Fibrinolysis inhibitors in plaque stability: a morphological association of PAI-1 and TAFI in advanced carotid plaque.

Essentials Fibrinolysis inhibitors are localized in advanced atheroma by immunohistology of endarterectomies. Neovascular endothelium/neocapillaries show thrombin-activatable fibrinolysis inhibitor (TAFI). Macrophage areas show free plasminogen activator inhibitor (PAI-1), notably in the vulnerable part. Free PAI-1 and TAFI stabilize active plaque area by inhibition of fibrinolysis and inflammation. SUMMARY: Background Fibrinolysis plays an important role in destabilization of atherosclerotic plaques and is tightly regulated by specific inhibitors. Objective The fibrinolysis inhibitors plasminogen activator inhibitor type-1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI) were quantified and described in the morphological context of advanced carotid plaques American Heart Association VI-VIII to elucidate their role in plaque stability. Methods Immunohistochemistry in serial sections along the longitudinal axis of endarterectomies from patients with symptomatic carotid stenosis (n = 19) were studied using an antibody specific for free PAI-1 (I205), an antibody with high affinity for TAFI/TAFIa (CP17) and established antibodies for smooth muscle cells (α-actin), endothelial cells (von Willebrand factor [VWF]), macrophages (CD68) and platelets (CD42). Results PAI-1 and TAFI show a specific distribution in these advanced plaques with a maximum corresponding to the internal carotid artery (ICA). Free PAI-1 was mainly detected in macrophages and in intravascular thrombi, and TAFI in endothelial cells (ECs) but also macrophages. The one-way ANOVA analysis with Bonferroni's correction showed a significant increase of macrophages and ECs, TAFI and PAI-1 in areas with high neovascularization in endarterectomy sections corresponding to ICA. High Spearman factors for TAFI, PAI-1 and VWF indicate neovascularization as the main source of plasma proteins, transported by platelets into the atheroma (PAI-1) or expressed by ECs (TAFI). CD68 was highly associated with VWF, PAI-1 and especially TAFI, underlining the role of macrophages in fibrinolytic activity and inflammation. Conclusion The abundance of free PAI-1 and TAFI in the plaque may inhibit plasmin generation and thereby counteract plaque destabilization by fibrinolysis, cell migration and inflammation.

Brefeldin A, but not monensin, completely blocks CD69 expression on mouse lymphocytes: efficacy of inhibitors of protein secretion in protocols for intracellular cytokine staining by flow cytometry.

Flow cytometry is increasingly used for cytokine detection where it serves to complement ELISA (enzyme-linked immunosorbent assay) and ELISPOT assays. Since it is possible to stain both extracellular epitopes and intracellular cytokines on the same cells, this is a powerful technique for analysing cytokine expression in defined cell populations. However unstimulated cells do not express cytokines. Thus, appropriate stimulation is a prerequisite for studying cytokine expression. Here phorbol 12-myristate 13-acetate (PMA)/ionomycin in vitro stimulation has been applied. In order to accumulate the cytokines within the cells, protein secretion needs to be inhibited, by the addition of reagents that inhibit protein secretion during the stimulation. The two most widely used reagents are monensin and brefeldin A (BFA). These reagents differ somewhat in their mode of action, which might explain their different effects. Monensin is an inhibitor of trans-Golgi function, while BFA inhibits protein transport between the endoplasmic reticulum (ER) and the Golgi. CD69, a very early activation marker on lymphocytes and neutrophils, was monitored in order to measure the efficacy of the protein secretion inhibition. Here we report that: (a) BFA, but not Monensin, is able to completely block extracellular CD69 expression on mice splenocytes after in vitro stimulation with PMA/ionomycin; (b) Monensin is more toxic than BFA and increases the relative amount of CD4+ cells due to a more profound increase in dead cells in the CD4- population; (c) CD69 is a useful marker when setting up intracellular staining of cytokines for flow cytometry.

A rat model of chronic Helicobacter pylori infection. Studies of epithelial cell turnover and gastric ulcer healing.

BACKGROUND: Our aim was to infect rats with Helicobacter pylori and to study the effects of the infection on the gastric mucosa in normal and in ulcer-operated rats. METHODS: A mouse-adapted H. pylori (cagA-, VacA-) strain was inoculated into 23 rats. Another 20 uninfected rats served as controls. Two months later a gastric ulcer was induced in some rats. The animals were killed 3, 6, or 15 days after the ulcer operation. Tissues were taken for histology and for culture of H. pylori. Serum antibodies were determined. RESULTS: All inoculated rats were infected by H. pylori after 2 months, mainly in the antrum. In these rats a mild to moderate chronic inflammation and a significantly increased frequency of apoptotic cells were observed in the antrum and in the ulcer margin, the ulcer healing was delayed, and the serum level of H. pylori-specific Ig was increased. CONCLUSIONS: H. pylori infection in rats was successful and was accompanied by a mild to moderate mucosal inflammation. Gastric ulcer healing was delayed in infected rats, probably due to the inflammation and the increased apoptosis in epithelium.

