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Biomedical subjects

G Wallukat

Publications and source records attributed to G Wallukat.

At least 19 recordsLinked to original sources
PubMed2006

Relation between circulating angiotensin II type 1 receptor agonistic autoantibodies and soluble fms-like tyrosine kinase 1 in the pathogenesis of preeclampsia.

CONTEXT: Placental and circulatory soluble fms-like tyrosine kinase 1 (sFlt1) has proven to be elevated in pregnant women with preeclampsia, a disease characterized by hypertension, proteinuria, and endothelial dysfunction. Recent studies also demonstrated an autoantibody against the angiotensin II type 1 (AT1) receptor (AT1-AA) in that disease. OBJECTIVE: Both factors are discussed as key players in the etiology of preeclampsia. However, it has not yet been clarified whether these two circulating factors correlate and whether synergy determines the severity of pathology. DESIGN: AT1-AA was retrospectively determined by a bioassay and sFlt1 by an ELISA. PATIENTS: Serum from second-trimester pregnancies with normal or abnormal uterine perfusion and in women at term with or without pregnancy pathology was analyzed. RESULTS: Most of the preeclamptic patients were characterized by high sFlt1 levels and the presence of AT1-AA, although the agonistic effects of the antibody did not correlate with the sFlt1 concentrations (P = 0.85). Although AT1-AA was also detected in second-trimester pregnancies evidencing abnormal uterine perfusion without later pathology, sFlt1 was not significantly elevated in these pregnancies, compared with those with normal uterine perfusion. However, whereas women with abnormal perfusion and later pregnancy pathology did not differ in AT1-AA, compared with those with normal outcome, sFlt1 was significantly increased. Again, the two factors did not correlate (P = 0.15). CONCLUSIONS: We conclude that AT1-AA bioactivity and sFlt1 concentrations do not correlate, are not mutually dependent, and are thus probably involved in distinct pathogenetic mechanisms. Both factors in combination may not be causative for the early impaired trophoblast invasion and pathological uterine perfusion.

Adult↗
PubMed2003

Norepinephrine-induced interleukin-6 increase in rat hearts: differential signal transduction in myocytes and non-myocytes.

Continuous i.v. infusion of norepinephrine in rats has been shown to induce early interleukin (IL)-6 mRNA expression in the left ventricle (LV) which was followed by hypertrophy and fibrosis. In this study, two approaches were used. In the first, NE (0.1 mg/kg per hour) was infused i.v. in rats for several time periods, and freshly obtained ventricular myocardium was dissociated into myocyte (MC) and non-myocyte (NMC) fractions. Second, isolated adult MCs and fibroblasts were treated with NE (10 microM). NE infusion (4 h, in vivo) caused an 11-fold increase in IL-6 mRNA in both cell populations. In vitro treatment of isolated adult MCs for 2 h and of fibroblasts for 1 h with NE induced a 3.5- and 23-fold maximum increase, respectively, in IL-6 mRNA. After in vivo NE treatment, the expression of the mRNA of the transcriptional factor of IL-6, C/EBP-beta, was elevated earlier (after 45 min of NE infusion) than IL-6 mRNA (after 4 h) and was seen in MCs and NMCs. The mRNAs of both receptors of IL-6, the soluble IL6R and gp130, were increased subsequently to IL-6 mRNA. Gp130 was elevated after 24 h and, like IL6R, predominantly in NMCs. In contrast, the IL6R protein and the downstream regulator STAT3 were increased only in MCs after 24 h of NE infusion. The mRNA of C/EBP-delta, which is regulated by STAT3, was elevated only in myocytes.

Adrenergic alpha-Agonists↗
PubMed2001

The upregulation of angiotensin II receptor AT(1) in human preeclamptic placenta.

