p53 autoantibodies in patients with autoimmune diseases: a quantitative approach.

With few exceptions, autoantibodies directed against the gene product of the tumor suppressor gene p53 are only detected in cancer patients. From 73 patients with various autoimmune diseases, we obtained 17 sera with elevated autoantibodies against the p53 protein comprising patients with SLE, Graves' disease, and immune vasculitis including Wegener's granulomatosis. The overall prevalence (23%) of p53 autoantibodies was comparable to that in various cancers; differences, however, were obvious with respect to the magnitude of antibody levels. Only 5% of seropositive colorectal cancer patients had levels within the critical range (150-180 U/ml) but nearly half (41%) of seropositive autoimmune disease patients were that low. None of the autoimmune disease patients exceeded 300 U/ml serum compared to more than 60% of seropositive colorectal cancer patients with higher levels. This remarkable difference in magnitude underlines the necessity of quantification of p53 autoantibodies over a mere qualitative determination. Patients with autoimmune diseases face an increased risk for malignancies. It still remains to be established whether p53 seropositivity in autoimmune diseases adds to the rare exceptions of p53AAb in non-malignant diseases or is indicative for a yet occult cancer.

The antibody reactivity of monoclonal lipid A antibodies is influenced by the acylation pattern of lipid A and the assay system employed.

The influence of the acylation pattern of lipid A on the reactivity of murine monoclonal antibodies (mAb) was tested in different assay systems with synthetic lipid A antigens. Both the number and type of fatty acids had an impact on the antigen amounts needed for optimal sensitization of sheep red blood cells, on the inhibition capacity of compounds and on the reactive antigen amounts in enzyme immunoassay and dot blot assay. Results obtained with two pentaacyl isomers indicated that the location of fatty acids is of no importance. Although all mAbs used recognized epitopes residing in the hydrophilic backbone of lipid A, their reactivities were greatly influenced by the number as well as the type of acyl chains present. In the various assays, the mAbs reacted either similarly or discrepantly suggesting that epitopes are exposed differently in the test systems. We conclude that for the determination of the reactivity of lipid A mAbs it is useful and sometimes necessary to run various assays in parallel and to compare mAbs on the basis of reaction patterns.

Cross-reactivity of monoclonal antibodies and sera directed against lipid A and lipopolysaccharides.

The cross-reactive capacity of monoclonal and polyclonal lipid A antibodies was tested with rough and smooth lipopolysaccharides (LPS). The antibodies represented different specificities recognizing epitopes in the hydrophilic lipid A backbone. In none of the various assay systems applied did the antibodies react with complete rough or smooth-form LPS. Cross-reactions, in general, were only detected with the most rudimentary rough LPS tested, i.e. Re-LPS. A variety of reactivities with other LPS was shown not to be related to lipid A antibodies; such reactivities were present in rabbit sera as well as in crude ascites. These results underline the need for careful checks on the origin of reactivities observed. In addition, rabbit antisera raised with R- and S-LPS were screened for lipid A reactivity using synthetic lipid A and partial structures as antigens. No cross-reactivity of LPS antibodies with lipid A was detected in these sera.

Immune response of rabbits to lipid A: influence of immunogen preparation and distribution of various lipid A specificities.

Sixty-two rabbit anti-lipid A serum samples were compared with respect to the immunogens used (synthetic lipid A and partial structures, natural lipid A, or acid-treated bacteria). Immunoglobulin (Ig) type-specific differences in rabbit response between liposomal membrane-embedded (LME) and other lipid A immunogens were found: LME lipid A elicited predominantly IgM antibodies. Previous findings of equally good immune responses to synthetic lipid A and acid-treated bacteria (L. Brade, E.T. Rietschel, S. Kusumoto, T. Shiba, and H. Brade, Infect. Immun. 51:110-114, 1986, and L. Brade, E.T. Rietschel, S. Kusumoto, T. Shiba, and H. Brade, Prog. Clin. Biol. Res. 231:75-97, 1987) turned out to be restricted to complement-fixing antibodies; IgG titers of sera against free lipid A (whether synthetic or natural) were significantly lower than those raised with bacteria. The results indicated an increase in IgG content of sera from LME lipid A over other free lipid A immunogens to acid-treated bacteria. These data underline the importance of the physicochemical environment for the immunogenicity of lipid A. As a second objective, the presence of various lipid A antibody specificities was tested with synthetic lipid A antigens. Antibodies to monophosphoryl lipid A were detected only in sera raised with monophosphoryl immunogens. Reactivity with monosaccharide partial structures of lipid A was found both in sera against monophosphoryl lipid A and in 60% of sera against bisphosphoryl lipid A. In the former, monosaccharide reactivity was of a magnitude similar to that of reactivity with lipid A; in sera against bisphosphoryl lipid A, it was lower. No reactivity or only marginal reactivity was found with phosphate-free lipid A, thus emphasizing the role of phosphate substitution for the lipid A epitopes recognized.