Isolation of human NuMA protein.

NuMA is a protein involved in maintenance of nuclear structure and in the assembly of the mitotic spindle. Expression of amino-terminal deletion mutants results in a phenotype identical to that caused by a temperature-sensitive defect of RCC1 (regulator of chromosome condensation). Here we describe the isolation of NuMA protein from HeLa cells under mild conditions as a prerequisite to study its interactions with elements of the RCC1-Ran regulatory pathway. In an overlay assay, NuMA did not bind Ran.[gamma-32P]GTP. Thus it is clearly different from Ran.GTP binding proteins of similar M(r).

Characterization of a 58- and a 78-kD monocytic membrane protein with affinity to the acetylcholine receptor in myasthenia gravis patients.

The autoimmune disease myasthenia gravis (MG), caused by the effect of specific antibodies, directed towards the nicotinic acetylcholine receptor, is triggered by autoantigen-specific T cells. In order to investigate cellular parts of the immune response in MG, the authors investigated the binding of the nicotinic acetylcholine receptor (AChR) to peripheral blood mononuclear cells (PBMC) from MG patients. AChR binding cells were identified by rosetting experiments using AChR-coated fluorescein beads. Applying this technique, a significant percentage of PBMC (21.2 +/- 7.65%) from MG patients formed rosettes with AChR-coated beads. Membrane preparations of nycodenz- or percoll-separated monocytes from MG patients or T-cell depleted monocytic subpopulations were applied to SDS-PAGE under reducing conditions. Ligand-blotting studies with biotinylated AChRs revealed two cell-membrane proteins with molecular weights of 58- and 78-kD. In parallel the same results were obtained by affinity chromatography of monocytic membrane proteins using AChR-sepharose. A possible interference of anti-AChR IgG was excluded. The 58- and the 78-kD proteins are detectable under reducing conditions by ligand blotting with AChR-biotin, while under non-reducing conditions only the 58-kD protein can be detected. Furthermore, in experiments using Endoglycosidase-H, the 58-kD protein appears to be non-glycosylated, while the 78-kD protein bears carbohydrates. These findings suggest that monocytes which bind the AChR via specific membrane proteins on their surface might act as antigen-presenting cells and may lead to an induction of the T-cell response, in the early phase of the disease.

Tumor-cell killing by MAbs against fucosyl GM1, a ganglioside antigen associated with small-cell lung carcinoma.

Monoclonal antibodies (MAbs) reactive with the ganglioside fucosyl GM1 (Fuc-GM1), an antigen associated with small-cell carcinoma of the lung (SCLC), were tested for their ability to mediate tumor-cell killing in vitro in conjunction with humoral and cellular effectors and to inhibit tumor engraftment in nude mice in vivo. MAbs F12 and F15, both IgG3k, induced human complement-mediated cytolysis of 3 Fuc-GM1-expressing tumor cell lines: one rat hepatoma cell line, H4-II-E, and 2 human SCLC cell lines, NC1 H69 and NC1 H128. F12 and F15 also induced ADCC of these cell lines in the presence of either murine or human effector cells. Addition of sub-cytolytic amounts of fresh human serum as complement source resulted in enhanced ADCC induced by MAb F12 (IgG3). Also a Fuc-GM1-reactive MAb of IgM isotype, F9, was able to induce such complement-aided ADCC (CADCC). F12 and F15 both proved to effectively inhibit engraftment of H4-II-E tumors in nude mice. A single dose of a modest amount (40 micrograms) of MAb conferred 65 to 100% protection against development of tumors. Our results demonstrate that Fuc-GM1 can act as a target antigen on tumor cells for specific immunotherapy in vitro and in a mouse model in vivo. Complement and murine and human mononuclear effector cells were effective mediators of tumor cytolysis in vitro in the presence of murine Fuc-GM1-reactive MAbs. Our results also suggest that humoral and cellular effectors may co-operate in specific tumoricidal reactions and that these may be induced by antibodies of both IgG and IgM isotypes.

Monoclonal antibodies monospecific to single-chain urokinase-type plasminogen activator (scu-PA).