The human placenta has been considered to possess a locally generated renin-angiotensin system (RAS), which may play a physiological role in the regulation of uteroplacental blood circulation. The changes in the expression of such a placental RAS during pregnancy could be important for the physiological and pathophysiological aspects of some clinical disorders, such as pregnancy-induced hypertension, preeclampsia. In the present study, the alterations of expression and localization of placental angiotensin II receptor subtypes, namely AT(1) in patients with preeclampsia (elective caesarean delivery) were investigated and compared with controls (vaginal delivery and elective caesarian delivery) using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry respectively. Results from RT-PCR analysis revealed an upregulated expression of placental mRNA for AT(1) receptor subtype in patients with preeclampsia when compared with those in controls. In addition, there was also a significant activation of placental expression of angiotensinogen mRNA in patients with preeclampsia. Results from Western blot showed that the expression of AT(1) receptor was also upregulated. Immunohistochemical results further demonstrated that increased immunoreactivity for placental AT(1) receptor was predominantly localized to the thin layers of syncytiotrophoblasts and, to a less extent, the capillaries of the term placental villi. These data indicate that upregulation of placental RAS components, notably AT(1) receptor in the syncytiotrophoblasts, could play a pathophysiological role in patients with preeclampsia.

Angiotensinogen↗
PubMed2001

Immunohistological changes in dilated cardiomyopathy induced by immunoadsorption therapy and subsequent immunoglobulin substitution.

BACKGROUND: Immunoadsorption (IA) and subsequent immunoglobulin (Ig) G substitution represent an additional therapeutic approach in dilated cardiomyopathy (DCM). It remains to be elucidated whether this treatment modulates myocardial inflammation, which is possibly a causal factor of ventricular dysfunction. METHODS AND RESULTS: From 25 DCM patients (EF <30%), 12 patients were randomized for IA therapy and subsequent IgG substitution at 1-month intervals until month 3. Before (<7 days) and after IA therapy, right ventricular biopsies were obtained from all patients. Biopsies were also obtained at intervals of 3 months from 13 patients without IA/IgG treatment (controls). IA/IgG treatment induced improvement in left ventricular ejection fraction from 21.3+/-1.7% (+/-SEM) to 27.0+/-1.3% (P<0.01 versus baseline/controls) and reduction of the beta-receptor autoantibody serum levels (P<0.01 versus baseline/controls). The number of CD3 cells decreased from 5.7+/-0.8 to 2.9+/-0.5 cells/mm(2) (P<0.01 versus baseline/controls). This decline was paralleled by a decrease in CD4 (P<0.01 versus baseline/controls) and CD8 (P<0.05 versus baseline/controls) lymphocytes. The number of leukocyte common antigen-positive cells (leukocytes) was reduced from 20.0+/-3.2 to 9.9+/-2.8 cells/mm(2) (P<0.01 versus baseline/P<0.05 versus controls). HLA class II expression decreased from 2.1+/-0.7% to 1.1+/-0.4% (P<0.05 versus controls/baseline). The number of immunopositive cells and the expression of HLA class II in controls remained stable. In both groups, the degree of fibrosis remained unchanged. CONCLUSIONS: IA and subsequent IgG substitution mitigate myocardial inflammation in DCM.

Autoantibodies↗
PubMed2001

A monoclonal antibody against the immunodominant epitope of the ribosomal P2beta protein of Trypanosoma cruzi interacts with the human beta 1-adrenergic receptor.

Monoclonal antibodies were raised against a recombinant ribosomal P2beta protein of Trypanosoma cruzi. One of these reacted with the C terminus of this protein (peptide R13, EEEDDDMGFGLFD) and epitope mapping confirmed that this epitope was the same as the one defined by the serum of immunized mice, and similar to the previously described chronic Chagas' heart disease (cChHD) anti-P epitope. Western blotting showed that the monoclonal antibody recognized the parasite ribosomal P proteins, as well as the human ribosomal P proteins. Electron microscopy showed that it stained different structures in parasite and human cells. Interestingly, surface plasmon resonance measurements indicated that the affinity for the parasite ribosomal P protein epitope (R13) was five times higher than for its human counterpart (peptide H13, EESDDDMGFGLFD). Since the human epitope contained an acidic region (EESDD) similar to the AESDE peptide recognized by cChHD patients in the second extra-cellular loop of the human beta1-adrenergic receptor, the biological activity of the antibody was assessed on neonatal rat cardiomyocytes in culture. The monoclonal antibody had an agonist-like effect. These results, together with the fact that the monoclonal reacted in Western blots with the different isoforms of the heart beta1-adrenergic receptor, confirm the possible pathogenic role of antibodies against the parasite ribosomal P protein based on their cross-reaction with the human beta1-adrenergic receptor.

Animals↗
PubMed2001

Autoantibodies against the beta1 adrenoceptor from patients with dilated cardiomyopathy prolong action potential duration and enhance contractility in isolated cardiomyocytes.

