgM, an activator of the classical pathway, are ingested when cultured serum‐free with mouse peritoneal macrophages. We found a significantly higher binding of labelled protein to EIgM than to E kept in macrophage cultures in the presence of tritiated leucine, showing that de novo synthesis of macrophage‐derived protein with affinity to EIgM takes place. A fraction of the bound protein is C3b and iC3b, since anti‐mouse C3 antibodies bound to the co‐cultured EIgM. Cycloheximide or anti‐Mac‐1 in the cultures inhibited macrophage attachment and uptake of EIgM. The phagocyte uptake of EIgM coated with complement by serum pretreatment was not inhibited by cycloheximide. This shows that the phagocytosis of the EIgM is dependent on erythrocyte‐bound complement proteins made by the macrophage.

Bjerve, K.S.; Espevik, T.; Kildahl‐Andersen, O.; Nissen‐Meyer, J.

doi: 10.1111/j.1699-0463.1987.tb00004.xpmid: 3577767

The effect of different fatty acids on tumour necrosis factor (TNF)‐induced cytolysis of TNF‐sensitive WEHI 164 murine fibrosarcoma cells has been determined. The saturated fatty acids palmitic (16:0) and stearic (18:0) acid strongly potentiated the cytolytic activity of TNF. They also had a slight cytotoxic effect in the absence of TNF. Contrary to this, the unsaturated fatty acids oleic (18:1), linoleic (18:2), alpha‐linolenic (18:3), arachidonic (20:4), and eicosapentaenoic (20:5) acid were neither cytotoxic by themselves, nor did they have any significant effect on the cytolytic activity of TNF. Addition of either of the unsaturated fatty acids 18:2 or 20:5 together with 18:0 eliminated the potentiating effect 18:0 by itself had on TNF‐induced cytolysis. A mixture of fatty acids resembling that found in cell phospholipids had no cytotoxic effect by itself, nor any effect on the cytolytic activity of TNF. The potentiating effect of saturated fatty acids on TNF thus seems to depend on their relative amount compared to the amount of unsaturated acids. The results indicate that TNF does not exert its cytotoxic effect simply by suppressing hydrolysis of triglyceride, thereby inducing an insufficient cellular supply of fatty acids.

Pekkola‐Heino, Kirsi; Viljanen, Matti K.; Ståhlberg, Tom H.; Granfors, Kaisa; Toivanen, Auli

doi: 10.1111/j.1699-0463.1987.tb00005.xpmid: 3554894

In order to obtain specific tools for studying the alterations of the immunochemical structure of Yersinia enterocolitica lipopolysaccharide in various conditions, we have produced monoclonal antibodies reacting with core and O‐polysaccharide chains of Yersinia enterocolitica O:3 LPS. Immunizations were made with whole bacterial cells and outer membrane preparation, respectively. Monoclonal antibody 2B5 reacted in enzyme immunoassay with purified core‐lipid A complex, and its binding was not inhibited by Polymyxin B, suggesting that the target determinant is in the outer core. 2B5 reconized 100% of all tested Y. enterocolitica O:3 strains (n=152) and reacted to some extent also with many other gram‐negative bacteria. In immunoblotting with 2B5, a band corresponding to core‐lipid A complex was visualized both with Y. enterocolitica. Brucella abortus and Haemophilus influenzae. In immunofluorescence assay, the only positive reaction was seen with Y. enterocolitica. Monoclonal antibody A6 reacted in enzyme immunoassay with purified O‐polysaccharide chains, recognized 100% of tested Y. enterocolitica O:3 strains, and showed no cross‐reactions with other bacteria. A typical ladder pattern was not seen in the immunoblotting analysis with A6. This suggests that the O‐chain of Y. enterocolitica O:3 may be different from those in other gram‐negative bacteria. These two antibodies will make it possible to study the structural variations of Yersinia enterocolitica LPS more precisely than described before, because of their fine specificity against important immunogenic components of LPS. They will also be useful in serology measuring the immune response against the target determinants of these antibodies.

Lehtonen, O.‐P. J.; Tenovuo, J.; Aaltonen, A. S.; Vilja, P.

doi: 10.1111/j.1699-0463.1987.tb00006.xpmid: 3577768

Serum and salivary total IgA, IgG and IgM as well as salivary innate non‐immunoglobulin antimicrobial factors (lysozyme, lactoferrin, salivary and leukocyte peroxidase systems) were measured in 13 children prone to recurrent respiratory infections and compared to their age‐matched healthy controls. Serum IgG and IgM levels were significantly elevated and salivary IgA remarkably low in infection‐prone children as compared to the controls. However, the levels of secretory piece‐bearing IgA were about the same in both groups. There were no significant differences between the two groups in serum IgA levels or in any of the non‐immunoglobulin factors. The results indicate that low salivary IgA is associated with recurrent respiratory infections.

