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Publications and source records attributed to K Drössler.
Protected mercaptoalkylpyrimidinones: synthesis and test for immunostimulating activity 3-Hydroxyalkyl-pyrimidine 1 reacts with phosphoroxychloride and thioglycolic acid or thiourea to yield pyrimidin-3-ylalkylthioacetic acids 3 or pyrimidin-3-ylalkylthiouroniumsalts 5 respectively. Some of the pyrimidines 3 and 5 showed immunomodulatory activity.
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An antigen-capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies (mAb) was set up and evaluated for selective detection of salmonid antibody responses to the antigen P1, which is a weakly immunogenic exoprotease of typical Aeromonas salmonicida. This new assay permits a specific determination of anti-protease-antibodies, without antigen purification. Serum antibodies induced by the strongly immunogenic lipopolysaccharide could reliably be discriminated from anti-P1-antibodies. Antibody titres of 45 experimental antisera recorded by cELISA were moderately correlated with titres determined by routinely used indirect ELISA (iELISA) by detecting partially different antibody populations (r=0.753). Substitutions of immunoreactants and confirmatory immunoblotting strongly suggest that the mAb-based assay selectively recognises antibodies directed to epitopes of native protease. A conjugate of inhibited protease and cationized bovine serum albumin (cBSA) was found to engender a significant anti-protease-response in three salmonid species (P<0.05), whereas the unconjugated antigen and Apoject 1-Fural were proved to be ineffective. Recorded specific antibody titres were as high as 1:381,400, indicating a considerable enhanced immunogenicity of cBSA-conjugated P1 and high assay sensitivity. The established cELISA offers a promising approach to further improvement of monitoring fish humoral immune response to surface accessible epitopes of the immunosuppressive exoprotease, P1, and to scrutinize its protective significance.
A novel membrane proteinase of the nosocomial important bacteria species Bacillus cereus (synonyms: camelysin, CCMP) was purified up to homogeneity as was shown by mass spectrometry in its amphiphilic form. Camelysin is a neutral metalloprotease with a molecular mass of 19 kDa. Its unique N-terminus Phe-Phe-Ser-Asp-Lys-Glu-Val-Ser-Asn-Asn-Thr-Phe-Ala-Ala-Gly-Thr-Leu-Asp-Leu-Thr-Leu-Asn-Pro-Lys-Thr-Leu-Val-Asp-(Ile-Lys-Asp)- was not detected in the protein data bases during BLAST searches, but in the partially sequenced genome of Bacillus anthracis, coding for an unknown protein. Cleavage sites of the membrane proteinase for the insulin A- and B-chains were determined by mass spectrometry and N-terminal sequencing. Camelysin prefers cleavage sites in front of aliphatic and hydrophilic amino acid residues (-OH, -SO3H, amido group), avoiding bulky aromatic residues. The internally quenched fluorogenic substrates of the matrix metalloproteases 2 and 7 were cleaved with the highest efficiency at the Leu-decrease-Gly or Leu-decrease-Ala bond with the smaller residue in the P1' position. The protein specificity is broad--all various kinds of casein were cleaved as well as acid-soluble collagen, globin and ovalbumin; intact insulin was destroyed only to a low extent. Actin, collagen type I, fibrinogen, fibrin, alpha2-antiplasmin and alpha1-antitrypsin were cleaved. The protease formed SDS-stable complexes with Glu-plasminogen and antithrombin III, visible after SDS electrophoresis by gold staining and Western blot. The CCMP-plasminogen complex caused a partial activation of plasminogen to plasmin. Camelysin interacts with proteins of the blood coagulation cascade and could facilitate the penetration of fibrin clots and of the extracellular matrix during bacterial invasion.