Autoantibodies against the beta1-adrenoceptor (beta1-AAB) from patients with dilated cardiomyopathy (DCM) increase the beating frequency of cultured neonatal rat cardiomyocytes. This effect is accompanied by only a small increase in cAMP production. Here we have investigated whether beta1-AAB affect electrophysiological properties and cell shortening of isolated cardiomyocytes by interacting with the beta1-adrenoceptor. Beta1-AAB were obtained during immunoadsorption of patients with DCM and were used for experiments in isolated myocytes cultured from neonatal rat hearts, or freshly isolated from adult rat ventricles or from human right atria. The unselective beta -adrenoceptor agonist (-)-isoprenaline was studied for comparison. Immunoglobulin G (IgG) antibodies increased the spontaneous beating frequency of neonatal rat cardiomyocytes to a lesser degree than (-)-isoprenaline, but both effects were maximum and stable after 2 min. In rat ventricular and human atrial myocytes, IgG increased action potential duration (APD) in a concentration-dependent manner with larger effects on late than on early repolarization phases. Similar effects were obtained with purified beta1-AAB, whereas flow through of the chromatography column was ineffective. (-)-isoprenaline prolonged APD to the same extent during plateau and late phase of repolarization. beta1-AAB increased L-Type Ca2+ current in correspondence with the prolongation of APD. The effects of beta1-AAB and (-)-isoprenaline on APD were strongly attenuated after preincubation of the myocytes with the selective beta1-adrenoceptor antagonist (-)-bisoprolol. In addition, beta1-AAB increased cell shortening in ventricular myocytes from adult rat hearts. Beta1-AAB enhancing the beating frequency of cultured cardiomyocytes, increase L-Type Ca2+ current, APD and contractility in freshly isolated cardiomyocytes mediated via beta1-adrenoceptors. These effects may contribute to beta1-adrenoceptor-mediated cardiotoxicity in heart failure.

Action Potentials↗
PubMed2001

HELLP syndrome in the 18th week of gestation in association with elevated angiotensin AT(1)-receptor autoantibodies.

We present a 24-year-old woman with a twin pregnancy who was with a typical HELLP syndrome at the 18th week of pregnancy. One fetus was dead, while the other was severely growth retarded. Our patient had agonistic autoantibodies directed at the angiotensin AT(1)-receptor. Termination of the pregnancy proved necessary. This report is the first to our knowledge associating HELLP syndrome with angiotensin AT(1)-receptor antibodies. Since the antibodies may have a pathogenic significance, their removal could permit the prolongation of pregnancy in preeclamptic and HELLP syndrome patients.

Adult↗
PubMed2001

Decreased oxidative stress in patients with idiopathic dilated cardiomyopathy one year after immunoglobulin adsorption.

OBJECTIVES: In a substudy to a recently reported investigation that demonstrated the benefit of immunoglobulin adsorption (immunoadsorption) for patients with idiopathic dilated cardiomyopathy (IDC), we tested whether this benefit is associated with a reduction of oxidative stress. BACKGROUND: The progression of cardiomyopathy is believed to be related to the increase of oxidative stress. Therefore, reduction of oxidative stress could be one of the effects of immunoadsorption for improvement of cardiac performance and clinical status. METHODS: Plasma markers for oxidative stress-thiobarbituric acid-reactive substances (TBARS), lipid peroxides (LPO), anti-oxidized low-density lipoprotein-autoantibodies (anti-oxLDL-AB), thiol groups and vitamin E-were compared in 31 patients, of whom 16 underwent immunoadsorption and 15 received conventional treatment (controls). All patients received a daily supplement of vitamins, minerals and trace elements. RESULTS: After one year, TBARS (p = 0.026), LPO (p = 0.026) and anti-oxLD

Adsorption↗
PubMed2001

Inducible nitric oxide synthase in the myocard.