Schenck, Karl; Michaelsen, Terje E.

doi: 10.1111/j.1699-0463.1987.tb00007.xpmid: 3604686

Serum IgG1, IgG2, IgG3 and IgG4 antibody levels directed against lipopolysaccharide (LPS) from Bacteroides gingivalis were measured in the sera from systemically healthy subjects with and without periodontitis. An enzyme‐linked immunosorbent assay was used that included coating of microtiter plates with LPS, and subsequent incubation with patient sera followed by mouse monoclonal subclass‐specific antibodies, biotinylated sheep anti‐mouse IgG and alkaline phosphatase conjugated to streptavidin. Anti‐LPS IgG antibodies were dominated by IgG2, and moderate amounts only of IgG1, IgG3 and IgG4 were found. The periodontitis patients had significantly higher anti‐LPS IgG1, IgG2 and IgG3 levels when compared to the subjects with healthy periodontium (p < 0.05, Mann‐Whitney test).

Ternowitz, Thomas; Herlin, Troels; Fogh, Karsten

doi: 10.1111/j.1699-0463.1987.tb00008.xpmid: 3037849

Comparable investigations of the chemotactic and chemokinetic responses of purified monocytes (MO) and polymorphonuclear leukocytes (PMN) to leukotriene B4 (LTB4) and N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) were made in this study. Using a sensitive and objective 51Cr‐chemotactic assay, it was shown that both MO and PMN showed a bell‐shaped response to LTB4 and FMLP, with a maximum response at 10‐8M for both drugs. For PMN, the maximal response elicited by LTB4 was similar in magnitude to that produced by FMLP, whereas the MO chemotaxis induced by 10‐8M FMLP was significantly higher than the response evoked by 10‐8M LTB4. For both cell types, LTB4 at low concentrations (< 10‐9M) gave rise to higher chemotactic responses than FMLP. Chemokinesis was differentiated from chemotaxis, using a checkerboard system. At concentrations < 10‐9M the LTB4‐evoked contribution of chemokinesis to the total migrational response was significantly higher than the chemokinetic contribution of FMLP. Preincubation with LTB4 produced only homologous chemotactic deactivation to subsequent LTB4 stimulation, whereas preincubation with FMLP resulted in diminished secondary response to both FMLP and LTB4. The degree of deactivation was dependent upon the dose of attractant used, with a LTB4 concentration of 10‐7M leading to about 40% and 25% deactivation of PMN and MO, respectively. Preincubation with 10‐7M FMLP led to about 50% and 32% suppression of the subsequent chemotactic response of PMN and MO, respectively.

Mandrup‐Poulsen, T.; Egeberg, J.; Nerup, J.; Bendtzen, K.; Nielsen, J. H.; Dinarello, C. A.

doi: 10.1111/j.1699-0463.1987.tb00009.xpmid: 3300162

Previous electron‐microscopic studies of isolated islets of Langerhans exposed to the monokine interleukin‐1 for 7 days have indicated that interleukin‐1 is cytotoxic to all islet cells. To study the time‐course and possible cellular specificity of interleukin‐1 cytotoxicity to islets exposed to interleukin‐1 for short time periods, isolated rat or human islets were incubated with or without 25 U/ml highly purified human interleukin‐1 for 24 h. Samples of rat islets were taken after 5 min, 30 min, 1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 h and samples of human islets after 5 min, 30 min and 24 h of incubation and examined by electron microscopy in a blinded fashion. Already after 30 min, accumulation of opaque intracytoplasmic bodies without apparent surrounding membranes, and autophagic vacuoles were seen in about 20% of the beta cells examined in rat islets exposed to interleukin‐1. After 16 h of incubation with interleukin‐1, more than 80% of rat beta cells showed signs of degeneration. Beta cell specific changes similar to those observed in rat islets exposed to IL‐1 for 30 min were seen in human islets exposed to IL‐1 for 24 h. The described changes were not observed in alpha cells in interleukin‐1‐treated rat or human islets, or in alpha and beta cells in control islets. Passing interleukin‐1 over columns containing Sepharose‐coupled anti‐interleukin‐1 antibody completely removed the beta cell cytotoxic action on rat islets. Thus, within minutes following exposure to interleukin‐1, beta cell specific ultrastructural changes were observed in isolated rat islets, whereas human islets required longer exposure to IL‐1 to display equivalent morphological abnormalities.

Heilmann, Carsten

doi: 10.1111/j.1699-0463.1987.tb00010.xpmid: 3037850

In order to study the regulation of the human B cell response to pneumococcal polysaccharides (PPS), anti‐PPS‐secreting cells (SC) and Ig‐SC were quantitated in unstimulated and EBV‐stimulated cultures of blood mononuclear cells (MNC) established before and one and two weeks after vaccination. In unstimulated cultures established one week after vaccination, significant numbers of anti‐PPS‐SC were detected; however, they were observed only in cultures depleted of T cells (mean: 62/106), suggesting the presence of T cells suppressing in vivo‐activated PPS‐specific B cells. In EBV‐stimulated cultures depleted of T cells, low numbers of anti‐PPS‐SC were observed before the vaccination, and the numbers of these cells increased significantly two weeks after the immunization (from 28 to 48/106). In contrast, the numbers of anti‐PPS‐SC and total Ig‐SC decreased in EBV‐stimulated cultures of unseparated MNC established after the vaccination, suggesting T cell‐mediated suppression. The findings were not explained by changes in the numbers of blood T helper (Leu 3+) or T suppressor (Leu 2+) cells following the vaccination. Possibly the vaccination induces the appearance in the blood of activated suppressor cells responsible for the suppression of B cells in unstimulated and EBV‐stimulated cultures.