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V Brade

Publications and source records attributed to V Brade.

At least 73 records · Page 4Linked to original sources
PubMed1989

Influence of surface modulations by enzymes and monoclonal antibodies on alternative complement pathway activation by Yersinia enterocolitica.

Effector mechanisms resulting from alternative complement pathway (ACP) activation cannot act efficiently against Yersinia enterocolitica serotype O3, as indicated by poor C3 to C9 consumption and by survival in EGTA (ethyleneglycoldiaminetetraacetic acid) Mg-serum. These results were not influenced by the lack or presence of plasmid-encoded outer membrane proteins or lipopolysaccharides (LPS) with different amounts of side chains or by treatment of the bacteria with pronase or neuraminidase. Surface modulation of Y. enterocolitica with polyclonal immunoglobulin G or the immunoglobulin G fragments F(ab')2 and Fab always converted Y. enterocolitica to a high ACP activator, with strong C3 to C9 consumption and surface deposition of activated C3. Killing of Y. enterocolitica as a result of antibody-mediated ACP activation was observed only with bacteria grown at 22 degrees C but not with bacteria from 37 degrees C cultures. The expression of complement resistance in Y. enterocolitica grown at 37 degrees C was not influenced by the presence or absence of plasmids. Using different monoclonal antibodies (MAb), we found that MAb with LPS specificity mediated ACP activation, whereas MAb specific for different plasmid-encoded outer membrane proteins were ineffective, despite surface binding. These results suggest a major inhibitory role of LPS on ACP activation which was neutralized by LPS-specific antibodies.

Animals↗
PubMed1989

[Serodiagnosis of Lyme borreliosis: antibody titer and specificity in the IFT and western blot].

Out of 334 sera from healthy persons from northern Bavaria 6% reacted positive in the indirect immunofluorescence test (IFT, titre greater than or equal to 1:64). The percentage of positive cases in patients with erythema chronicum migrans (ECM), meningoradiculitis (M. Bannwarth), or acrodermatitis chronica atrophicans (ACA) was 43, 83 and 78%, respectively. In other patients with less characteristic symptoms in which an infection with B. burgdorferi was suspected the number of positive cases was only about 20%. A positive IgM-IFT was most often associated with ECM and M. Bannwarth; however, even in these cases IgM was only found in 30%. The serodiagnosis of Lyme disease required simultaneous serum testing of T. pallidum antibodies (TPHA) to exclude a syphilis with cross-reacting antibodies. In cases with a positive B. burgdorferi-IFT and TPHA the final diagnosis of Lyme disease was possible on the basis of Western blot analysis. The prominent antigens detected by sera from patients with Lyme disease--but not with syphilis--had molecular weights of 18, 22, 41 and 60 K; IgM antibodies were mainly directed against the 22 K and 41 K antigens. The antigen pattern was not characteristic for different disease stages.

Acrodermatitis↗
PubMed1988

[Congenital toxoplasmosis: value of modern IgM serodiagnosis is from the example of problem cases].

Congenital toxoplasmosis may be symptomatic or asymptomatic at the time of birth. In the latter case late manifestations may occur which can be avoided by early diagnosis and treatment. In comparison with the IgM-IFT we demonstrate the specificity and high sensitivity of a recently available immunosorbent agglutination assay (ISAGA) for the detection of IgM against toxoplasma. We also present evidence for the high diagnostic value of the ISAGA in cases where congenital toxoplasmosis is suspected. Based on the presented data we propose a pattern of diagnostic methods for the diagnosis or exclusion of congenital toxoplasmosis in newborns.

Antibodies, Protozoan↗
PubMed1987

[Congenital toxoplasmosis with delayed immune response in children. Diagnostic problems].

A newborn, delivered at term, developed a rapidly increasing hydrocephalus with intracranial calcifications and seizures during the first week of life. Clinical suspicion of congenital toxoplasmosis was at first not confirmed serologically (serum titer in the immunofluorescence test [IFT] of 1:1024 and complement-fixation reaction [CFR] of 1:40 equalling those of the mother; IgM-IFT being negative and the IFT and CFR titers significantly falling within two weeks). But in the further course of the disease the diagnosis of congenital toxoplasmosis was confirmed: 1. Microscopic tachyzoits in CSF when aged six weeks; 2. positive IgM-IFT in serum and CSF from the seventh weeks onwards; 3. steep IFT titer rise in serum and CSF from the 16th week onwards. This case demonstrates that with delayed immune response in the infected child only serial serological tests will exclude or confirm the diagnosis of congenital toxoplasmosis.

Antibodies, Bacterial↗
PubMed1986

Inactivation of C3a by a monocarboxypeptidase present in culture supernatants of stimulated guinea pig peritoneal macrophages.

