Eberhard Nissen

Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor.

Immune mechanisms and the renin-angiotensin system are implicated in preeclampsia. We investigated 25 preeclamptic patients and compared them with 12 normotensive pregnant women and 10 pregnant patients with essential hypertension. Antibodies were detected by the chronotropic responses to AT1 receptor-mediated stimulation of cultured neonatal rat cardiomyocytes coupled with receptor-specific antagonists. Immunoglobulin from all preeclamptic patients stimulated the AT1 receptor, whereas immunoglobulin from controls had no effect. The increased autoimmune activity decreased after delivery. Affinity-column purification and anti-human IgG and IgM antibody exposure implicated an IgG antibody directed at the AT1 receptor. Peptides corresponding to sites on the AT1 receptors second extracellular loop abolished the stimulatory effect. Western blotting with purified patient IgG and a commercially obtained AT1 receptor antibody produced bands of identical molecular weight. Furthermore, confocal microscopy of vascular smooth muscle cells showed colocalization of purified patient IgG and AT1 receptor antibody. The protein kinase C (PKC) inhibitor calphostin C prevented the stimulatory effect. Our results suggest that preeclamptic patients develop stimulatory autoantibodies against the second extracellular AT1 receptor loop. The effect appears to be PKC-mediated. These novel autoantibodies may participate in the angiotensin II-induced vascular lesions in these patients.

Immunoglobulin Adsorption in Patients With Idiopathic Dilated Cardiomyopathy

BACKGROUND Idiopathic dilated cardiomyopathy (IDC) frequently is a progressive disease without causative therapy options. Following the hypothesis that in certain patients autoantibodies against cardiac structures may induce, maintain, or promote the progression of the disease, we investigated whether the elimination of these autoantibodies through immunoadsorption would improve cardiac function. METHODS AND RESULTS This prospective case-control study included 34 patients with IDC. Each patient presented with moderate to severe heart failure and evidence of autoantibodies directed against beta(1)-adrenoceptors (beta(1)-AABs). Seventeen patients received standard medical therapy (control group), whereas 17 were also treated with immunoadsorption (treatment group) to eliminate beta(1)-AABs. A 1-year follow-up included echocardiographic assessment of left ventricular ejection fraction and internal diameters, beta(1)-AAB levels, and clinical status every 3 months. Within 1 year, the mean+/-SD left ventricular ejection fraction rose from 22.3+/-3.3% to 37.9+/-7.9% (P=0.0001) in the treatment group, with a relative increase of 69.9%. However, in the control group, no overall increase was seen (from 23.8+/-3.0% to 25.2+/-5.9%, P=0. 3154). Left ventricular diameter in diastole decreased by 14.5% from 74.5+/-7.1 to 63.7+/-6.0 mm in the treatment group (P=0.0001) and by 3.8% (P=0.2342) in the control group. In the treatment group, the NYHA functional rating improved after immunoadsorption (P=0.0001). beta(1)-AABs did not increase anew. CONCLUSIONS In IDC, the use of immunoadsorption is superior to the use of standard medical therapy. It significantly improves cardiac performance and clinical status.

Agonist-like beta-adrenoceptor antibodies in heart failure

Anti-beta1-adrenoceptor antibodies may play a harmful role, and the elimination of these antibodies could have beneficial effects for some patients with dilated cardiomyopathy. In vitro experiments showed that the antibody was able to influence the function of cultured cardiomyocytes. In these experiments, the antibody prevented the down-regulation of the beta-adrenoceptor-mediated chronotropic response. This lack of desensitization, which resulted in permanent stimulation, could also influence the Ca2+ homeostasis of cardiomyocytes. However, in longer-term (72 hours)-treated cells, the antibodies were able to decrease subtype-specific expression of the beta1 adrenoceptor. In animal experiments, it was shown that long-term immunization with a peptide corresponding to the second extracellular loop of the beta1 adrenoceptor induced a failing heart similar to that in dilated cardiomyopathy. In humans, we observed a remarkable correlation between disappearance of the antibodies and improvement of heart function. Furthermore, in anti-beta1-adrenoceptor-positive patients with dilated cardiomyopathy treated with the immunoadsorption technique, removal of the antibodies also led to improvement of cardiac function and quality of life. This finding indicates that autoimmune processes may be involved in some patients with dilated cardiomyopathy.

