Refine
Has Fulltext
- yes (22)
Is part of the Bibliography
- yes (22)
Year of publication
Document Type
- Journal article (17)
- Doctoral Thesis (5)
Keywords
- antibodies (22) (remove)
Institute
- Neurologische Klinik und Poliklinik (6)
- Theodor-Boveri-Institut für Biowissenschaften (5)
- Institut für Molekulare Infektionsbiologie (4)
- Institut für Virologie und Immunbiologie (3)
- Medizinische Klinik und Poliklinik I (2)
- Institut für Anatomie und Zellbiologie (1)
- Institut für Hygiene und Mikrobiologie (1)
- Institut für Klinische Neurobiologie (1)
- Institut für Pharmakologie und Toxikologie (1)
- Julius-von-Sachs-Institut für Biowissenschaften (1)
Sonstige beteiligte Institutionen
Despite recent therapeutic advances the prognosis of heart failure remains poor. Recent research suggests that heart failure is a heterogeneous syndrome and that many patients have stimulating auto-antibodies directed against the second extracellular loop of the \(β_1\) adrenergic receptor \((β_1EC2)\). In a human-analogous rat model such antibodies cause myocyte damage and heart failure. Here we used this model to test a novel antibody-directed strategy aiming to prevent and/or treat antibody-induced cardiomyopathy. To generate heart failure, we immunised n = 76/114 rats with a fusion protein containing the human β1EC2 (amino-acids 195–225) every 4 weeks; n = 38/114 rats were control-injected with 0.9% NaCl. Intravenous application of a novel cyclic peptide mimicking \(β_1EC2\) (\(β_1EC2-CP\), 1.0 mg/kg every 4 weeks) or administration of the \(β_1-blocker\) bisoprolol (15 mg/kg/day orally) was initiated either 6 weeks (cardiac function still normal, prevention-study, n = 24 (16 treated vs. 8 untreated)) or 8.5 months after the 1st immunisation (onset of cardiomyopathy, therapy-study, n = 52 (40 treated vs. 12 untreated)); n = 8/52 rats from the therapy-study received \(β_1EC2-CP/bisoprolol\) co-treatment. We found that \(β_1EC2-CP\) prevented and (alone or as add-on drug) treated antibody-induced cardiac damage in the rat, and that its efficacy was superior to mono-treatment with bisoprolol, a standard drug in heart failure. While bisoprolol mono-therapy was able to stop disease-progression, \(β_1EC2-CP\) mono-therapy -or as an add-on to bisoprolol- almost fully reversed antibody-induced cardiac damage. The cyclo¬peptide acted both by scavenging free \(anti-β_1EC2-antibodies\) and by targeting \(β_1EC2\)-specific memory B-cells involved in antibody-production. Our model provides the basis for the clinical translation of a novel double-acting therapeutic strategy that scavenges harmful \(anti-β_1EC2-antibodies\) and also selectively depletes memory B-cells involved in the production of such antibodies. Treatment with immuno-modulating cyclopeptides alone or as an add-on to \(β_1\)-blockade represents a promising new therapeutic option in immune-mediated heart failure.
The pathogenic role of endogenous antibodies in a mouse model for Charcot-Marie-Tooth 1B neuropathy
(2015)
Charcot-Marie-Tooth (CMT) type 1 neuropathies are a genetically heterogeneous group of non-treatable inherited disorders affecting the peripheral nervous system that lead to sensory and motor dysfunction. Secondary low grade inflammation, implicating the innate and adaptive immune system, could previously be identified as a substantial disease modifier in two mouse models for CMT1, CMT1B and 1X, respectively. However, the exact mechanism how the adaptive immune system contributes to disease pathogenesis is not completely understood. Based on observations that the accumulation of endogenous antibodies to myelin components is important for rapid myelin clearance after nerve injury during Wallerian degeneration, a possibly similar mechanism was considered for endogenous antibodies as disease amplifier in mice heterozygously deficient for P0 (P0het), mimicking some typical features of CMT1B.