Until now, single-chain urokinase-type plasminogen activator (scu-PA) could not be distinguished from urokinase (UK) by immunological methods. Therefore, in our study monoclonal antibodies (mAbs) specific for scu-PA were raised in mice. These mAbs proved to discriminate between scu-PA, UK and plasmin-activated scu-PA. The clones producing these mAbs could be cultivated in a serum-free medium. Furthermore, very pure preparations were obtained by a one-step purification procedure on protein-A-sepharose. The mAbs described provide a future potential for immuno-affinity chromatography and for the specific determination of scu-PA.

Characterization of a protein with an acetylcholine receptor epitope from myasthenia gravis-associated thymomas.

Immunohistochemical studies have shown that almost all thymomas of myasthenia gravis patients contain at least one protein sharing an antigenic determinant with the nicotinic acetylcholine receptor (AchR) of human muscle. We describe the characterization of this protein (p153) which has a molecular weight of 153 and an isoelectric point of 5.0. By treatment of p153 with endoglycosidases, no significant glycosylation has been detected. Immunologically, p153 crossreacts with monoclonal antibodies against the amino acid sequence 371-378 of the alpha-chain of the AchR. No cross-reactivity to the main immunogenic region of the AchR nor an alpha-bungarotoxin binding site are found. By Western blotting, p153 was generally neither detectable in normal tissues nor extrathymic tumors with the exception of paraganglioma and neuroblastoma. In conclusion, the structure of p153 is apparently unrelated to the AchR from muscle or the alpha-bungarotoxin binding proteins from thymoma. Since there is no evidence for an AchR expression in thymoma, the antigenic homology of p153 with the nicotinic AchR might be relevant for triggering an intrathymomatous autosensitization of maturing T cells and could be responsible for the high association of thymomas with myasthenia gravis.

Acetylcholine receptor epitope in proteins of myasthenia gravis-associated thymomas and non-thymic tissues.

Immunohistochemical studies have shown that almost all thymomas of myasthenia gravis (MG) patients contain proteins which share antigenic determinants with the nicotinic acetylcholine receptor (AChR) of human muscle. Here we describe one of the proteins (p153) which (1) is not part of a compound structure, (2) has a MW of 153 kd, (3) has an isoelectric point of 5.0, and (4) is probably free of sugar residues. The protein does not bind mAb to the main immunogenic region of the AChR and has no alpha-bungarotoxin (alpha-btx) binding site. p153 was not found in both normal tissues and a variety of tumours. However, the epitope defined by mAb155 also occurs in at least two other proteins (from muscle and TE671 cells) which have MW different from 153 kd and which are unrelated to AChR. Experiments presented elsewhere [Geuder et al., this volume] show that there are no proteins in thymomas which share an extensive molecular homology with the AChR. All these findings suggest that p153 is unrelated to the AChR. As p153 is the only protein demonstrated in thymomas which is significantly correlated with MG and which shares an antigenic determinant with AChR, p153 is a candidate protein determining the AChR-specificity of the autoimmune process in MG.

Constant isotype pattern of anti-dsDNA antibodies in patients with systemic lupus erythematosus.

The isotype profile, particularly emphasizing IgG subclass distribution, of dsDNA antibodies in patients with systemic lupus erythematosus was evaluated using an especially adapted ELISA technique. Anti-dsDNA antibodies were quantified with class-specific antisera and subclass-specific monoclonal antibodies. IgG subclass specificity was proven with 20 myeloma proteins differing in light chains and allotypes. The standardization with myeloma proteins proved to be useful and reliable. Results from more than 100 anti-dsDNA positive sera from SLE patients showed specific antibodies within the three subclasses (IgG1: 52-100%, IgG2: 0-39%, IgG3: 0-48%). IgG4 was not detected in significant amounts. No correlation to the subclass distribution of total IgG was found. Each patients' serum displayed an individual isotype pattern that remained constant in longitudinal studies, independent of anti-dsDNA titre fluctuations. These results suggest a stable population of autoreactive clones in the progress of the disease.

Antigen-gelonin conjugates. Preparation and application in experimental myasthenia gravis.

