Altogether, the results suggest a dominant role of IL-1β-induced NETosis in AAA formation.

Objective The purpose of this study was to establish a reliable, chronic model of abdominal aortic aneurysm (AAA). Materials and methods 120 eight-week old C56BL/6 male mice were equally divided into three groups: 1) BAPN Group: 0.2% 3-aminopropionitrile fumarate salt (BAPN) drinking water was provided to mice two days before surgery until the end of study. Sham aneurysm induction surgery was performed using 5 μl of heat de-activated elastase. 2) Elastase Group: Mice were given regular drinking water without BAPN. During aneurysm induction surgery, 5 μl of active form elastase (10.3mg protein/ml, 5.9units/mg protein) was applied on top of adventitia of infrarenal abdominal aorta for 5 minutes. 3) BAPN+Elastase Group: Mice were given both BAPN drinking water and active form of elastase application as above. On post-operative days 7, 14, 21, 28 and 100, aortic samples were collected for histology, cytokine array and gelatin zymography after aortic diameter measurement. Results Compared with Elastase group, BAPN+Elastase group had higher AAA formation rate (93% vs 65%, P < .01) with more advanced-staged AAAs (25/42 vs 1/40 for Stage II & III, P < .001). Aneurysms from the BAPN+Elastase Group demonstrated persistent long-term growth (221.5 ± 36.6%, 285.8 ± 78.6%, 801 ± 160% on day 21, 28 and 100 respectively, P ˂ .001), with considerable thrombus formation (54%) and rupture (31%) at the advanced stages of AAA development. Cytokine levels (pro-MMP9, IL-1β, IL-6, CCL-5, TREM-1, MCP-1 and TIMP-1) in BAPN+Elastase Group were higher than Elastase Group on day 7. After day 7, cytokine levels returned to baseline with the exception of elevated MMP2 activity. By histology, CD3⁺T cells in the BAPN+Elastase Group were elevated on days 28 and 100. Conclusions A combination of oral BAPN administration and peri-aortic elastase application induced a chronic, advanced staged AAA with characteristics of persistent aneurysm growth, thrombus formation, and spontaneous rupture. Future studies should utilize this model, especially for examining tissue remodeling during the late stages of aneurysm development.

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The role of resolvins in abdominal aortic aneurysm (AAA) has not been established. We hypothesized that treatment with D-series resolvins (RvD2 or RvD1) would attenuate murine AAA formation through alterations in macrophage polarization and cytokine expression. Male C57/B6 mice (n = 9 per group) 8 to 12 wk old received RvD2 (100 ng/kg/treatment), RvD1 (100 ng/kg/treatment), or vehicle only every third day beginning 3 d before abdominal aortic perfusion with elastase as prevention. Aortas were collected 14 d after elastase perfusion. Cytokine analysis (n = 5 per group) or confocal microscopy (n = 4 per group) was performed. In a separate experiment, RvD2 was provided to mice with small AAAs 3 d after elastase treatment (n = 8 per group). Additionally, apolipoprotein E knockout mice treated with angiotensin II (1000 ng/kg) were treated with RvD2 or vehicle alone (n = 10 per group) in a nonsurgical model of AAA. To determine the effect of RvD2 on macrophage polarization, confocal staining for macrophages, M1 and M2 macrophage subtypes, α-actin, and DAPI was performed. Mean aortic dilation was 96 ± 13% for vehicle-treated mice, 57 ± 9.7% for RvD2-treated mice, and 61 ± 11% for RvD1-treated mice (P < 0.0001). Proinflammatory cytokines macrophage chemotactic protein 1, C-X-C motif ligand 1, and IL-1β were significantly elevated in control animals compared to RvD2- and RvD1-treated animals (P < 0.05), resulting in a reduction of matrix metalloproteinase 2 and 9 activity in resolvin-treated mice in both elastase and angiotensin II models. Treatment of existing small AAAs with RvD2 demonstrated a 25% reduction in aneurysm size at d 14 compared to vehicle alone (P = 0.018). Confocal histology demonstrated a prevalence of M2 macrophages within the aortic medium in mice treated with RvD2. Resolvin D2 exhibits a potent protective effect against experimental AAA formation. Treatment with RvD2 significantly influences macrophage polarization and decreases several important proinflammatory cytokines. Resolvins and the alteration of macrophage polarization represent potential future targets for prevention of AAA.-Pope, N. H., Salmon, M., Davis, J. P., Chatterjee, A., Su, G., Conte, M. S., Ailawadi, G., Upchurch, G. R., Jr. D-series resolvins inhibit murine abdominal aortic aneurysm formation and increase M2 macrophage polarization.