A complement-dependent enzyme immunoassay (C-EIA) with increased sensitivity for IgM-rich rabbit sera.

An enzyme immunoassay involving activation of complement (C-EIA) was developed for rabbit polyclonal IgM antibodies against lipid A and lipopolysaccharide antigens. C-EIA was significantly higher in sensitivity for IgM-rich rabbit sera compared to EIA using anti-immunoglobulin secondary antibodies. Hence, C-EIA should be useful for the detection of weak IgM reactivities in rabbit sera, especially after short-time immunizations. Selective inhibition of both complement pathways indicated that C-EIA measures activation of the classical pathway.

Characterization of the epitope specificity of murine monoclonal antibodies directed against lipid A.

A series of monoclonal antibodies directed against lipid A was characterized by using synthetic lipid A analogs and partial structures. These compounds vary in phosphate substitution, acylation pattern (type, number, and distribution of fatty acids), and, in the case of monosaccharides, in their backbone glycosyl residue. The monoclonal antibodies tested could be subdivided into five groups according to their reactivity patterns. One group reacted exclusively with 1,4'-bisphosphoryl lipid A, and a second also reacted with 4'-monophosphoryl lipid A. Two further groups recognized either 4-phosphoryl or 1-phosphoryl monosaccharide partial structures of lipid A. The fifth group reacted with 4-phosphoryl monosaccharide structures and with phosphate-free compounds. Antibodies reactive with monosaccharide structures also recognized their epitopes in corresponding phosphorylated disaccharide compounds. Both groups of monosaccharide and monophosphoryl lipid A-recognizing antibodies have access to their epitopes in bisphosphoryl compounds as well. Because of this unidirectional reactivity with more complex structures, the various specificities cannot be distinguished by using bisphosphoryl lipid A (e.g., Escherichia coli lipid A) as a test antigen. The epitopes recognized by the various monoclonal antibodies all reside in the hydrophilic backbone of lipid A, and there was no indication that fatty acids were part of the epitopes recognized. Nevertheless, the reactivities of compounds in the different test systems are strongly influenced by their acylation patterns; i.e., acyl groups may modulate the exposure of lipid A epitopes.

Determination of the epitope specificity of monoclonal antibodies against the inner core region of bacterial lipopolysaccharides by use of 3-deoxy-D-manno-octulosonate-containing synthetic antigens.

Partial structures of enterobacterial lipopolysaccharides (LPS) of the Rechemotype, consisting of lipid A and 3-deoxy-D-manno-2-octulosonic acid (Kdo), as well as oligosaccharides and derivative of Kdo were synthesized and used to characterize the epitope specificity of monoclonal antibodies against Re-mutant LPS. High-molecular-weight antigens, obtained after copolymerization of the respective allyl glycosides with acrylamide, and the haptenic oligosaccharides were used in immunoprecipitation, immune hemolysis, and in inhibition assays. A monoclonal antibody (clone 20, igM) recognizing a terminal Kdop group was shown to require for its binding the alpha-anomeric configuration and OH-4 and OH-5 groups, whereas the C-7 - C-8 chain was of minor importance. Another monoclonal antibody (clone 25, IgG3), which recognizes a (2--4)-linked Kdo disaccharide, was shown to require for its binding the alpha-anomeric configuration of both residues. The isomer having a reducing beta-Kdo residue was significantly less active, and that with a terminal beta-Kdo group was completely inactive. The OH-5 group of the reducing residue was shown to be not important for the specificity of this antibody, since it could be replaced by a hydrogen atom without loss of serological reactivity. The alpha-(2--8)-linked Kdo disaccharide was strongly cross-reactive with its (2--4)-linked isomer. The antibody recognized also parts of the 2-amino-2-deoxy-D-glucose backbone of lipid A.

ECA, the enterobacterial common antigen.