Application of surfactin for mycoplasma inactivation in virus stocks.

Dear Editor: A novel procedure for the purification of virus stocks from mycoplasma contaminations was established using the lipopeptide antibiotic and biosurfactant surfactin, composed of a 3-hydroxy-13-rnethylmyristic acid forming a lactone ring system with an anionic heptapeptide as antimycoplasma agent. We combined the mycoplasma inactivation procedure for cell cultures with the results of antiviral studies (Vollenbroich et al., submitted for publication). The enveloped viruses SHV-1 and BVDV and the nonenveloped viruses EMCV and PPV were investigated. Mycoplasma contamination of ceils and virus stocks were monitored by poly chain reaction (PCR) (7,11). For the inactivation of mycoplasmas in cell cultures, different more or less efficient techniques are available, but the most effective procedure published is the treatment with antibiotics (2,9,10). In virological research, one of the common sources for contaminations are virus stocks harvested from mycoplasma-contaminated cell cultures, collected in the field, or passaged through animals. Therefore~ in virology not only the cell cultures but also the virus stocks must be freed from mycoplasmas. Only a few techniques are published for the treatment of virus stocks (4,5,6). Source ofsurfactin. Surfactin was obtained from culture supematants of Bacillus subtilis OKB105 by acid precipitation, extraction with methanol, charcoal treatment, and geI filtration on Pharmacia LH20 as described previously (1). Otherwise, suffaetin can be purchased from Biomol (Hamburg, Germany) or Sigma Chemical Co. (Deisenhofen, Germany). In our experiments, we used an autoclaved suffaetin solution of 1 nel4 in phosphate-buffered saline (PBS). Virus, cells, and culture conditions. The following cell virus systems were used: nonenveloped viruses: murine eneephalomyoearditis virus (EMCV) / Hep2 cells (ATCC CCL 231), porcine parvovirus (PPV strain NADL) 1 ST cells {ATCC CRL 1746); enveloped viruses: bovine diarrhea virus (BVDV) / KL cells (embryonal calf lung), swine herpes virus type 1 (SHY-l, Pseudorabies) / ML cells (mink lung). In the last, both cell lines are propagated in our institute for a long time. Virus stocks were a gift from Behring Research Laboratories (Mannheim, Germany) and prepared by infecting subconfluent celt monolayers with a multiplicity of infection (m.o.i.) of 10 -3. The culture supernatants were harvested when a pronounced cytopathic effect (CPE) approximately 2 to 6 d postinfection was visible. Aliquots of the cell free supernatants were stored at 70 ° C. Cell lines were propagated as monolayers in Dulbeccos modified Eagles medium (DMEM; ICN Bioinedicals GmbH, Eschwege, Germany) supplemented with 8% heat-inactivated fetal calf serum (FCS; GIBCO, Great Britain) in 25 cm 2 tissue culture flasks (Greiner GmbH, Heidelberg, Germany). Virus titration. The virus titers were determined by a standard microtitration assay. Approximately 1 to 2 × 104 cells in 100 }.tl medium were plated into each well of a 96-wetl mierotiter plate (Nunc, Copenhagen, Denmark). The virus solution was serially diluted 1:5 or 1:10 in culture medium and 100 pl of each dilution were added to each of 8 wells of a 96-well microtiter plate. The microtiter plates were incubated at 37 ° C and evaluated microscopically for CPE. When a pronounced CPE was visible, the 50% tissue culture infectivity doses (TC1Dso) were calculated according to Reed and Muench (8). M)coplasma detection. Mycoplasma contamination was detected by the highly sensitive PCR technique with a mycoplasma groupspecific primer pair as outlined elsewhere (7,11). M)roplasma inactivation procedure with surfactin. 0.5 ml of the virus stocks were diluted in 4.5 ml cell culture medium without FCS supplemented with 80 ~M surfactin for nonenveloped and 20 btM for enveloped viruses. The reaction mixtures were incubated at room temperature by gentle shaking for 2 h. Thereafter, subconfluent cultures of mycoplasma-free cells were infected with the surfactintreated virus. The procedure was repeated with viruses harvested from these cultures to ensure that all mycoplasmas have been removed. Mycoplasma detection and virus titer determination of samples from the culture supernatant were performed when a pronounced CPE was visible. Mycoplasma-free virus stocks were subcultivated