The human NK gene complex encodes for the leucocyte C-type lectins, CD69, AICL (activation-induced C-type lectin), LLT1 (lectin-like transcript), CD161/NKR-P1A, CD94, and for NKG-2 molecules. These gene products have been implicated in the regulation of the function of natural killer (NK) cells and other lymphocytes. In this study the expression of C-type lectins during the early activation of PMA-stimulated peripheral blood lymphocytes was examined. To investigate the influence of de novo protein synthesis on activation-dependent expression of C-type lectins, cells were cultured in presence of cycloheximide (CHX) and mRNA levels were analyzed by semi-quantitative reverse transcription-polymerase chain reaction. Upregulated levels of CD69, AICL, and LLT1, but less pronounced changes of CD161/NKR-P1A and CD94 mRNA were found at early time points of cellular activation. CD69 was superinduced by CHX at the nuclear precursor transcript and the mRNA level suggesting that regulation of transcriptional activity and mRNA stability contribute to extent of CD69 mRNA accumulation. CHX treatment resulted also in an overexpression of AICL, LLT1, and CD161/NKR-P1A mRNAs. Conversely, CHX blocked CD94 mRNA expression in PMA-stimulated cells, demonstrating that this process is dependent on new protein synthesis. Expression kinetics in context with susceptibility to CHX indicate that the mechanisms responsible for upregulated CD69, AICL, and LLT1 expression are distinct from those which control CD161/NKR-P1A or CD94 expression. J. Cell. Biochem. Suppl. 36: 201-208, 2001.
Two monoclonal antibodies (Mabs) binding to a toxic extracellular metallo-proteinase of Aeromonas salmonicida subsp. achromogenes, AsaP1, were produced. Both reacted with common epitopes of the native enzyme and recognized this 20 kDa antigen on Western blots. One of these Mabs had an inhibitory effect on the caseinase activity of the exotoxin. A Mab-based ELISA was set up and evaluated for serological detection of AsaP1 in bacterial culture filtrates. The exotoxin was identified serologically in the extracellular products of 11 of 26 atypical Aer. salmonicida isolates, including the type strain for subsp. achromogenes NCIMB 1110. The ELISA was approximately 100-fold more sensitive in detecting AsaP1 compared with an azocasein assay. The established serological test enables AsaP1 to be quantified reliably with a lower detection limit of about 0.12 ng ml-1 and has a potential use for the phenotypic differentiation of atypical Aer. salmonicida isolates.
Four monoclonal antibodies (MAbs) directed to native glycerophospholipid:cholesterol acyltransferase (GCAT) epitopes of Aeromonas salmonicida were isolated using an esterase capture assay. The molecular mass of this MAb-defined antigen was estimated to be 26 kDa in SDS-PAGE. Three different epitope specificities of these MAbs were demonstrated. It was shown that all 4 MAbs recognize GCAT in culture filtrates of the strain MT004 excluding the simultaneous trapping of other components. None of the MAbs react with the denatured GCAT in Western blots.
PURPOSE: To investigate the role of a thiol-containing biologically active compound in lipid peroxidation of membranes. MATERIALS AND METHODS: Thiyl radicals were generated from 3-(2-mercaptoethyl)quinazoline-2,4(1H,3H)-dione (MECH) using pulse radiolysis and gamma-radiolysis in aqueous and alcoholic solutions saturated with N2O. The products were analysed by 1H NMR and by HPLC. RESULTS: THE thiyl radicals abstract bisallylic hydrogens from [cis-9, cis-12]-methyl linoleate, yielding a pentadienyl radical. In the absence of oxygen, a thiyl radical-induced cis/trans-isomerization leads to linoleic-type isomers. These chain-type isomerization reactions can occur with the long living pentadienyl radical, followed by a 'repair' reaction of the attached thiol, and with the thiyl radical adduct with a double bond of the fatty acid residue. CONCLUSIONS: The results show that the mechanism of cis/trans-isomerization is an integral part of the thiyl radical attack on polyunsaturated fatty acids in homogeneous solutions and in bilayers.