Enterobacterial common antigen (ECA) is a family-specific surface antigen shared by all members of the Enterobacteriaceae and is restricted to this family. It is found in freshly isolated wild-type strains as well as in laboratory strains like Escherichia coli K-12. The family specificity of ECA can be used for taxonomic and diagnostic purposes. ECA is located in the outer leaflet of the outer membrane. It is a glycophospholipid built up by an aminosugar heteropolymer linked to an L-glycerophosphatidyl residue. In a few rough mutants, in addition, the sugar chain can be bound to the complete lipopolysaccharide (LPS) core. Recently, for Shigella sonnei a lipid-free cyclic form of ECA was reported. The genetical determination of ECA is closely related to that of lipopolysaccharide. For biosynthesis of ECA and LPS partly the same sugar precursors and the same carrier lipid is used.

The immunogenicity and antigenicity of lipid A are influenced by its physicochemical state and environment.

We investigated the immunogenicity and antigenicity of synthetic lipid A and partial structures thereof. Included in the study were compounds which varied in the position of phosphate (1-mono-, 4'-mono-, and 1,4'-bisphosphates) and in the acylation (type, number, and distribution of fatty acids) and, in the case of monosaccharide compounds, the nature of the backbone sugar (D-glucosamine, D-glucose, 3-amino-3-deoxy-D-glucose, and 2,3-diamino-2,3-dideoxy-D-glucose). With the aid of the passive-hemolysis and passive-hemolysis-inhibition assays and by absorption experiments, five distinct antibody specificities were detected in polyclonal rabbit antisera raised against sheep erythrocyte-coated lipid A and lipid A incorporated into the membrane of liposomes (liposome-incorporated immunogens). Three antibody specificities reacted with disaccharide antigens specific for a 1-mono-, 4'-mono-, and 1,4'-bisphosphorylated beta-1,6-linked D-glucosamine disaccharide. Two antibodies reacted with either 1- or 4-phosphates of acylated D-gluco-configured monosaccharides and exhibited no cross-reaction with each other. However, they cross-reacted with disaccharide antigens with phosphate groups in the appropriate positions. We found that the physicochemical state and the environment of lipid A modulated its immunoreactivity. The immunogenicity was best expressed by erythrocyte-coated and liposome-incorporated immunogens. The antigenicity of lipid A was also greatly influenced by its physical surroundings. The reaction pattern of the above antibodies was highly specific in the hemolysis assay and in absorption experiments (the antibody reacted with antigen embedded in a cell membrane), whereas some cross-reactivities were observed in inhibition studies (the antibody reacts with antigen in aqueous solution). By using liposome-incorporated antigens as inhibitors, nonspecific reactions were avoided and specific ones were enhanced. Thus the antibodies described above against lipid A recognize epitopes in the hydrophilic backbone, the exposure of which depends on the intrinsic physicochemical properties of lipid A on the one hand and the physical environment on the other.

Chemical characterization of enterobacterial common antigen isolated from Plesiomonas shigelloides ATCC 14029.

Serologically characterized samples of enterobacterial common antigen (ECA) from Plesiomonas shigelloides, Salmonella montevideo and Shigella sonnei were investigated by chemical methods including methylation and NMR techniques. All showed the same sugar composition and contained a lipid moiety with palmitic acid as main fatty acid and with a phosphodiester group. Additional enzymatic studies, reported in the preceding paper, provided evidence that the lipid moiety is an L-glycerophosphatidyl residue attached via a phosphodiester linkage to C-1 of GlcNAc as the reducing end of the ECA sugar chain. ECA of P. shigelloides showed the best-resolved 13C-NMR spectra, especially after the removal of non-stoichiometric O-acetyl groups at C-6 of GlcNAc of the ECA repeating unit and of the lipid moiety by mild acid hydrolysis (0.01 M HCl, 100 degrees C, 10 min). Subsequent 13C-NMR studies were therefore carried out with the mild-acid-treated ECA of P. shigelloides which allowed a tentative assignment of all resonances of the ECA repeating unit. 13C-NMR spectra of Salmonella and Shigella ECA were essentially the same as those obtained with Plesiomonas ECA. The same trisaccharide repeating unit was encountered as demonstrated previously in the cyclic form of ECA isolated from S. sonnei by Dell et al. [Carbohydr. Res. 133, 95-104 (1984)]. Methylation analysis, however, afforded small amounts of terminal GlcNAc thus proving, in combination with the demonstration of the attached lipid moiety, an acyclic nature of ECA from P. shigelloides and from the two enterobacterial species. The question of whether the cyclic form co-exists in S. sonnei phase I and possibly in other enterobacterial species or, whether it had been formed during extraction as an artifact, has not yet been answered. The way in which ECA was isolated in our studies would preclude the presence of a non-amphiphilic (cyclic) polysaccharide. The finding that the sugar chain of ECA is attached to an L-glycerophosphatidyl residue is in full corroboration with serological, enzymatic and gel electrophoretic studies shown in the preceding paper and with the character of ECA as a surface antigen being anchored by hydrophobic interactions in the outer membrane of Enterobacteriaceae and P. shigelloides.

