Integrins and their ligands in rheumatoid arthritis

Integrins play an important role in cell adhesion to the extracellular matrix and other cells. Upon ligand binding, signaling is initiated and several intracellular pathways are activated. This leads to a wide variety of effects, depending on cell type. Integrin activation has been linked to proliferation, secretion of matrix-degrading enzymes, cytokine production, migration, and invasion. Dysregulated integrin expression is often found in malignant disease. Tumors use integrins to evade apoptosis or metastasize, indicating that integrin signaling has to be tightly controlled. During the course of rheumatoid arthritis, the synovial tissue is infiltrated by immune cells that secrete large amounts of cytokines. This pro-inflammatory milieu leads to an upregulation of integrin receptors and their ligands in the synovial tissue. As a consequence, integrin signaling is enhanced, leading to enhanced production of matrix-degrading enzymes and cytokines. Furthermore, in analogy to invading tumors, synovial fibroblasts start invading and degrading cartilage, thereby generating extracellular matrix debris that can further activate integrins

Slit3 inhibits Robo3-induced invasion of synovial fibroblasts in rheumatoid arthritis

Introduction: The repellent factor family of Slit molecules has been described to have repulsive function in the developing nervous system on growing axons expressing the Robo receptors. However, until today no data are available on whether these repellent factors are involved in the regulation of synovial fibroblast (SF) activity in rheumatoid arthritis (RA). Methods: mRNA expression in primary synovial fibroblasts was quantified by quantitative reverse transcription PCR and protein expression was measured by fluorescence activated cell sorting (FACS) analysis. Different functional assays were performed with rheumatoid arthritis synovial fibroblasts (RASF): proliferation, migration and a novel in-vitro cartilage destruction assay. Results: First, we found increased expression of Robo3 expression in RASF compared to normal SF. Interestingly, analysis of data from a recently published genome-wide association study suggests a contribution of ROBO3 gene polymorphisms to susceptibility of RA. Functional assays performed with RASF revealed induction of migration and cartilage destruction by Robo3 and increased matrix metalloproteinase (MMP)1 and MMP3 expression. Treatment of RASF in early passages with Slit3 led to inhibition of migration whereas RASF in later passages, having reduced Robo3 expression in cell culture, were not inhibited by Slit3 treatment. Here, reduction of Robo3 expression from passage 3 to 10 might reflect an important step in losing repulsive activity of Slit3. Conclusions: Taken together, our data showed that deregulation of the Robo3 receptor in synovial fibroblasts in RA correlates with aggressiveness of the fibroblasts. Slit3 reduces the migratory activity of synovial cells from patients with RA, potentially by repulsion of the cells in analogy to the neuronal system. Further studies will be necessary to prove Slit activity in vivo

A sustained high fat diet for two years decreases IgM and IL-1 beta in ageing Wistar rats

Background The immune system undergoes several alterations of innate and adaptive immunity during ageing. The main features of the aged immune system are a reduced diversity of T cell receptors and a reduced activity of innate immune cells with subsequent changes in adaptive immunity resulting in a less effective, less specific, and dys-regulated immune response and in an increased susceptibility towards infection, malignancy, and autoimmunity. The process is referred to as immunosenescence and is also modulated by environmental modifiers, such as dietary factors. High fat diet (HFD), via direct modulation of immune cell function by fatty acids and/or increased body fat mass, influences immune function. However, it is not clear whether HFD is beneficial or detrimental for the functioning of the ageing immune system. Methods Male Wistar rats fed with either a high fat diet (HFD 43 en% of fat) or control diet (SD, 25 en% of fat) over up to 24 month and were analyzed for plasma IL-1β, IL-6, TNF, IgM, IgG1, IgA, IgG2a, IgG2b, IgG2c, light chains lambda and kappa, testosterone, prolactin and percentage of splenic B cells and apoptosis rate, respectively. Results In general, all analyzed immunoglobuline isotypes increased with age, except for IgA. This increase was attenuated by HFD. In HFD and SD rats the percentage of B cells in the spleen and also their apoptotic rate was lower in aged as compared to young animals with no additional diet-induced effect. Testosterone and prolactin levels were lower in old animals, as expected. There was a statistical trend towards an increased prolactin/testosterone ratio in middle aged (6–12 monthsnth) HFD rats as compared to SD. IL-6 was neither affected by HFD nor age. On the other hand, HFD rats showed a decrease in IL-1β as compared to SD, which correlated with the above-mentioned suppressive effect on immunoglobulin isotypes, especially IgM. Conclusion In Wistar rats, HFD reveals an immunosuppressive effect in ageing animals by decreasing immunoglobulins, especially IgM, and IL-1β when compared to SD

