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Biomedical subjects

U Hadding

Publications and source records attributed to U Hadding.

At least 55 records · Page 3Linked to original sources
PubMed1988

In vivo effects of C3a on neutrophils and its contribution to inflammatory lung processes in a guinea-pig model.

C3a, when injected intravenously in guinea-pigs, caused a rapid drop of circulating neutrophils and platelets. The neutropenia was reversible and followed by a neutrophilia, which reached about 200% of baseline values. Upon challenge with octa- and hexapeptide, mimicking the C-terminal sequence of C3a, neutrophils and platelets reacted in the same manner. The hexapeptide-desArg (pentapeptide without the C-terminal arginine of hexapeptide) induced no neutropenia but a significant neutrophilia. Likewise, when injected in animals with a genetic deficiency or dysfunction of the C3a-receptor, the hexapeptide caused no drop of the neutrophils, but a neutrophilia, indicating that both neutrophil reactions are mediated by different mechanisms. With the octapeptide in vivo dose-response studies were performed. Despite maximal doses of octapeptide about 40% of the neutrophils remained in circulation, indicating that some but not all PMNs are susceptible to C3a. By pretreating the animals with an inhibitor of the serum carboxypeptidase N (SCPN-Inh) the C3a-induced neutropenia could be significantly augmented. But intravenous application of the inhibitor itself caused a 20-40% reduction of neutrophils during the first hour after injection, followed by a neutrophilia. In histological studies the timecourse of neutrophil sequestration in the lung was established, showing that the initial high neutrophil content of the lung lasted for at least 1 h and declined thereafter. Structural derangements could not be detected. These observations stress the importance of C3a besides C5a as an important mediator of inflammatory processes in species, where the C3a-receptor is present on inflammatory cells such as granulocytes.

3-Mercaptopropionic Acid↗
PubMed1987

Inhibition of interleukin 3 function by a fragment of the third component of complement.

A C3d-like (C3d-1) fragment of 33 kDa was isolated and its biological activity studied. The fragment was generated from guinea pig C3b by porcine pancreas kallikrein and purified by fast protein liquid chromatography. The C3d-like fragment inhibited interleukin (IL) 2-dependent T lymphocyte proliferation. The suppressive activity of the described C3d-1 fragment was not restricted to lymphocytes as targets but inhibited in addition the proliferation of a nonlymphocyte mast cell line which was strictly IL3-dependent in its proliferative capacity. Kinetic studies implied early stages of cellular proliferation to be influenced. Furthermore, the C3d-1 fragment was not only an inhibitor of cellular proliferation but was also a potent inducer of leukocytosis.

Animals↗
PubMed1987

Suppressive effects of C3b on monocyte-dependent T cell proliferation.

The effect of C3b treatment of human monocytes on secondary antigen-dependent T cell response was studied. When antigen-specific T cell blasts were cultivated together with C3b-treated monocytes the proliferative response was inhibited in a dose-dependent fashion. This suppressive effect was specific for C3b because heat-inactivated C3b or buffer alone had no influence on T cell proliferation. In part, this suppressive effect is mediated through a C3b-induced decreased expression of class II antigens on the surface of treated monocytes, but another suppressive mechanism exists because the C3b pretreatment of monocytes also led to an inhibition of the proliferative response in a class II antigen-independent T cell proliferation system. In addition to the C3b data, our finding that treatment of monocytes with C3d resulted in a lower T cell proliferation, while C3c has no effect, suggested that C3d, which could be generated from C3b in the culture, may induce the second inhibitory mechanism.

Cells, Cultured↗
PubMed1987

Influence of lysophospholipids and PAF on the oxidative burst of PMNL.

Lysophosphatidylcholine (LC), platelet activating factor (PAF) and its precursor lysophosphatidalcholine (LP) enhance O-2-release by polymorphonuclear leucocytes (PMNL) triggered by PMA whereas lysophospholipids with other polar headgroups fail to do so. The generation of these lysophosphatidylcholine-like molecules appears to represent an essential step in the activation of the oxidative burst of the PMNL triggered by PMA since inhibition of phospholipase A2 (PLA2) by p-bromophenacylbromide (BB) or mepacrine results in an inhibition of the O-2 release. This inhibition seems to be due to the reduced generation of the phospholipids studied as it could be reversed by LP. In addition, stimulation of the oxidative burst of the PMNL by the chemotactic stimuli, N-formyl-methionyl-leucylphenylalanine (FMLP), and the complement fragment C5a could also be significantly enhanced by LP as shown by chemiluminescence. However, the response to the phagocytic stimulus, opsonized zymosan (Zx), is not affected by LP. These data provide evidence for the participation of phospholipid metabolism in the initiation of the oxidative burst of PMNL induced by the soluble monomeric stimuli PMA, FMLP and C5a.