A series of 3-mercaptoalkylthieno[2,3-d]pyrimidine-2,4(1H,3H)-diones 3 was prepared and their immuno-stimulating activity was examined. The title compounds were obtained conveniently by hydrolytic ring cleavage of fused thiazolo- or 1,3-thiazino-thienopyrimidines 1 under alkaline or acidic reaction conditions. The ms fragmentation of the thieno[2,3-d]pyrimidine-2,4-diones 3 is discussed. In the delayed type hypersensitivity (DTH) test some compounds 3 showed immuno-stimulating activities in the range of isoprinosine.
A 3-step synthesis, starting from substituted isatoic anhydride was used to prepare substituted 3-(2-mercaptoethyl)quinazoline-2,4(1H,3H)-diones 4. Reaction of 1 with cystamine afforded bis[2-(2-amino-benzoyl-amino)ethyl]disulfanes 2. Reaction of 2 with ethyl chloroformate and subsequent reduction of the heterocyclic disulfanes 3 gave mercaptoethylquinazoline-2,4-diones 4a-f.N-1 methyl and benzyl substituted derivatives 4b and 4c, respectively, show immuno-stimulating activity in various tests.
A modification of the MTT based tetrazolium colorimetric assay is described. Using the electron-coupling agent menadione formazan formation by murine splenocytes and P-815 cells was significantly increased whereas dye reduction by macrophages was hardly influenced. These observations suggest that it should be possible to improve the tetrazolium based cytotoxicity assays of murine macrophages against cells of the syngeneic tumour cell line P-815.
The presence of substance P (SP) and vasoactive intestinal peptide (VIP) in auricular lymph nodes of normal and sensitized guinea pigs and mice has been investigated using polyclonal antibodies directed against these neurotransmitters and the unlabelled peroxidase-antiperoxidase procedure. Positive immunoreactivity could be observed only in draining lymph nodes of sensitized animals. SP-immunoreactivity was found in mice and guinea pigs but a VIP-positive reaction only in mice. The immunopositive sites were always associated with blood vessels. The present study suggests that the neurotransmitters SP and VIP are involved in mechanisms of sensitization taking place in secondary lymphoid organs.
Reactions of 1,2,4-dithiazolidine-3,5-diylidene-bis(o-benzoquinone-met hid) 3 with alkyl and substituted benzyl or phenacyl halides yield 1,2,4-dithiazol-3-ylidene-quinone-methides 4a-4i. Mass spectral fragmentations of 4a, 4c, and 4f are discussed. Tests for immunomodulating properties of 4a-c, 4e, and 4g are described. Some of these compounds show immuno-stimulatory activity.
Guinea pigs immunized with DNP30-Ficoll produced IgM antibody only. No IgG1, IgG2, IgE antibodies or delayed hypersensitivity were detected in these animals. However, Arthus reactions, induced by the hapten coupled to a foreign carrier or the whole antigen, were found. The time course of the IgM response was limited and the response to reinjection of the antigen reduced. Cyclophosphamide (CY), given 3 days before primary immunization, prolonged the IgM response. Given on the day of immunization or 3 days after CY reduced this response. CY given on days +3 or +7 after primary immunization completely suppressed the response to antigen reinjected 42 days later. Arthus reactions were totally suppressed by CY given on the day of immunization, or 3 or 7 days later.
In guinea pigs primed by injection of bovine gammaglobulin (BGG) anti-BGG immune complexes (IC) prepared in antibody excess, in Freund's complete adjuvant, the effect of this treatment on the immune response induced at various days after IC priming and by injection of DNP-BGG has been investigated. The IC injection did not result in a generation of measurable anti-BGG antibodies but it led to a BGG-specific priming and sensitization. The latter was already there after 5 days, showed its maximum on day 9 and remained for two month. Priming for sub-class-specific anti-carrier and anti-hapten antibody synthesis revealed a different kinetics. While the enhancing mechanims for IgG2 antibody production had their optimum already after 9 days, the same phenomenon for the IgG1 response could be recognized much later (17th day for anti-BGG IgG1, 60th day for anti-DNP IgG1).