Hog C3a, as well as its derivative C3a-desArg were not found to act cytotoxically on starch gel-induced guinea pig peritoneal macrophages. Likewise, neither peptide significantly modified the secretion of N-acetyl-beta-D-glucosaminidase from these cells. However, C3a rapidly lost its spasmogenic activity during incubation in serum-free macrophage cultures and less rapidly in cellfree supernatants collected from cultured macrophages. The following results indicate that C3a is converted into its spasmogenically inactive derivative C3a-desArg by a macrophage-derived monocarboxypeptidase. The inactivated C3a product does not differ from native C3a in sodium dodecyl sulfate-polyacrylamide gel electrophoresis; it elutes from CM cellulose in the same position as purified C3a-desArg; and it is devoid of the carboxyl-terminal arginyl residue of C3a, but still contains the carboxyl-terminal sequence of C3a-desArg as determined by analysis after treatment with carboxypeptidases B or Y. Furthermore, inactivation of C3a in supernatants of macrophage cultures is completely blocked by the specific carboxypeptidase inhibitors guanidinopropylsuccinic acid and 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid in final concentrations of 10 mM and 2.1 mM, respectively. The monocarboxypeptidase is apparently supplied by biosynthesis of new material but is not stored as a preformed enzyme because cycloheximide markedly inhibits its expression.

Acetylglucosaminidase↗
PubMed1986

Lipopolysaccharides as complement inhibitors by complex formation with the purified third complement component (C3).

Lipopolysaccharides (LPS) from different bacteria in smooth or rough form (Y. enterocolitica, Y. pseudotuberculosis, E. coli, S. typhimurium, S. marcescens) strongly inhibited hemolytic C3 in incubation mixtures with purified C3. LPS from a core deficient mutant was still reactive, whereas lipid A no longer affected C3 activity. The physical state of LPS was critical for its effect on C3. Strand-like LPS structures formed by Ca++-induced aggregation of solubilized LPS, as shown by electron microscopy, demonstrated the highest reactivity with C3. Inhibition of hemolytic C3 was found to be due to complex formation between LPS and C3 by a hydrophobic reaction. The binding capacity of 1 microgram LPS-R and LPS-S was as high as 125 ng C3 and 56 ng C3, respectively. The C3b fragment required different reaction conditions for maximal binding. The strong binding capacity of LPS for the complement component C3 raises the possibility that LPS act as inhibitors of complement by interruption of the reaction cascade at local infectious sites with gram-negative bacteria.

Centrifugation, Density Gradient↗
PubMed1986

C3 activation by a new factor B-dependent enzyme detected in culture supernatant from guinea-pig peritoneal macrophages.

Culture supernatants (c.s.) collected from thioglycollate-elicited macrophages were concentrated and incubated with purified C3. In this reaction mixture loss of haemolytic C3 according to classical enzyme kinetics was observed. As revealed by SDS-PAGE, c.s.-catalysed fragmentation of the C3 alpha-chain occurred. The cleavage products were identified by size and function as C3a and C3b. The apparent molecular weight of this C3-activating enzyme in c.s. was approximately 220,000 according to ultracentrifugation studies. This large enzyme showed the following characteristics: it had no activity against C5; it was inhibited by EDTA; Mg2+ was required for its optimal function; its half-life at 37 degrees was approximately 35 min; it was completely inhibited by anti-B IgG. Thus, we were able to detect a C3-activating enzyme in c.s. containing B but differing otherwise from a preformed C3 convertase of the alternative pathway. The exact component composition of this new enzyme is under further investigation.

Animals↗
PubMed1984

Formation of EAC142 and EAC1423 with macrophage culture supernatant containing the secreted complement components C1 to C3.

Culture supernatants of thioglycollate-elicited guinea pig peritoneal macrophages contained hemolytic C1, C4, C2 and C3, whereas hemolytic C5, C6, C7, C8 or C9 were not detected. Activity of C1, C2 and C3 increased up to a 48 h culture period, whereas C4 activity already declined in 2 day old cultures. After secretion, the hemolytic activity of C1 was least stable in culture supernatant. Sensitized sheep erythrocytes (EA) when incubated with culture supernatant initiated activation and functional cooperation of secreted C1 to C3 as indicated by formation of EAC142 and EA1423 intermediates. Decay and regeneration with purified C2 was shown for EAC142 and deposition of C3 fragments on EAC1423 was demonstrated with anti-C3. On an average, supernatants of 2 day old macrophage cultures were most suitable for formation of EAC142 and EAC1423 . The rate of EAC142 and EAC1423 formation, and also of C2 and C3 inactivation, during incubation of EA with culture supernatant was slow; addition of purified C1 to culture supernatant, however, greatly enhanced the same reactions of EA with supernatant which indicated that C1 was the rate limiting factor. Local secretion of hemolytic C1, C4, C2 and C3 by macrophages may have an important role in antimicrobial defense mechanisms due to the well-known functional cooperation between macrophages and activated C3.