Acetophenones↗
PubMed1987

Purification of two high molecular weight toxins of Clostridium difficile which are antigenically related.

Two Cl. difficile toxins were isolated from cultures of Cl. difficile strain VPI 10463. A purification procedure to prepare homogenous Cl. difficile toxins is given. This procedure allows purification of high molecular weight toxins A and B without using immunaffinity chromatography. The main step of the purification is the separation of a partially purified toxin preparation over a FPLC-Mono Q column by anion exchange chromatography. The experimental conditions for a rechromatography were determined to prepare the two major toxic activities as homogenous high molecular weight proteins. Our toxin A has a molecular weight (Mr) of ca. 300 kDa and an IP of 4.7. The Mr of our toxin B is ca. 250 kDa, the isoelectric focusing gives rise to two bands one at 4.7 and the other at 4.8. The two bands represent charge isomers as have been described for other bacterial toxins. Both toxins differ in cytotoxicity testing by a factor of 1000 but have the same activity when tested in vivo. Toxin specific monoclonal antibodies (mabs) were elicited by separate immunization of mice either with toxin A or toxin B, respectively. All of our mabs cross react with pure toxin A and toxin B when tested by ELISA or Western Blotting. Some mabs strongly cross react indicating that both toxins have major epitopes in common. A hypothesis for the structural and possible functional relatedness between the two toxins is discussed.

Animals↗
PubMed1986

The role and mechanism of cobra venom factor-induced suppression of the humoral immune response in guinea pigs.

The cobra venom factor (CVF)-mediated suppression of the humoral immune response to bacteriophage phi X 174, a T cell-dependent antigen, was investigated in guinea pigs. The suppression was markedly dependent on the amount of antigen used: decomplementation of animals diminished the humoral immune response when immunizing with low doses of antigen, whereas increasing the antigenic dose resulted in little or no diminution by CVF. In contrast to the results with normal animals, CVF had no influence on the humoral immune response of genetically C2-deficient guinea pigs; i.e., no suppression occurred above that due to the inherited complement deficiency state alone. We therefore postulate that CVF suppresses the humoral immune response by the depletion of C3, with no measurable role for complement split products under these experimental conditions.

Animals↗
PubMed1986

An inherited deficiency of the third component of complement, C3, in guinea pigs.

Hereditary deficiency of the third component of complement, C3, is found very seldom in the human. C3 deficiency is associated with severe bacterial infections revealing the central role of C3 in complement activation via the classical or alternative pathway. We describe a new hereditary C3 deficiency in strain 2 guinea pigs. Serum from these animals had a markedly reduced lytic activity in a standard assay for complement-dependent, antibody-mediated cytotoxicity. In functional assays of individual components, the hemolytic activity of the components C4, C2, C5 and of factors B, D and H was in the normal range. The functional C3 titer, and similarly C3 antigenic activity in the serum of these C3-deficient animals (C3D) was on average only 5.7% of normal activity. Typing the animals with alloantisera or monoclonal antibodies to guinea pig Ia-antigens revealed that the C3D animals had the major histocompatibility complex-haplotype of inbred strain 2 guinea pigs (B.1, Ia.2,4). The C3 defect is not linked to the major histocompatibility complex and, in addition, is not linked to a C3a receptor deficiency. Macrophages and hepatocytes of the C3D animals have an unimpaired capacity for synthesis and secretion of C3 as measured by enzyme-linked immunosorbent assay. There was no indication for hypercatabolism of normal C3 by the animals as shown by plasma clearance of 125I-radiolabeled C3. Thrombocytes of the C3D animals responded normally to stimulation with purified C3a in an ATP-release assay without an indication for a desensitization in vivo. Possibly the fault resides in an enhanced susceptibility of their own C3 to proteolysis. However, C3 partially purified from the plasma of the C3D animals or secreted by hepatocytes exhibited no obvious structural differences to purified normal C3 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis or in immunoblotting. The C3D serum had a reduced bactericidal activity compared to normal or to C4-deficient serum. Nevertheless, the animals are apparently healthy without an indication for increased frequency of bacterial infections. These guinea pigs provide an unique model for analysis of the biological functions of C3 in vivo and in vitro without the need for artificial C3-depletion procedures with all their known and unknown side-effects.