Anti-inflammatory effects of cell-based therapy with tyrosine hydroxylase-positive catecholaminergic cells in experimental arthritis

Objectives: Studies in rheumatoid arthritis (RA), osteoarthritis (OA) and mice with arthritis demonstrated tyrosine hydroxylase-positive (TH+) cells in arthritic synovium and parallel loss of sympathetic nerve fibres. The exact function of TH+ cells and mode of TH induction are not known. Methods: Synovial cells of RA/OA were isolated and cultured under normoxic/hypoxic conditions with/without stimulating enzyme cofactors of TH and inhibitors of TH. We studied TH expression and release of cytokines/catecholamines. In vivo function was tested by cell therapy with TH+ neuronal precursor cells (TH+ neuronal cells) in DBA/1 mice with collagen type II-induced arthritis (CIA). Results: Compared with normoxic conditions, hypoxia increased TH protein expression and catecholamine synthesis and decreased release of tumour necrosis factor (TNF) in OA/RA synovial cells. This inhibitory effect on TNF was reversed by TH inhibition with α-methyl-para-tyrosine (αMPT), which was particularly evident under hypoxic conditions. Incubation with specific TH cofactors (tetrahydrobiopterin and Fe2+) increased hypoxia-induced inhibition of TNF, which was also reversed by αMPT. To address a possible clinical role of TH+ cells, murine TH+ neuronal cells were generated from mesenchymal stem cells. TH+ neuronal cells exhibited a typical catecholaminergic phenotype. Adoptive transfer of TH+ neuronal cells markedly reduced CIA in mice, and 6-hydroxydopamine, which depletes TH+ cells, reversed this effect. Conclusions: The anti-inflammatory effect of TH+ neuronal cells on experimental arthritis has been presented for the first time. In RA/OA, TH+ synovial cells have TH-dependent anti-inflammatory capacities, which are augmented under hypoxia. Using generated TH+ neuronal cells might open new avenues for cell-based therapy

P136 SLEDAI response prediction to belimumab therapy by baseline levels of BLyS, APRIL and CD8+ effector memory T-cells

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Association Between Changes in BLyS Levels and the Composition of B and T Cell Compartments in Patients With Refractory Systemic Lupus Erythematosus Treated With Belimumab

Introduction: Belimumab is a monoclonal antibody against soluble BLyS used for treatment of refractory Systemic Lupus Erythematosus (SLE). Although B cells are the main target of this therapy, a BLyS-dependent T cell activation pathway has also been demonstrated. The aim of the study is to analyze B and T cells phenotype modifications in a cohort of SLE patients treated with belimumab in correlation with serum BLyS levels.Materials and Methods: Fourteen SLE patients were enrolled in the study. Lymphocyte immunophenotyping by flow cytometry and determination of serum BLyS levels by high sensitivity ELISA were performed before the first infusion of belimumab, after 6 and 12 months of treatment. Sex and age-matched healthy controls were enrolled for the comparisons.Results: Baseline number of total B cells, especially switched memory B cells, were lower in SLE patients compared to control subjects. After 6 months of treatment, the total number of B cells, particularly, naive and transitional B cells, was significantly reduced in correlation with the reduction of BLyS levels. No significant association was found between baseline counts of B cells and reduction of SLEDAI-2K over time. In terms of response prediction, a significant association between SLEDAI-2K improvement at 12 months and the decrease of total number of B cells within the first 6 months of therapy was observed. Concerning the T cell compartment, the baseline percentage number of CD8+ effector memory was associated with SLEDAI-2K at baseline and with its improvement after 12 months of therapy. Furthermore, T cell lymphopenia and low number of circulating recent thymic emigrants were also observed compared to control subjects measured at baseline.Discussion: The effects of belimumab on B cell subpopulations could be explained by the direct blockage of soluble BLyS, while the mild effects on T cells might be explained indirectly by the reduction of disease activity by means of therapy. B cell immunophenotyping during belimumab might be useful for monitoring the response to treatment