In this study an increased antibody deposition was detected in the affected peripheral nerves of P0het myelin mutant mice. By crossbreeding P0het mutants with mice specifically lacking B-lymphocytes, and therefore antibodies (JHD-/-), a decline of endoneurial macrophages together with a substantially ameliorated demyelination could be demonstrated in 6-month-old mutant mice. Moreover, reconstitution with murine IgGs reverted the neuropathic phenotype, substantiating that endogenous antibodies are potentially pathogenic at this early stage of disease. Unexpectedly, in 12-months-old P0het mutants, JHD deficiency resulted in disease aggravation accompanied by an increased inflammatory reaction and M2-polarized macrophage response.
These observations suggest that in a mouse model for CMT1B, the lack of endogenous antibodies has a dichotomous effect: ameliorating early macrophage-mediated demyelination, as opposed to increasing inflammatory reactions leading to disease aggravation at older ages.
Virotherapy on the basis of oncolytic vaccinia virus (VACV) infection is a promising approach for cancer therapy. In this study we describe the establishment of a new preclinical model of feline mammary carcinoma (FMC) using a recently established cancer cell line, DT09/06. In addition, we evaluated a recombinant vaccinia virus strain, GLV-5b451, expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as an oncolytic agent against FMC. Cell culture data demonstrate that GLV-5b451 virus efficiently infected, replicated in and destroyed DT09/06 cancer cells. In the selected xenografts of FMC, a single systemic administration of GLV-5b451 led to significant inhibition of tumor growth in comparison to untreated tumor-bearing mice. Furthermore, tumor-specific virus infection led to overproduction of functional scAb GLAF-2, which caused drastic reduction of intratumoral VEGF levels and inhibition of angiogenesis.
In summary, here we have shown, for the first time, that the vaccinia virus strains and especially GLV-5b451 have great potential for effective treatment of FMC in animal model.
Background: Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes.
Results: Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves.
Conclusions: We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission.
Autoimmune myositis encompasses various myositis-overlap syndromes, each being identified by the presence of serum marker autoantibodies. We describe a novel myositis-overlap syndrome in 4 patients characterized by the presence of a unique immunologic marker, autoantibodies to nuclear pore complexes. The clinical phenotype was characterized by prominent myositis in association with erosive, anti-CCP, and rheumatoid factor-positive arthritis, trigeminal neuralgia, mild interstitial lung disease, Raynaud phenomenon, and weight loss. The myositis was typically chronic, relapsing, and refractory to corticosteroids alone, but remitted with the addition of a second immuno-modulating drug. There was no clinical or laboratory evidence for liver disease. The prognosis was good with 100% long-term survival (mean follow-up 19.5 yr). By indirect immunofluorescence on HEp-2 cells, sera from all 4 patients displayed a high titer of antinuclear autoantibodies (ANA) with a distinct punctate peripheral (rim) fluorescent pattern of the nuclear envelope characteristic of nuclear pore complexes. Reactivity with nuclear pore complexes was confirmed by immunoelectron microscopy. In a cohort of 100 French Canadian patients with autoimmune myositis, the nuclear pore complex fluorescent ANA pattern was restricted to these 4 patients (4%). It was not observed in sera from 393 adult patients with systemic sclerosis (n = 112), mixed connective tissue disease (n = 35), systemic lupus (n = 94), rheumatoid arthritis (n = 45), or other rheumatic diseases (n = 107), nor was it observed in 62 normal adults. Autoantibodies to nuclear pore complexes were predominantly of IgG isotype. No other IgG autoantibody markers for defined connective tissue diseases or overlap syndromes were present, indicating a selective and highly focused immune response. In 3 patients, anti-nuclear pore complex autoantibody titers varied in parallel with myositis activity, suggesting a pathogenic link to pathophysiology. The nuclear pore complex proteins, that is, nucleoporins (nup), recognized by these sera were heterogeneous and included Nup358/RanBP2 (n = 2 patients), Nup90 (n = 1), Nup62 (n = 1), and gp210 (n = 1). Taken together the data suggest that nup autoantigens themselves drive the anti-nup autoimmune response. Immunogenetically, the 4 patients shared the DQA1*0501 allele associated with an increased risk for autoimmune myositis. In conclusion, we report an apparent novel subset of autoimmune myositis in our population of French Canadian patients with connective tissue diseases. This syndrome is recognized by the presence of a unique immunologic marker, autoantibodies to nuclear pore complexes that react with nups, consistent with an "anti-nupsyndrome.''