Recognition of significance of nitric oxide synthases (NOS) in cardiovascular regulations has led to intensive research and development of therapies focused on NOS as potential therapeutic targets. However, the NOS isoform profile of cardiac tissue and subcellular localization of NOS isoforms remain a matter of debate. The aim of this study was to investigate the localization of an inducible NOS isoform (NOS2) in cardiomyocytes. Employing a novel immunocytochemical technique of a catalyzed reporter deposition system with tyramide and electron microscopical immunocytochemistry complemented with Western blotting and RT-PCR, we detected NOS2 both in rat neonatal and adult cultured cardiomyocytes and in the normal myocard of adult rats as well as in the human myocard of patients with dilative cardiomyopathy. NOS2 was targeted predominantly to a particulate component of the cardiomyocyte--along contractile fibers, in the plasma membrane including T-tubules, as well as in the nuclear envelope, mitochondria and Golgi complex. Our results point to an involvement of NOS2 in maintaining cardiac homeostasis and contradict to the notion that NOS2 is expressed in cardiac tissue only in response to various physiological and pathogenic factors. NOS2 targeting to mitochondria and contractile fibers suggests a relationship of NO with contractile function and energy production in the cardiac muscle.

Animals↗
PubMed2001

Expression of alpha1-adrenergic receptor subtypes in heart cell culture.

Three alpha1-AR subtypes have been cloned so far and are designated as alpha1a, alpha1b,, and alpha1d. Organ-specific distribution pattern and subtype-specific effects are known but not fully understood. To address a cell-type specific expression pattern in the heart we investigated expression pattern of alpha1-AR subtypes on RNA- and protein-level in heart tissue, cultured cardiomyocytes and non-myocytes of the rat. Each alpha1AR-subtype mRNA was present in neonatal and adult rat heart culture but the relative distribution pattern was significantly different. While the alpha1a-AR subtype is preferentially expressed in adult cardiomyocytes, the alpha1b-AR subtype was preferentially expressed in the non-myocyte cell fraction. The RT-PCR results were confirmed by Western-blotting (alpha1b) and immunocytochemical studies. Incubation with an alpha1-agonist (phenylephrine) for 72 h led to a significant reduction of the alpha1b-AR in neonatal heart cell culture on both mRNA and protein level. In contrast, incubation with an alpha1-antagonist (prazosin) induced a 1.6 fold upregulation of the alpha1a-AR mRNA without significant effects on radioligand binding and functional assay. The results indicate a distribution pattern of the alpha1-AR subtype which is specific for cell type and ontogeny of the rat heart and may be regulated by adrenergic agents.

Animals↗
PubMed2001

Acute hemodynamic effects during immunoadsorption in patients with dilated cardiomyopathy positive for beta 1-adrenoceptor autoantibodies.

In two recent studies immunoadsorption improved left ventricular function in patients with dilated cardiomyopathy (DCM) who were positive for cardiostimulatory autoantibodies against the human beta 1-adrenoceptor (beta 1-AAB). In this study we invasively measured acute hemodynamics during immunoadsorption. Three patients with DCM, who were positive for beta 1-AAB (NYHA-class III, left ventricular ejection fraction < 0.3), were treated with 1 immunoadsorption session daily on 5 consecutive days. Immunoadsorption induced a strong decrease of cardiac index, whereas systemic vascular resistance increased. Baseline levels were reached again several hours after therapy ended. We conclude that during immunoadsorption substantial hemodynamic changes occur in patients positive for beta 1-AAB.

Adult↗
PubMed2000

AT(1) receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor.

BACKGROUND: We recently described autoantibodies (angiotensin-1 receptor autoantibodies, AT(1)-AA) directed at the AT(1) receptor in the serum of preeclamptic patients, whose placentas are commonly infarcted and express tissue factor (TF). Mechanisms of how AT(1)-AA might contribute to preeclampsia are unknown. We tested the hypothesis that AT(1)-AA cause vascular smooth muscle cells (VSMC) to express TF. METHODS AND RESULTS: IgG from preeclamptic patients containing AT(1)-AA was purified with anti-human IgG columns. AT(1)-AA were separated from the IgG by ammonium sulfate precipitation. We transfected Chinese hamster ovary cells overexpressing the AT(1) receptor with TF promoter constructs coupled to a luciferase reporter gene. VSMC were obtained from human coronary arteries. Extracellular signal-related kinase activation was detected by an in-gel kinase assay. AP-1 activation was determined by electromobility shift assay. TF was measured by ELISA and detected by immunohistochemistry. Placentas from preeclamptic women stained strongly for TF, whereas control placentas showed far less staining. We proved AT(1)-AA specificity by coimmunoprecipitating the AT(1) receptor with AT(1)-AA but not with nonspecific IgG. Angiotensin (Ang) II and AT(1)-AA both activated extracellular signal-related kinase, AP-1, and the TF promoter transfected VSMC and Chinese hamster ovary cells, but only when the AP-1 binding site was present. We then demonstrated TF expression in VSMC exposed to either Ang II or AT(1)-AA. All these effects were blocked by losartan. Nonspecific IgG or IgG from nonpreeclamptic pregnant women had a negligible effect. CONCLUSIONS: We conclude that AT(1)-AA and Ang II both stimulate the AT(1) receptor and initiate a signaling cascade resulting in TF expression. These results show an action of AT(1)-AA on human cells that could contribute to the pathogenesis of preeclampsia.