The antigen-specific immune suppression by gelonin-antigen conjugates was tested in two different systems: (i) the horseradish-peroxidase-stimulated T-cell proliferation in vitro and (ii) in vivo with experimental autoimmune myasthenia gravis (EAMG) in the rat. For this, the phytotoxin gelonin, a glycoprotein from Gelonium multiflorum, was purified and linked to the respective antigens. For the in-vitro assay a lymph node cell suspension from rats immunized with horseradish peroxidase was cultured in the presence of this protein and proliferation was measured by [3H]thymidine uptake. In-vitro proliferation was significantly inhibited by adding gelonin-horseradish peroxidase conjugates. The therapeutic effects of antigen-gelonin conjugates were tested in the rat model EAMG. For these experiments rats were immunized with purified nicotinic acetylcholine receptor from electric fish in order to develop EAMG. The success of the immunization was monitored by the change in physical performance tests, the change in anti-acetylcholine receptor antibody titer, and by the change in the number of ionic endplate channels using a novel electrophysiological method. The latter method permits a very accurate assay of functional damage of acetylcholine receptor at the endplate and correlates well with the clinical severity of the disease. Rats were conventionally immunized with acetylcholine receptor from electric fish. After the onset of EAMG as measured by physical performance tests and rise in antibody titer a group of the animals was injected with an acetylcholine receptor-gelonin conjugate and this treatment was repeated seven days later. The loss in functional acetylcholine receptor was significantly smaller in the therapy group than in the untreated EAMG group.(ABSTRACT TRUNCATED AT 250 WORDS)

Patient-specific heterogeneity of antinuclear antibodies as revealed by an isoelectric focusing immunoblot system.

Ninety-nine sera from patients with different rheumatic diseases (systemic lupus erythematosus, rheumatoid arthritis, progressive systemic sclerosis, and mixed and unidifferentiated connective tissue disease) were applied to a newly developed isoelectric focusing (IEF) immunoblot system for the demonstration of antinuclear antibodies. Nucleoproteins were separated according to their isoelectric points (pI) and immobilized onto nitrocellulose, and binding of serum antibodies was determined by an alkaline phosphatase labelled second antibody. 89.8% of all sera positive in indirect immunofluorescence assays with Hep 2 as substrate showed positive reactivity in IEF immunoblot. Furthermore, 88% of patients' sera negative on Hep 2 cells gave a positive reaction in IEF immunoblot. The predominant antibody banding pattern observed showed parallel bands in the acidic as well as the neutral pH ranges. Antibody specificities found in the IEF immunoblot system turned out to be patient-specific, but no marker antibody for a discrete disease entity was obtained. Even when monoclonal antibodies or WHO standard sera were applied to nuclear antigen they exhibited heterogeneity in their binding pattern. Bands with the same pI were observed using sera from patients with different rheumatic disease entities. Immunodeletion experiments suggest the recognition of identical antigenic proteins by the different patients' sera.

Myasthenia gravis: stimulation of antireceptor autoantibodies by autoreactive T cell lines.

We studied autoreactive acetylcholine receptor (AChR)-specific T cell lines from two patients with myasthenia gravis. Anti-AChR autoantibody production by peripheral blood mononuclear cells (PBM) from the donors of the T cell lines was measured with an enzyme-linked immunoadsorbent assay, using purified human AChR as antigen. Freshly isolated PBM produced barely detectable amounts of anti-AChR autoantibodies. If, however, autologous AChR-specific T cells were added to the cultures, the production of anti-AChR autoantibodies, and of total IgM and IgG, was markedly stimulated, depending on the number of T line cells and on the amount of AChR present in the cultures. AChR-specific functional helper T-lymphocytes may have a role in the immunoregulation of myasthenia gravis.

Genetic restriction of autoreactive acetylcholine receptor-specific T lymphocytes in myasthenia gravis.

Human autoreactive helper T lymphocytes with specificity for acetylcholine receptor (AChR) were isolated from three HLA-DR3-positive patients who had myasthenia gravis (MG), an autoimmune disease known to be associated with HLA-DR3 in the North European population. The antigen-specific T cells were evaluated for genetic restriction. Antigen presentation studies were performed with mitomycin C-treated accessory cells from a panel of HLA-typed unrelated donors. AChR-induced proliferation of the autoreactive T cells was maximal in the presence of autologous or HLA-DR-compatible antigen-presenting cells. In two DR-heterozygous patients both parental DR specificities served as restriction elements of the polyclonal AChR-reactive T cell populations. Preferential restriction to HLA-DR3 was observed in one patient, but this was also seen with PPD-specific T cells from the same donor. A series of monoclonal antibodies against HLA class II molecules was used for inhibition experiments. The inhibitory effects of the antibodies were not due to unspecific toxicity and could be observed after separate treatment of the antigen-presenting cells but not of the responding T cells. Several monoclonal antibodies against monomorphic HLA-DR determinants (DA 231, MAS 53, MAS 54, L243, OKIa1) had pronounced inhibitory effects. Anti-HLA-DQ(DC) monoclonal antibodies (Leu-10; TU 22) had only mild or no inhibitory effects in two patients but significantly inhibited AChR-specific T cells in one patient. A monoclonal antibody against HLA-DR3 (antibody 16.23) was not or was only weakly inhibitory in the DR3-positive patients, although it bound to autologous T line cells and B cells by indirect immunofluorescence. In one patient it was possible to compare the inhibition patterns of AChR-specific and PPD-specific T cells. Most of the monoclonal antibodies affected AChR- and PPD-specific T cells to a similar extent, but three antibodies (TU 22, 36, 39) inhibited PPD-specific T cells more than AChR-specific T cells, indicating the possibility of differential restriction of antigen- and autoantigen-specific T cells. It is suggested that the in vitro system described here may be helpful for the evaluation of anti-HLA class II antibodies as potential immunotherapeutic reagents.