CD46 and CD134 mediate attachment of Human Herpesvirus 6A (HHV-6A) and HHV-6B to host cell, respectively. But many cell types interfere with viral infection through rapid degradation of viral DNA. Hence, not all cells expressing these receptors are permissive to HHV-6 DNA replication and production of infective virions suggesting the involvement of additional factors that influence HHV-6 propagation. Here, we used a proteomics approach to identify other host cell proteins necessary for HHV-6 binding and entry. We found host cell chaperone protein GP96 to interact with HHV-6A and HHV-6B and to interfere with virus propagation within the host cell. In human peripheral blood mononuclear cells (PBMCs), GP96 is transported to the cell surface upon infection with HHV-6 and interacts with HHV-6A and -6B through its C-terminal end. Suppression of GP96 expression decreased initial viral binding but increased viral DNA replication. Transient expression of human GP96 allowed HHV-6 entry into CHO-K1 cells even in the absence of CD46. Thus, our results suggest an important role for GP96 during HHV-6 infection, which possibly supports the cellular degradation of the virus.
Functionally active (conformational) autoantibodies directed against the β1-adrenergic receptor (β1-AR) are supposed to have a pathogenic relevance in human heart failure, particularly in idiopathic dilated cardiomyopathy (DCM). Prevalence of anti-β1-autoantibodies (anti-β1-aabs) in the healthy population is almost negligible, whereas it amounts to up to 30% in heart failure patients with idiopathic DCM. As β1-ARs are not restricted to the heart and are also highly expressed in particular segments of the nephron, it is conceivable that such autoantibodies might also affect kidney function to some extent through the activation of renal β1-ARs.
In the kidney, β1-ARs are highly abundant in the juxtaglomerular apparatus, the distal convoluted tubules, the collecting duct, and the renal arteries. However, the functional significance of β1-ARs at these particular sites along the nephron is poorly understood, as are the effects of conformational stimulating anti-β1-aabs on renal β1-ARs. From the available literature, it is well known that the β1-adrenergic system is involved in, e.g., the regulation of renin-secretion from juxtaglomerular cells. In addition, the β1-adrenergic system is thought to be involved in the regulation of the urine pH via type B-intercalated cells in the collecting duct. In contrast, the regulation of salt- and fluid-secretion in the medullary collecting duct appears to occur independently from the SNS.
As a consequence, the present work aimed to unravel the potential pathophysiological links between renal function, alterations in the cardiovascular system, and circulating agonist-like anti- β1-abs. We analyzed possible renal effects of anti-β1-abs in a human-analogous rat model. After immunization with a GST-fusion protein containing the second extracellular loop (β1-ECII) of the human β1-AR, Lewis-rats develop functionally active, stimulating, conformational anti-β1-ECII-abs. Within the first 6 months, anti-β1-ECII-ab-positive animals develop a hypertensive phenotype, which after 9 months evolves into a DCM phenotype.
In n=40 GST/ β1-ECII-immunized Lewis rats and n=40 age-matched, 0.9% NaCl-injected control animals, we sequentially (i.e. at months 1, 2, 3, 6, 9, 12, 15, and 18 after start of immunization) analyzed the changes in renal function on a molecular, functional, and structural level. We could show that the presence of stimulating anti-β1-ECII-abs – even though having detrimental effects on the heart – has only a minor impact on kidney function and structure. Within the first 3 months after induction of anti-β1-ECII-abs, the levels and activity of renin were significantly increased in immunized compared to corresponding control animals, which was confirmed by experiments on isolated perfused kidneys, in which anti-β1-ECII-abs were able to directly induce the liberation of renin. However, within several weeks the initial anti-β1-ECII-ab-mediated RAAS activation was counter-regulated by auto-regulatory mechanisms activated in the kidney. Similarly, glomerular filtration rate (GFR) and renal blood flow (RBF) were initially decreased in the presence of the stimulating anti-β1-ECII-abs, but returned to control values within 3 months after immunization of the animals. Although expression of several pro-fibrotic markers was significantly up-regulated in anti-β1-ECII-ab-positive rats, no significant differences were noted on a histomorphological level with regard to the occurrence of renal fibrosis, glomerular damage, tubular damage, and perivascular fibrosis. Only a mild decrease in glomerular filtration function was observed in the kidneys of anti-β1-ECII-ab-positive animals from immunization-month 12 on, apparent by increased levels of urinary protein.