Animals↗
PubMed1986

Evaluation of low dose anaphylatoxic peptides in the pathogenesis of the adult respiratory distress syndrome (ARDS). Monitoring of early C5a effects in a guinea-pig in vivo model after i.v. application.

A guinea-pig in vivo model is presented that allows the infusion of purified C5a via a central vein catheter and the monitoring of its effects on granulocytes and platelets, the most important cells in the pathogenesis of several lung disorders, e.g. shock lung. After the infusion of C5a, which was adjusted to a quantity that caused slight and transient alterations of lung physiology, granulocytes disappeared from circulation within 1 min. Simultaneously the granulocyte content of the lung increased about three-fold as judged by histological evaluations. Morphologic destructions were not observed. After the drop a rebound of circulating Polymorpho-nuclear leucocytes (PMN) occurred, which was significantly higher than control values and the appearance of banded cells indicated a mobilization from bone marrow stores. Studies with 51-chromium labelled PMNs revealed that most, but not all, of the granulocytes returned to circulation after transient sequestration. The number of platelets also decreased after C5a infusion, but the rebound was delayed compared with the PMNs and did not exceed control values. The changes in circulating cells, lung histology, and lung physiology are comparable to those occurring during the onset of shock lung and thus strengthen the supposed importance of C5a concerning the pathogenesis of that syndrome.

Animals↗
PubMed1986

Guinea pigs with inherited deficiencies of complement components C2 or C4 have characteristics of immune complex disease.

Guinea pigs genetically deficient in the second (C2) or fourth component of complement (C4) generally appear healthy in contrast to humans with a C2 or C4 deficiency. However, upon investigation of these genetic deficiencies in guinea pigs for signs of dysregulation in the humoral immune system and especially autoantibodies, many complement-deficient guinea pigs (greater than 50%) had elevated levels of serum IgM and higher concentrations of anti-hapten (dinitrophenyl) antibodies as signs of polyclonally stimulated antibody synthesis. In addition, a significant number of the complement-deficient animals, on average 30%, had IgM rheumatoid factors in their sera compared with less than 1% of the normal animals. These observations, therefore, indicate that guinea pigs, genetically deficient in C2 or C4, show characteristics of immune complex disease in general.

Aging↗
PubMed1985

Interaction of Ross River virus with the complement system.

In the absence of virus-specific antibody, Ross River virus failed to activate either the classical or alternative complement pathways. Instead, it inhibited the cleavage of C3 via both pathways. The virus did not appear to act by disrupting C3bBb complexes or by preventing cleavage of factor B by factor D. Instead Ross River virus was found to interfere with the actual cleavage of C3 by activated factor B (C3bBb) of the alternative pathway and C4b2a of the classical pathway.

Alphavirus↗
PubMed1985

An in vitro system to study listericidal capacity of macrophages from separate mice: resident macrophages exhibit different activation patterns.

An in vitro system with macrophages from individual mice was established to study their listericidal capacity. Because no antibiotics were used, bacterial killing was really due to macrophages in short-term culture. To restrict the extracellular growth of bacteria, cell culture medium was changed at 1-h intervals. We demonstrated that intracellular growth of listeria in macrophage pools from untreated animals varies considerably. Obviously, preactivated macrophages are constantly present, so that the common procedure of using macrophage pools from several animals is no longer acceptable. In addition, we demonstrated that in vitro mixtures of listeria-immune macrophages of one animal with cells from untreated animals at different ratios exhibit enhanced bacterial killing above a mere additive effect. Consequently, by using macrophages from individual untreated mice, we found that cells of different animals exhibited various activation stages, although unstimulated, inbred specific-pathogen-free mice of the same age, weight, and sex were used. When equal numbers of macrophages from untreated separate animals were mixed in vitro, intracellular growth of listeria was only moderate; that is, the number of preactivated macrophages of the individual animals determined listerial growth in the pooled preparation. Furthermore, we showed that identical doses of phorbol myristate acetate exerted different effects on the listericidal activities of macrophages as a function of their preactivation states. These experiments clearly demonstrate the advantage of using macrophages from individual mice for in vitro studies of macrophage activation.