ANTI β1-ADRENOCEPTOR AUTOANTIBODIES ANALYZED IN SPONTANEOUSLY BEATING NEONATAL RAT HEART MYOCYTE CULTURES—COMPARISON OF METHODS

Dear Editor: Spontaneously beating cultured neonatal rat cardioinyocytes are a very useful model to investigate 13~-adrenoceptor autoantibodies (131-AR AAB) (Wallukat et al., 1995). AABs against G-protein-coupled receptors were observed in several cardiovascular diseases (Fu et al., 1994). Antibodies against the cq-adrenoceptor were observed in patients with essential hypertension. In sera of patients with idiopathic dilated eardiomyopathy (DCM) and endemic Chagas disease of south and central America autoantibodies were observed, which recognize the musearinic M2and the 13~-adrenoeeptor. All these AABs are directed against the second extracellular loop and, in cultured neonatal cardiomyocytes, exert a functional effect. The anti-131-AR AABs act like ~3-adrenergic agonists, exerting a positive chronotropie effect, which was blocked by the f3j-adrenoceptor antagonists bisoprolol and metoprolol (Magnusson et al., 1994; Wallukat et al., 1995). These agonist-like effects may elevate the adrenergic overdrive in these patients and could be involved in the pathogenesis of these diseases. This idea was supported by the finding that removal of the AABs by imnmnoadsorption improves the cardiac function in patients with DCM (Wallukat et al., 1996; DOfffel et al., 1997; Mtiller et al., 2000). There are different methods for determining these AAB levels (Magnusson et al., 1994; Wallukat et al., 1995; Jahns et al., 1999). Determination of ~ A R AAB levels is helpful for follow-up of therapy or assessment of time for weaning from mechanical support in patients with DCM (Mailer et al., 1997; Miiller et al., 2000). Here we compare a nonradioligand method of coimmunoprecipitation with the measurement of beating frequency of cultured rat eardiomyocytes and computer imaging systems (LUCIA and IMAGOQUANT). The eoimmunoprecipitation of ~t-AR was performed as follows. Lysed membranes (Daub et al., 1996) of 1-3 d old neonatal rat heart tissues or cultures (Wallukat et al., 1995) or C6 cells (glial tumor, rat, American Type Culture Collection, CCL 107) were incubated with anti-[31-AAB (5:1; v/v) for 1 h at room temperature. Antibody-antigen complexes were captured by Protein A sepharose (fast flow, Pharmacia; 2 h, 4 ~ C) and eluted with 60 txl sodium dodecyl sulfate (SDS) sample buffer (2 rain, 95 ~ C). SDS polyacrylamide gel electrophoresis (10% SDS gel) was performed. Proteins were identified by an antibody directed against a peptide with the sequence of the N-terminal part of 13t-AR produced in chicken (Affina GmbH, Germany) and detected by Western blot and ECL system with anti-chicken IgG peroxidase conjugates (whole molecule, Sigma). It is important to rinse all plastic vessels with bovine serum albumin (10 mg/ml). Table 1 shows results of coimmunoprecipitation. The two bands (molecular weight 47.5 and 65 kDa) could be accurately detected using internal standards of specific molecular weight. High optical density was measured in [3t-AR AAB-positive patients with DCM. The difference from healthy controls was significant (P < 0.05). Furthermore, f31-AR AABs were effectively eliminated through immunoadsorption (Wallukat et al., 1996). The intensity of the bands is significantly reduced after this treatment. The microscopic registration of the chronotropic effects of [3~-AR AABs on primary cultures of neonatal rat cardiomyocytes is a very sensitive bioassay. We found good correlation among the functional bioassay, the computer imaging systems and the immunoblot technique (Table 2). Prior to immunoadsorption, an increase of beating frequency/min (>15.0) and a high optical density (47.5 kDa: >1.000; 65 kDa: >0.200) were determined. After immunoadsorption, the beating frequency/min with all three methods (<3.0) and