Animals↗
PubMed1983

[Results with the IgM-FTA-Abs and IgM-TPHA tests in the study of seropositive syphilitic sera].

The IgM fraction was separated by gel filtration chromatography from patient sera which were positive in the FTA-abs-test as well as in the TPHA-test. After absorption the isolated IgM fraction was tested for specific IgM antibodies against Treponema pallidum in the IgM-FTA-abs- and also in the IgM-TPHA-test. The following results were obtained: Both IgM tests often showed positive reactions even with negative results in the cardiolipin complement fixation assay (CFA) (Tables 1 and 2). In most positive cases specific IgM was detectable with both IgM tests in the same sample (Table 3). The CFA titers were low (less than 1:20) in the majority of cases which did not have detectable IgM (Tables 1 and 3). The CFA titers were prevailingly high (greater than 1:20) where the IgM tests gave positive results (Table 1 and 3). Clinical symptoms of active syphilis were only reported for cases with high CFA titer and positive IgM tests in the same sample. Therefore only these latter results of diagnostic tests can be clearly interpreted to indicate a requirement for specific therapy against syphilis.

Antibodies, Bacterial↗
PubMed1983

Effect of cycloheximide and of anti-C3 Fab' on the intrinsic synthesis and secretion of lysosomal enzyme and of complement components by guinea-pig peritoneal macrophages.

Without any additional stimulus, the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (beta-GLU) is secreted immediately from starch gel induced guinea-pig peritoneal macrophages. A more than three-fold increase of the total enzyme activity in the course of 3 days and the reversible inhibition of this enzyme increase by cycloheximide prove the synthesis of beta-GLU. The synthesis rate of beta-GLU remains rather constant after the second day, despite rapid cell death in the same culture period which could be explained by heterogeneity of the macrophage population. Cycloheximide immediately inhibits secretion of C3 whereas inhibition of beta-GLU secretion is only observed after a lag phase of 24 h. Secretion of factor D and of beta-GLU is not altered if secreted C3 is totally neutralized by the addition of anti-C3 Fab' to macrophage cultures. Thus, endogenous C3 as well as its endogenously generated fragments do not influence these macrophage functions. In the same cultures with anti-C3 Fab', conversion of secreted factor B into its fragments is inhibited as indicated by the detection of functional B activity. These results indicate that the secretion of factor D and of beta-GLU is also independent of endogenous B-derived fragments such as Bb. Finally, the detection of functional B in cultures with anti-C3 Fab' proves that C3b is required for factor B activation. The apparent interaction of secreted C3 and factors D and B in regular macrophage cultures suggests the constant formation of the labile C3 convertase C3bBb of the alternative pathway.

Acetylglucosaminidase↗
PubMed1982

Functional active complement components secreted by guinea pig peritoneal macrophages.

In our studies on complement secretion functional C1, C4, C2, C3, P, D and B were clearly identified in the same cultures. Functional assays did not allow the detection of C5 to C9. Spontaneous C3 activation occurred at a very low level in culture supernatants. The responsible enzyme was identified as a metallo-enzyme. Upon addition of antibody-coated sheep erythrocytes (EA) to culture supernatant it was possible to induce C3 activation as indicated by the apparent formation of EAC1423. Zymosan was also able to activate C3 in culture supernatant after addition of purified functional factor B indicating efficient cooperation of factors of the alternative pathway. Thus in this in vitro system macrophages not only provide C3 but also all factors for spontaneous and induced C3 activation. If these secretory functions reflect in vivo properties of macrophages, our results may indicate that C3 and its activating systems are most relevant for local cooperation between macrophages and the complement system in inflammation and antimicrobial defense. Therefore availability of these essential factors at any time is secured by local production.

Animals↗
PubMed1980

[Mediators of inflammation and of antimicrobial activity secreted by macrophages (author's transl)].