The effect of 100 mg/kg cyclosporin-A (CS-A), given as a single dose either before or after immunization, on antibody levels and skin test reactivity was investigated. CS-A was found to suppress both primary and secondary anti-hapten and anti-carrier IgG1 and IgG2 antibodies. However, CS-A was also capable of inducing enhanced anti-hapten antibodies. CS-A showed a similar effect on contact sensitivity reactions to 2,4-dinitrofluorobenzene (DNFB) as has been shown for cyclophosphamide (CY) in that, given before sensitization, skin reactions were enhanced, whereas given after sensitization they were suppressed. However, the effect of CS-A on the T-cell proliferation in the lymph node, draining the site of sensitization to DNFB, differed from that of CY. Although CS-A induced a depression of T-cell proliferation, this suppression was more prolonged than that found in CY treated animals. Also, these draining lymph nodes never showed increased T-cell proliferation as did those in animals treated with CY before sensitization. This work demonstrates that a single dose of CS-A can both suppress or enhance antibody production and delayed hypersensitivity. The timing of the dose of CS-A in relation to the time of primary immunization is important. However, both IgG1 and IgG2 primary and secondary antibody responses can be altered. Comparison with the effect of CY on antibody levels and contact sensitivity would indicate that in some ways CS-A reacts similarly to antimitotic agents and in other ways is different.
The intradermal injection of bovine gammaglobulin (BGG) anti-BGG immune complexes, in antibody excess, in Freund's complete adjuvant, induced strong delayed hypersensitivity and a state of priming which was recognized by an antihapten and anticarrier humoral immune response following injection of DNP9-BGG nine days later. Cyclophosphamide (CY), given either as small multiple doses over the first seven days after sensitization, or as a single dose on various days, had a strongly selective effect on the antihapten and anticarrier response. The drug treatment, using multiple doses, suppressed the development of a priming for the antihapten IgG2, but not for IgG1 production. Pretreatment with a large dose of CY (250 mg/kg) led only to an enhanced production of anti-DNP IgG2 and not of IgG1 antibodies. This suggests that CY inhibits the suppressor cells regulating the IgG2, but not the IgG1, antibody response. CY, given on day +3 but not on days 0 and +7 impaired both the IgG1 and the IgG2 response. Anticarrier-specific IgG1 could not be detected and the IgG2 was completely suppressed by the drug, given either as multiple doses or as a single large dose administered on days 0, +3 or +7. The selective influence of CY given about the time of the initial immunization suggests that the IgG1 and IgG2 responses involve memory cells with different susceptibilities to CY. Moreover, these memory cells appear to have a different susceptibility to CY as compared with that of the effector cells involved in delayed hypersensitivity.
Cyclophosphamide (CY) 250 mg/kg was given before, at the same time or after immunization with dinitrophenylated bovine gammaglobulin (DNP47-BGG) in Freund's incomplete adjuvant (FIA). CY, given on day +3 or +7 strongly suppressed both IgG subclasses. CY, on the day of immunization, reduced the IgG2 but not IgG1 anti-DNP antibody synthesis. Carrier BGG, injected 42 days after initial immunization, caused a secondary anti-hapten IgG1 response. This was not altered by CY treatment. The secondary anti-BGG response in animals pretreated with CY was also not affected. However, IgG1 anti-BGG in the other groups given CY on day 0, +3 or +7 was delayed and reduced. IgG2 anti-BGG was suppressed in guinea-pigs treated with CY, 3 or 7 days after primary immunization. DNP-BGG-specific IgE serum antibodies, not detectable in CY untreated animals, were found in guinea pigs given CY on days 0, +3 or +7. It is suggested that the kinetics of the IgG1 and IgG2 primary immune response differ, as does the development of IgG1 and IgG2 specific immunological memory.