Comparison of enterobacterial common antigen from different species by serological techniques.

Enterobacterial common antigen (ECA) was isolated from a number of selected species (including Salmonella montevideo, Shigella sonnei and Plesiomonas shigelloides) using the extraction method described by Männel and Mayer [Eur. J. Biochem. 86, 361-370 (1978)]. ECA of all these species behaved identically in enzyme-linked immunosorption assay (ELISA) and in its inhibition using monoclonal anti-ECA antibodies. Immunoblotting showed a ladder-like pattern of at least 20 bands for all preparations tested. ECA modified at its lipid moiety (e.g. by phospholipases A2 and D or by mild acid hydrolysis) lost its coating capacity leaving, however, the serological reactivity as detected by inhibition assays intact. In contrast, reduction of the carboxylic groups of 2-acetamido-2-deoxy-D-mannopyranosyluronic acid destroyed the serological reactivity. Deacylated ECA was also not detectable in immunoblotting. Chemical reacylation restored the reactivity of deacylated ECA in ELISA and in immunoblot and thus proved the essential function of fatty acids for the physicochemical properties of the molecule. 2-Acetamido-2-deoxy-D-glucopyranose was identified as the reducing end of the ECA sugar chain after splitting off the lipid moiety by phospholipase D.

Biosynthesis of enterobacterial common antigen requires dTDPglucose pyrophosphorylase determined by a Salmonella typhimurium rfb gene and a Salmonella montevideo rfe gene.

In group C1 salmonellae, rfe and rff genes linked to the ilv locus specify the synthesis of a glycolipid called the enterobacterial common antigen. In contrast, in group B salmonellae the synthesis requires in addition some of the genes in the rfb cluster, the main genetic determinant of the O side chains of lipopolysaccharide. In an effort to define the biochemical functions of these rfb genes, we looked in Salmonella typhimurium LT2 (group B) for rfb mutants in which the synthesis of both enterobacterial common antigen and the O side chains would be blocked in a manner suppressible by the wild-type rfe cluster of S. montevideo, of group C1. We found one mutant with these characteristics. This rfb mutation affected the activity of dTDPglucose pyrophosphorylase (glucose-1-phosphate thymidylyltransferase, EC 2.7.7.24). Whereas the rfe cluster of S. montevideo contained a gene producing this enzyme activity, there was no evidence for the presence of such a gene in the rfe cluster of group B strains. These results also showed that the synthesis of dTDP-glucose is necessary for the biosynthesis of enterobacterial common antigen; this conclusion fits with the recent demonstration of 4-acetamido-4,6-dideoxy-D-galactose as a component of enterobacterial common antigen (Lugowski et al., Carbohydr. Res. 118:173-181, 1983), because the biosynthesis of the donor of this sugar, dTDP-4-acetamido-4,6-dideoxy-D-galactose, requires dTDPglucose pyrophosphorylase.

Production by various Pseudomonas species of a factor modifying the enterobacterial common antigen.

The enterobacterial common antigen (ECA) is a common determinant shared by almost all members of the family Enterobacteriaceae. The antigen modifies erythrocytes for agglutination by ECA antibodies. Previously it was reported that Pseudomonas aeruginosa produces a factor (PF) which destroys the erythrocyte-modifying capacity of ECA. The present investigation was undertaken to determine whether other species of this genus also produce PF. The passive bacterial hemagglutination and hemagglutination inhibition tests were used. It was observed that 47 strains belonging to 8 species of the genus Pseudomonas produce this factor and 34 strains representing 12 other species do not. Multiple strains of a given species gave concordant results. Mucoid variants of P. aeruginosa produced more of this factor than did nonmucoid isolates recovered from the identical sputum specimens from patients with cystic fibrosis. ECA treated with PF no longer modifies erythrocytes for agglutination by ECA antibodies and exerts less antibody-neutralizing capacity than untreated antigen.