Even though anti-β1-ECII-abs were able to induce mild changes in renal function, their effects were not strong enough to critically damage the kidneys in our rat-model. Differences between immunized anti-β1-ECII-ab-positive and corresponding control rats at later time-points (that is, from immunization-month 12 on) are most likely secondary to the progressive heart failure phenotype that immunized animals develop in the course of the experiment.
The present study is the first to focus on the effects of stimulating anti-β1-ECII-abs on the kidney, and on the prevalence of these effects for the heart (referred to as cardio-renal crosstalk). Although our results were obtained in a rat model, they might contribute to better understand the situation in anti-β1-AR-aab-positive human patients. Following the results of our experiments, treatment of such patients should focus on direct and specific neutralization/elimination of stimulating anti-β1-ECII-aab or at least comprise therapeutic strategies that counteract the anti-β1-ECII-aab-effects on the heart by standard treatment for heart failure (i.e. ACE inhibitors, AT1-receptor blockers, and β-blockers) according to current guidelines.
Many pathogenic bacteria cause local infections but occasionally invade into the blood stream, often with fatal outcome. Very little is known about the mechanism underlying the switch from local to invasive infection. In the case of Neisseria gonorrhoeae, phase variable type 4 pili (T4P) stabilize local infection by mediating microcolony formation and inducing anti-invasive signals. Outer membrane porin PorBIA, in contrast, is associated with disseminated infection and facilitates the efficient invasion of gonococci into host cells. Here we demonstrate that loss of pili by natural pilus phase variation is a prerequisite for the transition from local to invasive infection. Unexpectedly, both T4P-mediated inhibition of invasion and PorBIA-triggered invasion utilize membrane rafts and signaling pathways that depend on caveolin-1-Y14 phosphorylation (Cav1-pY14). We identified p85 regulatory subunit of PI3 kinase (PI3K) and phospholipase Cγ1 as new, exclusive and essential interaction partners for Cav1-pY14 in the course of PorBIA-induced invasion. Active PI3K induces the uptake of gonococci via a new invasion pathway involving protein kinase D1. Our data describe a novel route of bacterial entry into epithelial cells and offer the first mechanistic insight into the switch from local to invasive gonococcal infection.
Experimental Adaptation of Wild-Type Canine Distemper Virus (CDV) to the Human Entry Receptor CD150
(2013)
Canine distemper virus (CDV), a close relative of measles virus (MV), is widespread and well known for its broad host range. When the goal of measles eradication may be achieved, and when measles vaccination will be stopped, CDV might eventually cross the species barrier to humans and emerge as a new human pathogen. In order to get an impression how fast such alterations may occur, we characterized required adaptive mutations to the human entry receptors CD150 (SLAM) and nectin-4 as first step to infect human target cells. Recombinant wild-type CDV-A75/17red adapted quickly to growth in human H358 epithelial cells expressing human nectin-4. Sequencing of the viral attachment proteins (hemagglutinin, H, and fusion protein, F) genes revealed that no adaptive alteration was required to utilize human nectin-4. In contrast, the virus replicated only to low titres (102 pfu/ml) in Vero cells expressing human CD150 (Vero-hSLAM). After three passages using these cells virus was adapted to human CD150 and replicated to high titres (105 pfu/ml). Sequence analyses revealed that only one amino acid exchange in the H-protein at position 540 Asp→Gly (D540G) was required for functional adaptation to human CD150. Structural modelling suggests that the adaptive mutation D540G in H reflects the sequence alteration from canine to human CD150 at position 70 and 71 from Pro to Leu (P70L) and Gly to Glu (G71E), and compensates for the gain of a negative charge in the human CD150 molecule. Using this model system our data indicate that only a minimal alteration, in this case one adaptive mutation, is required for adaptation of CDV to the human entry receptors, and help to understand the molecular basis why this adaptive mutation occurs.
