Birgit Vogt

The influence on the immunomodulatory effects of dying and dead cells of Annexin V

Apoptotic and necrotic cells expose phosphatidylserine (PS). This membrane modification ensures a swift recognition and uptake by phagocytes of the dying and dead cells. Annexin V (AxV) preferentially binds to anionic phospholipids and thereby, modulates the clearance process. First, we analyzed the influence of AxV on the immunogenicity of apoptotic cells. The addition to apoptotic cells of AxV prior to their injection into mice increased their immunogenicity significantly. Next, we studied the influence of endogenous AxV on the allogeneic reaction against apoptotic and necrotic cells. To preserve heat‐labile, short‐lived “danger signals,” we induced necrosis by mechanical stress. Wild‐type mice showed a strong, allogeneic delayed‐type hypersensitivity (DTH) reaction. In contrast, AxV‐deficient animals showed almost no allogeneic DTH reaction, indicating that endogenous AxV increases the immune response against dead cells. Furthermore, AxV‐deficient macrophages had a higher immunosuppressive potential in vitro. Next, we analyzed the influence of AxV on chronic macrophage infection with HIV‐1, known to expose PS on its surface. The infectivity in human macrophages of HIV‐1 was reduced significantly in the presence of AxV. Finally, we show that AxV also blocked the in vitro uptake by macrophages of primary necrotic cells. Similar to apoptotic cells, necrotic cells generated by heat treatment displayed an anti‐inflammatory activity. In contrast, mechanical stress‐induced necrotic cells led to a decreased secretion of IL‐10, indicating a more inflammatory potent‐ial. From the experiments presented above, we conclude that AxV influences the clearance of several PS‐exposing particles such as viruses, dying, and dead cells.

CRP and the disposal of dying cells: Consequences for systemic lupus erythematosus and rheumatoid arthritis

C reactive protein (CRP) levels directly correlate with the disease activity of many inflammatory diseases, e.g. sepsis, infection, and various autoimmunopathies such as rheumatoid arthritis (RA) 1, 2, 3, 4, 5, 6, 7. In contrast, insufficient CRP levels are implicated in the development of systemic lupus erythematosus (SLE) 8, 9, 10, 11, 12, 13, 14, 15, 16. This article reports on the level-depended effects of CRP in various diseases. In detail we show that increased and decreased levels of CRP, as demonstrated in patients with RA and SLE, respectively can contribute to disease progression.

Specific Removal of C-Reactive Protein by Apheresis in a Porcine Cardiac Infarction Model

Background: C-reactive protein (CRP) is a possible causative factor of the destructive processes observed during the weeks after myocardial infarction. Methods: We developed a clinically relevant animal model including the removal of CRP from blood plasma utilizing a specific CRP adsorber and the visualization of the infarct scar in the living animal by cardiovascular magnetic resonance imaging as a tool to investigate the impact of CRP after acute myocardial infarction. Results: We describe the facets of this model system and kinetics of clinical blood parameters like CRP and troponin. In addition, we demonstrate the potency of CRP apheresis reducing CRP levels by ∼70% in the established treatment system. Conclusion: We showed for the first time that it is possible to conduct apheresis at the following 2 days after acute myocardial infarction in a porcine infarction model and to analyze the infarct by cardiovascular magnetic resonance imaging at day 1 and 14.

Detection of Low Level Cryoglobulins by Flow Cytometry

Several patients with cryoglobulin (CG) associated symptoms are seronegative for CG and other potentially causative biomarkers. We analyzed whether it is possible to detect cryoprecipitates by flow cytometry and whether the sensitivity of their demonstration can be increased as compared to visual inspection. Sera from 91 patients with suspected CG associated symptoms and 33 healthy controls were examined for the presence of CG by conventional visual testing and by flow cytometry for small diffracting particles. For calibration purposes we tested lipid micelle dilutions (positive controls) by both methods. The minimum concentrations of lipid micelles to be detected by visual inspection and flow cytometry were 128.5 and 2.0 pg ml−1, respectively. Among the 91 patients and 33 controls, only 1 patient serum was positive for CG by conventional testing. This sample was also positive on flow cytometry. In the serum of a patient known to be positive for CG, laser diffracting particles were quantified by flow cytometry after keeping serum at 4°C for 3 days. Of the 91 patients, 14 additional samples displayed cold precipitates which redissolved after rewarming during flow cytometry. All 15 (1 + 14) patients positive for CG on flow cytometry suffered from symptoms usually associated with CG. Some precipitates were labeled with anti IgG and IgM antibodies confirming that the particles detected by flow cytometry contained immunoglobulins. No small diffracting particles were detected in the sera of the 33 healthy controls. Flow cytometry is equally specific but much more sensitive in the detection of CG than visual inspection.

Intracellular capture of B7 in antigen-presenting cells reduces costimulatory activity

CTLA-4 gene constructs were designed to express CTLA-4 exclusively in the endoplasmic reticulum (ER). Four different CTLA-4 gene constructs were transfected into HEK 293 (human embryonic kidney) and A20 (Balb/c mouse B lymphoma) cells. All constructs contained an ER retention signal and coded for CTLA-4 expression in the ER. One of the constructs, which contained the membrane part of CTLA-4, coded for an expression both on the cell surface and in the ER. Three of the expressed CTLA-4 types (including the ER-membrane-expressed form) caused a reduced surface expression of B7 in the A20 cells. Only constructs which allow dimerization of CTLA-4 showed this effect. It is assumed that intracellular CTLA-4 bound B7 and inhibited therefore the transport of B7 to the surface. The binding obviously caused also an enhanced degradation of the complexes because both proteins showed a low concentration in the transfected cell lines. CTLA-4-transfected and B7-reduced A20 cells showed a diminished costimulating activity upon T cells. This was demonstrated by a reduced proliferation of T cells from ovalbumin-immunized Balb/c mice, incubated with ovalbumin peptide-primed CTLA-4-transfected A20 cells.

ELEVATION OF PLASMA COPEPTIN IN ACUTE MYOCARDIAL INFARCTION IN PIGS IS RELATED TO CHANGES IN MEAN ARTERIAL BLOOD PRESSURE BUT NOT TO MYOCARDIAL ISCHEMIA

Background: Copeptin increases early after myocardial infarction (MI). We used a porcine MI model with serial copeptin-testing and assessment of hemodynamic variables to evaluate the trigger for copetin-release and the liberation kinetics of copeptin in MI. Methods: AMI was induced by percutaneous transluminal coronary angioplasty (PTCA). Blood samples were taken in 30-minute intervals. Results: All animals (n=4) had a comparable infarction size (10.02 ± 2.11% of the left ventricle) and elevated troponin T levels. Highest copeptin levels were measured 30 minutes after the beginning of ischemia with a rapid decrease at 60 minutes, while coronary occlusion was still persisting. Animals with high copeptin values showed a significant drop of MAP (37.24% and 54.20% reduction from MAP-baseline). Conclusions: In experimental AMI in four pigs, the infarction-related decrease of MAP seemed to be the trigger for the copeptin increase. Remarkably, copeptin liberation was not correlated with infarction size and copeptin values already decreased while coronary artery occlusion was ongoing.