Charakterisierung rekombinant produzierter Virus-ähnlicher Partikel des humanen Parvovirus B19

Das Kapsid des ältesten bekannten humanpathogenen Vertreters innerhalb der Parvoviridae, Parvovirus B19, besteht aus zwei Strukturproteinen VP1 und VP2. Das VP2-Protein stellt im viralen Partikel einen Anteil von 95 %. Es spielt eine wichtige Rolle bei der T-Zell-Antwort und VP2-spezifische konformationelle Epitope induzieren eine lang anhaltende Immunantwort. Rekombinant hergestellte VP2-Kapside im Baculovirus-Expressionssystem sind für viele Tests zum Nachweis von Parvovirus-B19-spezifischen Antikörpern ein wichtiges Antigen. Ziel vorliegender Arbeit war es, ein alternatives Expressionssystem zu etablieren, das VP2- und VP1/VP2-Mischkapside in hoher Qualität und Ausbeute produzieren kann. Der Grund für die Etablierung eines alternativen Expressionssystems sind die hohen Kosten und die umständliche Handhabung die das Baculovirussystem auszeichnen. Die rekombinant produzierten Virus-ähnlichen Partikel sollten molekularbiologisch, histologisch sowie immunologisch charakterisiert und mit Kapsiden aus dem Baculovirus-Expressionssystem verglichen werden. Als eukaryontische Expressionssysteme standen die Bäckerhefe, Saccharomyces cerevisiae, Drosophila-Schneider-2-Zellen sowie die trypanosomenartigen Leishmania-tarentolae-Zellen zur Verfügung. Einzig in der Bäckerhefe war es möglich, VP2-Kapside zu produzieren. Als nächstes wurde die Kapsidsynthese optimiert. Dabei wurden verschiedene Anzuchtsbedingungen, Nährmediumzusammensetzungen und Hefestämme getestet. Der Protease-defiziente Saccharomyces-Stamm HT393 produzierte im Vergleich zu dem kommerziell erhältlichen Stamm INVSc2 deutlich mehr VP2-Protein und zeigte weniger Proteindegradation. Desweiteren wurden Methoden zur Reinigung der rekombinanten VP2-Kapside entwickelt und anschließend optimiert. Der Aufschluss der Bäckerhefe gelang über Hochdruck, der über 95 % der Zellen zerstörte. Die Aufreinigung der VP2-Kapside wurde über mehrere Ultrazentrifugationsschritte erreicht. Gewählt wurde eine Kombination aus Differential- und isopyknischer Zentrifugation. Diese beiden Ultrazentrifugationstechniken lieferten den besten Kompromiss aus Ausbeute und Reinheit. Im Elektronenmikroskop konnten keine Unterschiede zwischen Partikeln aus der Bäckerhefe, Baculovirus infizierten Insektenzellen oder nativen Viren festgestellt werden. Ähnlich waren auch die Ergebnisse aus vergleichenden ELISA-Tests und dem T-Zell-Proliferationsassay. Demnach haben in Bäckerhefe produzierte VP2-Kapside dieselbe Antigenizität und Immunogenität wie Partikel aus dem Baculovirus-Expressionssystem. Schließlich wurde überprüft, ob mittels eines dualen Vektorsystems beide Strukturproteine VP1 und VP2 in Saccharomyces cerevisiae produziert werden können. Die Ergebnisse zeigten eine Co-Synthese des VP1- und VP2-Proteins in selben Mengenverhältnissen. Zur Generierung von Mischkapsiden muß allerdings die Menge an VP1-Protein auf maximal 40 % der Gesamtmenge an Strukturprotein beschränkt werden