Macrophages are highly differentiated mononuclear phagocytes which originate from stem cells of the bone marrow. The secretory potential of these cells has been recognized in recent years. Major secretory products comprise lysosomal enzymes, complement proteins, prostaglandins and interferon. Secretion of lysosomal hydrolases and proteinases is most prominent in macrophages stimulated in vivo or in vitro (Fig. 4). Lysosomal enzyme secretion may be an important factor in the induction and maintenance of inflammatory reactions. Complement (C) proteins secreted by macrophages belong to the classical activation unit (C1, C4 and C2), alternative activation unit (C3, B, D, P) and to the group of delayed-acting C proteins (Fig. 7). Therefore macrophages produce at local sites the C component C3 from which biologically active C3 fragments (C3a, C3b, C3e) can be generated. These C3 fragments mediate inflammatory and cytotoxic reactions and also promote phagocytic processes (Fig. 6). Cleavage of secreted C3 into the active fragments may occur by enzymes derived from both C activation units or by secreted lysosomal proteinases (Fig. 8). Stimulated macrophages also synthesize and release prostaglandins. These compounds which have inflammatory as well as antiinflammatory effects (Fig. 12) may play an important regulatory role in inflammatory processes. Interferon has been also recognized as a secretory product of macrophages. This substance supports antimicrobial resistance by its phagocytosis-increasing effect and its antiviral activity. The secretory function of macrophages as well as the biological effects of secreted mediators are highly susceptible to modulation. Thus, C3 fragments stimulate the secretion of lysosomal enzymes (Fig. 6) whereas prostaglandins inhibit their release (Fig. 12). The inflammatory reactions induced by lysosomal enzymes may be further increased by the generation of C3b which stimulates additional lysosomal enzyme release (Fig. 4). These and other examples suggest that endogenous control mechanisms may have a strong influence on the secretory function of macrophages as well as on the biological activity of secreted mediators.

Animals↗
PubMed1980

Activation of human complement by Yersinia enterocolitica: ultrastructural alterations and C3b-deposition.

Rapid killing of Yersinia enterocolitica strain 75 in smooth form (Ye 75 S) was observed in the presence of serum or of lysozyme-free serum whereas the killing activity of EGTA-serum was slow, and absent in heated (30 min 56 degree C) serum. Similarly, complement (C) activation by Ye 75 S was rapid in serum and lysozyme-free serum but slow via the alternative pathway (EGTA-serum). These data suggest that C is sufficient for killing of the cells and most active via an intact classical pathway. Electronmicroscopic studies were performed on bacterial killed by serum (C + lysozyme) or by lysozyme-free serum (C). In these experiments cell fragmentation and spheroplast formation were seen after exposure of Ye 75 S to serum; in bacteria incubated with lysozyme-free serum "blebs" formation was observed as the most prominent alteration. These blebs most likely originate from the outer membrane as a result of C activation on the cell surface. The deposition of activated C (C3b) on Ye 75 S was analyzed kinetically in the presence of serum or EGTA-serum. With serum (30 vol%) massive C3b deposition was observed within 20--30 min whereas with EGTA-serum (30 vol%) the deposition of C3b was slower and less complete. Experiments with EGTA-serum also revealed that the deposition of C3b started at single sites mainly located in the region of the cell poles; from these sites spreading of C3b occurred until large areas of the cell surface were covered. These data suggest that C activation via the alternative pathway is restricted to certain regions of the bacterial surface.

Blood Bactericidal Activity↗
PubMed1979

[Complement: function. Biological significance and clinical aspects (author's transl)].

The term complement comprises approximately twenty serum proteins. Activation of these components can be started by antigen-antibody complexes or even without the participation of antibodies. The activated complement proteins bring about a number of important biological actions. They produce vigorous local inflammatory reactions, exert phagocytosis--increase effects, destroy microbial and animal cells and affect the immune response to antigen stimulus. Congenital lack of individual complement components may considerably impair the antimicrobial defense or even be the cause of excessive inflammatory reactions. The activation of complement may also have harmful effects on the organism.

Angioedema↗
PubMed1978

Requirements for the solubilization of immune aggregates by complement. The role of the classical pathway.

In this paper we examine the role of the classical pathway in the complement-mediated solubilization of immune precipitates (CRA). Serum reagents were depleted of the alternative pathway components properdin and factor D. Both depleted reagents lack CRA although they have almost intact hemolytic activity. Also, immune complexes were not solubilized when incubated with high concentrations of the classical pathway components (C1, C4, C2, and C3. We conclude that CRA is not mediated by the classical pathway alone. Activation of the classical pathway by the immune aggregates greatly enhances CRA. The effect of the classical pathway is to deposit C3b on the antigen-antibody lattice and promote the assembly of a lattice-associated, properdin-dependent C3-convertase. Although C3, C4, and properdin were detected on complexes solubilized by serum in the presence of Ca++ and Mg++, only C3 and properdin were found on the complexes when Ca++ had been chelated by ethylene glycol-bis-(beta-aminoethyl ether), N,N'-tetraacetic acid. In both situations the aggregates were capable of converting C5 in the fluid phase. However, no C5 was found on the solubilized complexes. These findings suggest that in contrast to nascent C3b and C4b, nascent C5-9 lacks binding affinity for immune aggregates.

Antigen-Antibody Complex↗