Angiotensin II↗
PubMed2000

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.

Autoantibodies↗
PubMed2000

Phosphorylation of phospholamban at threonine-17 in the absence and presence of beta-adrenergic stimulation in neonatal rat cardiomyocytes.

The site-specific phospholamban phosphorylation was studied with respect to the interplay of cAMP- and Ca(2+)signaling in neonatal rat cardiomyocytes. To elucidate the signal pathway(s) for the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII) we studied Thr17 phosphorylation of phospholamban in dependence of Ca(2+)channel activation by S(-)-Bay K8644 and in dependence of the depletion of the sarcoplasmic reticulum Ca(2+)stores by ryanodine or thapsigargin in the absence or presence of beta -adrenergic stimulation. The isoproterenol (0.1 microM)-induced Thr17 phosphorylation was potentiated 2.5-fold in presence of 1 microM S(-)-Bay K8644. Interestingly, S(-)-Bay K8644 alone was also able to induce Thr17 phosphorylation in a dose- and time-dependent fashion. Ryanodine (1.0 microM) reduced both the isoproterenol (0.1 microM) and S(-)-Bay K8644-(1 microM) mediated Thr17 phosphorylation by about 90%. Thapsigargin (1 microM) diminished the S(-)-Bay K8644 and isoproterenol-associated Thr17 phosphorylation by 53.5+/-6.3% and 92. 5+/-11.1%, respectively. Ser16 phosphorylation was not affected under these conditions. KN-93 reduced the Thr17 phosphorylation by S(-)-Bay K8644 and isoproterenol to levels of 1.1+/-0.3% and 8.6+/-2. 1%, respectively. However, the effect of KN-93 was attenuated (47. 8+/-3.6%) in isoproterenol prestimulated cells. Protein phosphatase inhibition by okadaic acid increased exclusively the Ser16 phosphorylation. In summary, our results reflect a cross-talk between beta -adrenoceptor stimulation and intracellular Ca(2+)at the level of CaMKII-mediated phospholamban phosphorylation in neonatal rat cardiomyocytes. We report conditions which exclusively produce Thr17 or Ser16 phosphorylation. We postulate that Ca(2+)transport systems of the sarcoplasmic reticulum are critical determinants for the activation of CaMKII that catalyzes phosphorylation of phospholamban.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2000

Autoantibodies against the beta- and muscarinic receptors in cardiomyopathy.

The sera of patients with idiopathic dilated cardiomyopathy and the Chagas' disease contain agonist-like autoantibodies directed against the beta 1-adrenoceptor and/or the muscarinic M2-receptor. The anti-beta 1-adrenoceptor antibodies could be directed against amino acid sequences of the first or second extracellular loop. In patients with dilated cardiomyopathy the first as well as the second extracellular loop was identified as an antibody epitope. In Chagas' disease the anti-beta 1-adrenoceptor antibody recognizes only 1 epitope on the second extracellular loop. The anti-beta 1-adrenoceptor antibodies acting like the beta-adrenergic agonist isoprenaline and exert a positive chronotropic effect in cultured rat cardiomyocytes. In contrast to isoprenaline the antibody caused no downregulation of the beta-adrenergic signal transduction cascade within 6 hours. The anti-M2 receptor antibodies recognize in both diseases an epitope on the second extracellular loop. The anti-M2-receptor antibody exert a negative chronotropic response in cultured cardiomyocytes. This antibody induced no downregulation of the muscarinic M2-receptor. The negative chronotropic effect was unabated for 6 hours. Based on these findings it is believed that the agonist-like autoantibodies that act against the beta 1-adrenoceptor and the muscarinic M2-receptor may play a role in the pathogenesis of dilated cardiomyopathy and Chagas' disease.

Autoantibodies↗