Physiochemical and immunological properties of acetylcholine receptors from human muscle.

The acetylcholine receptor protein from human muscle was extracted with the non-ionic detergent Triton X-100 and purified by affinity chromatography on alpha-Naja toxin sepharose 4B. Further purification on Dicap-MP sepharose 4B, a choline analog compound, led to ACHR preparations with specific activities of 2-7 nmol/mg protein. The isolated receptor, labeled with 125I-alpha-bungarotoxin was characterized by different methods and compared to ACHRs from Torpedo californica electroplax and rat-denervated skeletal muscle. Gel filtration on Ultrogel AcA 34 resulted in a stokes radius of 70 A for the receptor monomer and 99 A for the dimeric form. Sucrose density gradient centrifugation showed sedimentation coefficients of 9.1 S and 13.5 S. From these data the molecular weight of the ACHR monomer was estimated as 254 000 D and 540 000 D for the receptor dimer. The isoelectric point of the 125I-alpha-bgt-ACHR complex was determined by thin-layer isoelectric focussing to be pH 5. Purified ACHRs were used for immunization of rats and mice which developed an EAMG as verified by clinical observation and electrophysical measurements. Sera from the immunized animals as well as from myasthenia gravis patients were subsequently used to compare the cross-reactivity of ACHR preparations from different sources. While antibodies of rats immunized with Torpedo ACHRs cross-reacted with ACHR preparations from rat and human skeletal muscle, antibodies from mice immunized with rat ACHR only reacted with preparations from rats and mice. Antibodies from mice immunized with ACHR of human origin exhibited a broad cross-reactivity, as did antibodies from MG patients.

Autoimmune human T lymphocytes specific for acetylcholine receptor.

Myasthenia gravis is one of the best characterized human autoimmune disorders. Circulating autoantibodies to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction play a prominent part in the effector phase, that is, the immunoregulation. Indirect evidence, such as thymic abnormalities and the association with certain histocompatibility antigens (for example HLA-B8,-DR3) suggests a defect of immunoregulation at the level of thymus-dependent (T) lymphocytes. We report here on the isolation of autoreactive T cells from six patients with myasthenia gravis. From one of these patients, who is homozygous for HLA-DR3, we established a long-term T-cell line. The line cells are specific for purified fish and human AChR, display the surface phenotype of inducer/helper T cells and are genetically restricted to HLA-DR3. AChR-induced proliferation could be inhibited with two monoclonal antibodies against monomorphic DR determinants and also with DR3-specific alloantiserum.

[Heterogeneity of acetylcholine receptor antibodies in patients with myasthenia gravis]. / Heterogenität von Acetylcholinrezeptor-Antikörpern bei Myasthenia gravis Patienten.

Antibodies against the nicotinic acetylcholine receptor in sera of 21 myasthenia gravis patients were checked for their ability to block or split binding of alpha-bungarotoxin to the human acetylcholine receptor. Affinity-purified acetylcholine receptors from human skeletal muscle were used in parallel in the common precipitation assay and an inhibition assay. Cross-reactivity of acetylcholine receptor antibodies was analyzed with receptor preparation from different species (calf, rat, Torpedo c. and Electrophorus e.), purified identically to high specific activity. An antibody pattern was set up for each patient and related to the clinical state of the disease. alpha-Bungarotoxin-inhibiting antibodies were demonstrable in 74% of myasthenia gravis patients, alpha-bgt displacing antibodies were found in 39% of the investigated sera. Broad cross-reactivity with acetylcholine receptors from other mammalian muscle was evident (calf 75%, rat 90%) only very few sera reacted with acetylcholine receptors from electric fish (Torpedo c. 14%, Electrophorus e. 38%). Antibody concentrations determined by using xenoantigens were much lower than those obtained by human acetylcholine receptor. The lack of a clear-cut correlation between the amount of serum antibodies and the clinical state of myasthenia gravis can be explained by the established antibody-heterogeneity, shown by a constant antibody pattern characteristic for each patient. However, between this specific antibody pattern and the state of the disease no correlation could be established either.