SPONTANEOUSLY BEATING NEONATAL RAT HEART MYOCYTE CULTURE—A MODEL TO CHARACTERIZE ANGIOTENSIN II AT1 RECEPTOR AUTOANTIBODIES IN PATIENTS WITH PREECLAMPSIA

SummaryThis report focuses on angiotensin II AT1 receptor autoantibodies (anti-AT1-AABs) in preeclamptic women. An enzymelinked immunosorbent assay was described. Biotinylated peptide was incubated with anti-AT1-AABs. Streptavidin-coated magnetic particles bind the protein-autoantibody complex. Detection of anti-AT1-AABs was performed using anti-human IgG3 peroxidase-coupled antibody. The color reaction of tetramethylbenzidine solution was stopped by adding 0.5 M H2SO4. Optical density was measured at 450 nm (620 nm reference filter). Seventy-nine percent of anti-AT1-AAB-positive patients (measured by bioassay) showed an increase in optical density (>145%). The same biotinylated peptide was successfully used for purification of 6/6 anti-AT1-AABs. Chronotropic effects of purified antibodies were registered on primary cultured neonatal rat cardiomyocytes with the computer imaging system IMAGOQUANT. Western blot of coimmunoprecipitation of angiotensin II AT1 receptor shows one band (molecular weight >40.0 kDa) in potassium thiocyanate eluate.

Treatment of mycoplasma contamination

Dear Editor: Two papers (2,3) concerning treatment of mycoplasma-positive cell cultures were published in your journal during this year. Drexler et al. (2) recommend BM-Cyclin (BMC) hut Hlubinova et al. (3) propagate a more convenient method. This letter describes our experience with BMC (eight cell lines) and presents two representative examples. Cell cultures: MDCK/H (Mardine-Darby canine kidney) and NBE (human newborn kidney) cells were cultivated in Eagle s minimal essential medium (MEM) or Dulbecco s MEM containing 10% fetal bovine serum and standard antibiotics (penicillin, streptomycin). During the BMC treatment, medium without antibiotics was used. All cells were dispersed by 0.125% trypsin and 0.05% EDTA. Mycoplasma elimination: BMC treatment was carried out with 2.5-fold increase over the manufacturer s instructions (Boehringer Mannheim, Germany). Detection of mycoplasma: Detection was performed by DNA fluorochrome Bisbenzimide (1) or by enzyme-linked immunoassay (ELISA, Boehringer Mannheim). In addition to the discussion of the above-mentioned papers, the following points should be taken into consideration:

Effects of the Tryptase Receptor Activating Peptide and Antibodies Against the Tryptase Receptor Par-2 on Neonatal Rat Cardiomyocytes in Culture

We present data implicating mast cells within the myocardium in the development and progression of dilated cardiomyopathy (DCM). We show that the serine protease, mast cell tryptase, signals via the proteaseactivated receptor-2 (PAR-2). The receptor is expressed in abundance on fibroblasts and cardiomyocytes. We raised an antibody against the second extracellular loop of the PAR-2 receptor. This antibody had agonistic properties and acts in a fashion similar to the activating peptide SLIGKV. Our experiments implicate mast cells, mast cell tryptase, and PAR-2 receptor activation in DCM. They also shed light on possible autoimmune mechanisms contributing to DCM.