Animals↗
PubMed1985

Influence of genetically inherited complement deficiencies on humoral immune response in guinea pigs.

To assess the role of complement in the induction of the humoral immune response, we studied the antibody response of guinea pigs genetically deficient in the second component of the classical complement pathway (C2D-GP) to bacteriophage phi X 174--a T cell-dependent antigen--in comparison with normal guinea pigs and C4D-GP, for which a disturbance in induction of antibody response has been described. We were able to establish a clear dose-response relationship: with low doses of antigen (1 X 10(9) PFU/kg), the antibody response of both complement-deficient strains was grossly impaired as compared with normal guinea pigs. After primary immunization, the peak antibody titer was diminished (1 log10) and declined rapidly; after secondary immunization, the diminution became even more distinct. Both complement-deficient strains had unusual secondary antibody responses almost identical to their primary ones, and amplification of antibody titer, as well as regular isotype switch from IgM to IgG, was absent. By increasing the antigen dose (2 X 10(9) PFU/kg), the antibody responses of the complement-deficient guinea pigs tend to normalize, and when high doses of antigen (1 X 10(10) PFU/kg) were used, the behavior of the complement-deficient animals was nearly indistinguishable from that of normal animals. Partial restoration of the immune response was seen when substituting the genetic complement deficiency by giving serum as source of the missing complement component. The important contribution of the C2 deficiency is given by the now compelling evidence that it is not the missing individual component itself, but rather the common block in sequential activation of C3 via the classical pathway in both complement deficiencies, that is responsible for the impaired humoral immune response, especially at low antigen doses. We therefore postulate that an intact classical pathway contributes to reaching a normal humoral immune response.

Animals↗
PubMed1985

Cell surface antigens on the guinea-pig macrophage: identification by monoclonal antibodies and association with the activation state.

Ten monoclonal antibodies (moAb) directed against cell surface antigens of guinea-pig monocytes and macrophages (mph) were produced and characterized. The corresponding antigens are not present on granulocytes, T lymphocytes, an Ia-positive B-cell line or other haematopoietic cells. In binding or cytotoxicity assays, the moAb demonstrated characteristics patterns of reactivity, with mph being in different stages of differentiation or activation. Three moAb (342, 322, 249) recognized 'lineage antigens' (i.e. antigens continuously expressed during maturation of monocytes to mph and after stimulation or activation of the cells). MoAb 342 possibly defines a major cell surface determinant, being present on 90% of mph. The antigens detected by moAb 305, 320, 321 and 344 characterize mature mph. They were not expressed on monocytes, but were expressed on the majority of resident, elicited or activated peritoneal mph. MoAb 253, 310 or 257 defined discrete subpopulations of elicited and--with the exception of moAb 257--activated mph. The corresponding antigens were not present on monocytes or resident mph, but appeared on the cell surface during in vivo or in vitro stimulation of the cells. There was no indication of a contribution of the moAb-defined antigens to the presentation of antigen, mitogen or alloantigen by the mph to T cells. The functional significance of the antigens thus remains to be elucidated. Our studies indicate that cells committed to the monocyte/mph lineage share a family of differentiation antigens, distinguishing them from other cell lines. The moAb provide useful tools for further investigation of the activation of mph and allow the rapid detection of mph in different tissues.

Animals↗
PubMed1984

Release of prostaglandin E and thromboxane from macrophages by stimulation with factor H.

Recently novel actions of factor H of complement other than regulation of alternative pathway activation have been described. We examined the influence of H on the arachidonic acid (AA) metabolism of macrophages. Guinea-pig peritoneal macrophages cultured for up to 18 h under serum free conditions were supplied with homologous factor H. H, tested over a concentration range of 12.5-100 micrograms/ml, promoted an indomethacin sensitive release of prostaglandin E and thromboxane B2 in a dose-dependent manner. Stimulation of AA conversion to prostanoids in response to H was shown to be specific as evidenced by immunoabsorption experiments. This novel effect attests to the potential of H to act not only as regulatory protein of the complement pathway but also as an inducer of cellular release reactions. Moreover, these findings emphasize the close functional links that exist between the three main constituents of the inflammatory process: macrophages, the complement system and the AA cascade.