Using counterimmunoelectrophoresis, DNA may be detected at concentrations as low as 1.5 &ml In p h and 0.2 pg/ml in serum. Serum, however,was not suitable for this study because DNA was sporadically released into serum during the clotting process. Counterimmunoelectrophoresis is at least ten times more sensitive than simple immunodiffusion in the detection of DNA. DNA was found in plasma in a variety of illnesses which are delineated in this study. Tan et al, in 1966, using the method of gel diffusion, reported the detection of DNA in the sera of patients with systemic lupus erythematosus (SLE) and other diseases (1). In the same year, Barnett and Vaughan (2) suggested that normal human sera may contain small amounts of antigenic nuclear material, since rabbits immunized with whole human serum in adjuvant often developed antibodies to DNA. Barnett (3), in 1968, employed complement fixation to demonstrate the presence of DNA in sera and synovial fluids. The possibility that circulating DNA might induce pathogenetic antibodies (4) with a role in conditions such as lupus nephritis has been suggested (5). More recently, Hughes et a l , (6) diffusion in agarose gel, reported the detection of DNA in the sera and synovial fluids of patients with a variety of illnesses and suggested that the presence of DNA in blood may be a relatively nonspecific phenomenon. The level of DNA which Hughes was able to detect was approximately 10 rg/ml of serum. Using counterimmunoelectrophoresis (CIE), a technic previously described in this laboratory primarily for the determination of anti-DNA (7), we were able to detect DNA at concentrations as low as 1.5 rg/ml in plasma and 0.2 Pg/ml in serum. With the development of a rapid, reproducible and more sensitive screening technic in which multiple samples could be analyzed simultaneously with appropriate controls, we assayed a random hospital population as well as healthy subjects for circulating DNA. MATERIALS AND METHODS Blood was collected from the University of VirginiaClinical Laboratories in 7-ml tubes (100 x 13 mm) containing 9 mg of sodium EDTA. These blood specimens were obtained on a random basis, promptly refrigerated and processed within 18 hours of venapuncture. Plasma was separated by centrifugation at 2000 rpm for 15 minutes and was then heated at 56" C for 1 hour to destroy complement activity. (Heating at 56" C for 1 hour instead of 30 minutes increased our detection sensitivity from 3.0 to 1.5 pg/ml, whereas we were able to detect no less than 20 rg DNA/ml in fresh plasma.) The heated plasma was again centrifuged at 3000 rpm for 20 minutes in order to remove a flocculent 52

Unselected sera from patients with various rheumatic, inflammatory bowel, and autoimmune skin diseases (n = 268) were examined against human cell lysate by immunoblotting procedures, to determine the prevalence of autoantibodies to stress proteins (heat-shock proteins) hsp60 (homolog of Escherichia coli groEL and mycobacterial 65K antigens), hsp73, and hsp90. Using standard, sensitive and specific assay conditions, IgG and IgM autoantibodies to these stress proteins were not demonstrable, or were detected infrequently, in sera from control subjects (n = 36) and from patients with rheumatoid arthritis, Sjogren's syndrome, ankylosing spondylitis, Reiter's syndrome, systemic lupus erythematosus, and systemic sclerosis. Autoantibodies to

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