Modification of the lipid moiety of the enterobacterial common antigen by the "Pseudomonas factor".

Pseudomonas aeruginosa produces a factor (PF) which affects the enterobacterial common antigen (ECA); resulting in failure of the antigen to modify erythrocytes for hemagglutination by ECA antibodies. In the present study the nature of PF was determined. Pronase treatment abolished its activity, indicating the protein nature of PF. PF-treated ECA no longer coated erythrocytes but still reacted with ECA antibodies in immunoelectrophoresis tests with monospecific antiserum to ECA, although differences were noted between the precipitation patterns of PF-treated and untreated ECA. Therefore, PF does not significantly affect the antigenic determinant of ECA but rather affects its lipid carrier, an L-glycerophosphatide. Accordingly, differences in the sugar chain could not be detected by high-voltage paper electrophoretic examinations of partial hydrolysates of PF-treated and untreated ECA. PF liberates all fatty acids from ECA, similarly to commercial lipases, as evidenced by the liberation of unsubstituted glycerol upon HF degradation at 0 degrees C of PF-treated ECA. The lipase activity of PF is indicated also by the observation that a strain of P. aeruginosa with reduced lipase production and an exolipase-negative strain affect ECA either less or not at all. We conclude that PF is a lipase acting on the lipid moiety of ECA, which is responsible for the coating of erythrocytes, but not significantly on the serological determinant, the amino sugar chain.

[Fibrinolytic therapy of lower limb deep vein thrombosis with urokinase (author's transl)]. / Thrombolytische Therapie der tiefen Beinvenenthrombose mit Urokinase.

In 81 patients with deep vein thrombosis of the lower limb, urokinase therapy was performed in combination with heparin according to a new regimen at higher dosages. When urokinase was administered at an initial maintenance dosage of 1,000-2,000 IU/kg/h (loading dose 150,000-250,000 IU), phlebographically documented complete or partial recanalization could be observed in 68% of the cases. The higher dosage schedule induced a more pronounced deobliteration especially in treatment of iliac vein thromboses (67% recanalization) in comparison to the lower dosage regimen (only 43% recanalization). Nearly comparable therapeutic results could be achieved in therapy of popliteal or saphenous vein thromboses. The data suggest that the higher dosage schedule examined here is indicated in treatment of extensive and large volume thromboses. The dosage of urokinase was further adjusted to attain a reduction of fibrinogen to 50-100 mg/dl. The concentration should not fall below 50 mg/dl. Therapy with urokinase proved practicable. Serious side effects did not occur. 8.6% of the patients showed hematuria and 6% a decrease of the Hb by more than 2 g/dl. The high proportion of older thromboses and the only low rate of recanalization (23%) in these cases suggest the necessity of an early commencement of fibrinolyses in therapy of deep vein thrombosis.

Effect of proteins on the immunogenicity of enterobacterial common antigen.

Enterobacterial common antigen isolated by two independent extraction procedures was found to precipitate with a number of basic or hydrophobic proteins. Complexes of enterobacterial common antigen with protamine sulfate, with methylated bovine serum albumin or with a fraction of outer membrane proteins of two different Shigella wild types proved to be highly immunogenic in rabbits upon intravenous immunization, in contrast to the enterobacterial common antigen preparations by themselves. This explains why crude isolates of enterobacterial common antigen usually are good immunogens in contrast to the isolated antigen, which was described to be either not or only very poorly immunogenic.

Urokinase therapy of subclavian-axillary vein thrombosis.

In 18 cases with primary subclavian-axillary vein thrombosis fibrinolytic therapy was performed with urokinase in combination with heparin. The thrombolytic efficacy clearly depended on the thrombus age and the dose of urokinase applied. Under treatment with a median initial maintenance dosage of urokinase of 1,000-2,000 IU/kg/h (loading dose 150,000-250,000 IU urokinase) in combination with heparin (15-17 U kg/h) in mine of 11 patients (82%) with recently developed (8 days or less) thrombosis, a nearly complete deobliteration of the venous system was observed. In the case with thrombosis of more than 10 days no alteration of the venous occlusions could be seen. Relevant side effects did not occur. Our results emphasize urokinase therapy of acute subclavian-axillary vein thrombosis and permit general inferences concerning the efficacy and the dosage requirements of the thrombolytic substance urokinase.