Millionen Menschen weltweit leiden an den verschiedensten Autoimmunerkrankungen. Diese Krankheiten entstehen, wenn das Immunsystem gesundes körpereigenes Gewebe angreift und zerstört. An der Pathogenese sind sowohl Komponenten des angeborenen Immunsystems als auch Bestandteile des adaptiven Immunsystems, wie Lymphozyten und Antikörper, beteiligt. Da die Ursachen und molekularen Mechanismen der Pathogenese dieser Erkrankungen bis heute weitgehend unbekannt sind, wurden in dieser Arbeit autoaggressive Lymphozyten bei den humanen Autoimmunerkrankungen Polymyositis und Multiple Sklerose näher untersucht. Die Polymyositis ist eine chronisch entzündliche Erkrankung der Skelettmuskulatur. Die Muskelfasern werden dabei von zytotoxischen CD8+ gd-T-Lymphozyten infiltriert, attackiert und schließlich zerstört. In einem seltenen Fall der Polymyositis wurden die Muskelzellen hingegen in ähnlicher Weise von CD8- gd-T-Lymphozyten angegriffen. Die gd-T-Lymphozyten waren monoklonal expandiert und ihr Rezeptor, im Folgenden als M88 bezeichnet, wurde als Vg1.3+Vd2+ identifiziert. Frühere Untersuchungen der Antigenspezifität dieser Zellen zeigten, dass M88 mehrere funktionell und strukturell verschiedene Proteine aus unterschiedlichen Spezies erkennt. Die Bindung erfolgt spezifisch durch die Antigenerkennungsregionen beider Rezeptorketten von M88. In dieser Arbeit wurden verschiedene bakterielle und humane Proteine des Translationsapparates als Antigene von M88 identifiziert. Weitere ausführliche Untersuchungen eines paradigmatischen bakteriellen Antigens, dem Translationsinitiationsfaktor EcIF1, zeigten, dass M88 an Oberflächen-exponierte Konformationsepitope von Proteinen bindet. Interessanterweise erkennt M88 mehrere humane Aminoacyl-tRNA-Synthetasen, Antigene, die in anderen Formen der Myositis von Autoantikörpern angegriffen werden. Diese Beobachtung ergibt eine bemerkenswerte Verbindung zwischen T-Zell- und Antikörper-vermittelten B-Zell-Antworten bei der autoimmunen Myositis. Bei der Multiplen Sklerose ist das zentrale Nervensystem betroffen. Autoaggressive Lymphozyten greifen die Myelinschicht der Nervenzellen im Gehirn und Rückenmark an und zerstören sie. Im Liquor cerebrospinalis von Patienten lassen sich klonal expandierte und affinitätsgereifte B-Zellen sowie „oligoklonale Banden“ (OKB) Antikörper nachweisen. Obwohl diese Merkmale auf eine Antigen-induzierte Immunantwort hindeuten, sind die zugrundeliegenden Antigene und die Rolle der OKB bei der Pathogenese bis heute unbekannt. In dieser Arbeit wurde die Antigenspezifität von fünf IgG OKB-Antikörpern aus drei Patienten untersucht. Durch verschiedene proteinbiochemische Methoden konnten intrazelluläre Kandidatenantigene identifiziert werden. Interessanterweise sind darunter mehrere nukleäre Proteine, die an der Transkriptionsregulation oder der RNA-Prozessierung beteiligt sind. Reaktivitäten gegen intrazelluläre Antigene treten auch bei anderen Autoimmunerkrankungen, wie beispielsweise dem systemischen Lupus erythematodes, auf. Diese Ergebnisse könnten auf einen allgemeinen Mechanismus der Entstehung und Funktion von Autoantikörpern bei diesen humanen Autoimmunerkrankungen hindeuten.