Animals↗
PubMed1984

Regulation of the oxidative burst of macrophages by lipid mediators.

Macrophages, on stimulation, generate both cyclo-oxygenase and lipoxygenase products of arachidonic acid as well as the acetylated phospholipid, PAF-acether. The latter induces the oxidative burst in C. parvum-activated macrophages, but has little activity on resident macrophages. Mouse macrophages are generally rather insensitive to PAF-acether. Prostaglandin (PG)E2, on the other hand is a potent inhibitor of the macrophage oxidative burst, though its activity is also dependent upon the source of the macrophages and the stimulus used. Several lipoxygenase inhibitors inhibit the oxidative burst of mouse peritoneal macrophages, while other authors have reported stimulatory effects of lipoxygenase products on different macrophage populations. The differential actions of lipoxygenase and cyclo-oxygenase products on the macrophage oxidative burst are reflected by the effect of arachidonic acid on the zymosan induced response of mouse resident peritoneal cells: initial enhancement, followed by prolonged inhibition, the latter being indomethacin-sensitive. While PAF-acether is probably an important initiator of the macrophage oxidative burst in vivo, arachidonic acid modulates the response by a "see-saw mechanism", lipoxygenase products being stimulatory and cyclo-oxygenase products (mainly PGE2) being inhibitory.

Animals↗
PubMed1984

Expression of Ia antigens on macrophages is reduced after stimulation with homologous C3b.

The complement cleavage product C3b was examined for its ability to influence the expression of Ia antigens on macrophages. Monoclonal anti-Ia antibodies were applied for quantitation of Ia antigens by using FACS analysis. Albumin-elicited guinea pig peritoneal macrophages were cultured for 48 hr in the presence of C3b. C3b at final concentrations varying from 10 to 80 micrograms/ml reduced the proportion of Ia-positive macrophages from 87 to 25%. The C3b-stimulated loss of Ia antigens from the macrophage surface was mediated by metabolites of the cyclooxygenase pathway as shown by the inhibitory effect of indomethacin. Exposure of macrophages to C3b for 5 min was sufficient to induce Ia reduction, seen after subsequent 48-hr incubation. C3b generated in vivo in the course of inflammatory reactions might therefore influence the interaction of immunocompetent cells by modulating macrophage Ia expression.

Animals↗
PubMed1983

Synthesis of complement by macrophages and modulation of their functions through complement activation.

During the last decade considerable progress has been made to characterize intimate functional links between macrophages, a major cellular component of immunoinflammatory responses, and the complement system representing the major humoral mediator of inflammation. Macrophages of various species and tissue sites have been shown to synthesize and release most of the complement components providing these cells with their own "pericellular" complement system. Circumstantial evidence for the assembly of both classical and alternative pathway convertases has been adduced. An intricate network of feedback loops involving endogenous and extrinsic factors operates to adjust complement production to acute requirements, for example augmenting production in the face of accelerated turnover at sites of inflammation, and returning it to baseline levels once the inflammatory stimulus has subsided, in order to maintain a fine-tuned balance. The molecular mechanisms underlying regulation of complement synthesis by macrophages are beginning to be elucidated by use of gene technology. On the other hand, complement activation products exert a number of effects on macrophages via specific surface receptors causing internalization of offending agents, microbes, and immune complexes, promotion of intracellular killing, controlling migration behavior, inducing release of potent biologic substances such as lysosomal enzymes, arachidonic acid metabolites, and interleukin 1. In these interactions, two important humoral mediator systems of inflammation, the complement system and the arachidonic acid cascade, are functionally linked at the level of the macrophage. Stimulation of the release of immunomodulating compounds from macrophages invoke a role for complement in immune regulation. This multifaceted interplay is of particular importance considering the mobility of macrophages that allows them to gain almost unrestricted access to sites of ongoing immunoinflammatory responses. The time seems to have come to abandon the petrified thinking in socalled systems as, for instance, humoral versus cellular, specific versus unspecific, and to proceed to interlocking functions guided by physiology proper.

Anaphylatoxins↗