Mycoplasma pneumoniae and/or Chlamydophila pneumoniae inoculation causing different aggravations in cholesterol-induced atherosclerosis in apoE KO male mice

<p>Abstract</p> <p>Background</p> <p><it>Chamydophila pneumoniae </it>(CP) and/or <it>Mycoplasma pneumoniae </it>(MP) are two bacteria detected in vulnerable atheromas. In this study we aimed to analyze whether CP and/or MP aggravates atherosclerosis induced by cholesterol-enriched diet in C57BL/6 apoE KO male mice. Thirty male apoE KO mice aged eight weeks fed by a diet containing 1% cholesterol until 32 weeks of age were divided into four groups: the first was inoculated with CP (n = 7), the second with MP (n = 12), the third with both CP + MP (n = 5), and the fourth with saline (sham n = 6). The animals were re-inoculated at 36 weeks of age, and sacrificed at 40 weeks of age. Two ascending aorta and one aortic arch segments were sampled. In the most severely obstructed segment, vessel diameter, plaque height, percentage of luminal obstruction and the degree of adventitial inflammation were analyzed. The plaque area/intimal surface ratio was obtained by measuring all three segments. The adventitial inflammation was semiquantified (0 absent, 1 mild, 2 moderate, and 3 diffuse).</p> <p>Results</p> <p>The mean and standard deviation of plaque height, % luminal obstruction, external diameter, the plaque area/intimal surface ratio and the adventitial inflammation values are the following for each group: MP (0.20 +/- 0.12 mm, 69 +/- 26%, 0.38 +/- 0.11 mm, 0.04 +/- 0.04 and 0.22 +/- 0.67), CP (0.23 +/- 0.08 mm, 90 +/- 26%, 0.37 +/- 0.08 mm, 0.04 +/- 0.03, and 0.44 +/- 0.53), MP + CP (18 +/- 0.08 mm, 84 +/- 4.0%, 0.35 +/- 0.25 mm, 0.03 +/- 0.03 and 1.33 +/- 0.82) and sham (0.08 +/- 0.09 mm, 42 +/- 46%, 0.30 +/- 0.10 mm, 0.02 +/- 0.03 and 0.71 ± 0.76). A wider area of plaque/intimal surface was observed in MP + CP inoculated groups (p = 0.07 and 0.06) as well as an increased plaque height in CP (p = 0.01) in comparison with sham group. There was also an increased luminal obstruction (p = 0.047) in CP inoculated group in comparison to sham group. Adventitial inflammation in MP + CP inoculated group was higher than MP, CP and the sham groups (p = 0.02).</p> <p>Conclusion</p> <p>Inoculation of CP, MP or both agents in C57BL/6 apoE KO male mice caused aggravation of experimental atherosclerosis induced by cholesterol-enriched diet, with distinct characteristics. CP inoculation increased the plaque height with positive vessel remodeling and co-inoculation of MP + CP caused the highest adventitial inflammation measures.</p

Oral PTCTS (Particulated Transialidase) Removes Serum Microparticles and Decreases Inflammation in Atherosclerotic Plaques of Rabbits

Background: Previous studies showed that atherosclerotic plaque vulnerability was related with microparticles (MPs)-vesicles larger than 100 nm, which released MMP9 collagenase. In our pre- vious study, intramuscular injection of a new drug (PTCTS) normalized oxidized LDL serum levels and reduced rabbit atherosclerosis. Now, we studied administration of oral PTCTS in order to cla- rify anti-atherosclerotic mechanism of action, analyzing if the treatment removed MPs containing ox-LDL and Mycoplasma pneumoniae antigens and improved the immune response. Methods: We compared two groups of rabbits. Control group (CG, n = 6)—1% cholesterol enriched diet for 12 weeks; Treated group (TG, n = 8)—1% cholesterol enriched diet for 12 weeks with administration of PTCTS (400 μl/day) during the last 6 weeks of diet. The animals had their blood collected, in three different phases of the protocol before being fed with hypercholesterolemic diet, before be- ing treated with water or PTCTS and at the moment of sacrifice. The serum was submitted to im- munofluorescence technique to evaluate the quantity of microparticles marked with antibodies against Mycoplasma pneumoniae and ox-LDL. A fragment of aorta was submitted to immunohisto- chemical detection of antigens from MMP9, ox-LDL, NF-κB and IL-1β. Results: PTCTS showed significant reduction in MMP-9 (P = 0.001) and a tendency of reducing IL-1β (P = 0.09) in the aortic plaques compared with CG. In the serum, PTCTS was able to remove microparticles containing an- tigen of ox-LDL (P = 0.004) and Mycoplasma pneumoniae (P < 0.001). Conclusion: Oral treatment with PTCTS presented more adequate inflammatory response by reducing levels of ox-LDL, IL-1β and mycoplasma, as well as a better stabilization of the atheromatous plaque by reducing levels of MMP-9, avoiding plaque rupture, without causing mortality or toxicity.This work was supported by FAPESP (Fundation that supports research in the State of São Paulo, grant number 2012/12656-5) and Zerbini Foundation

Archaea Symbiont of T. cruzi Infection May Explain Heart Failure in Chagas Disease

Background: Archaeal genes present in Trypanosoma cruzi may represent symbionts that would explain development of heart failure in 30% of Chagas disease patients. Extracellular vesicles in peripheral blood, called exosomes (&lt; 0.1 μm) or microvesicles (&gt;0.1 μm), present in larger numbers in heart failure, were analyzed to determine whether they are derived from archaea in heart failure Chagas disease.Methods: Exosomes and microvesicles in serum supernatant from 3 groups were analyzed: heart failure Chagas disease (N = 26), asymptomatic indeterminate form (N = 21) and healthy non-chagasic control (N = 16). Samples were quantified with transmission electron microscopy, flow cytometer immunolabeled with anti-archaemetzincin-1 antibody (AMZ 1, archaea collagenase) and probe anti-archaeal DNA and zymography to determine AMZ1 (Archaeal metalloproteinase) activity.Results: Indeterminate form patients had higher median numbers of exosomes/case vs. heart failure patients (58.5 vs. 25.5, P &lt; 0.001), higher exosome content of AMZ1 antigens (2.0 vs. 0.0; P &lt; 0.001), and lower archaeal DNA content (0.2 vs. 1.5, P = 0.02). A positive correlation between exosomes and AMZ1 content was seen in indeterminate form (r = 0.5, P &lt; 0.001), but not in heart failure patients (r = 0.002, P = 0.98). Higher free archaeal DNA (63.0 vs. 11.1, P &lt; 0.001) in correlation with exosome numbers (r = 0.66, P = 0.01) was seen in heart failure but not in indeterminate form (r = 0.29, P = 0.10). Flow cytometer showed higher numbers of AMZ1 microvesicles in indeterminate form (64 vs. 36, P = 0.02) and higher archaeal DNA microvesicles in heart failure (8.1 vs. 0.9, P &lt; 0.001). Zymography showed strong% collagenase activity in HF group, mild activity in IF compared to non-chagasic healthy group (121 ± 14, 106 ± 13 and 100; P &lt; 0.001).Conclusions: Numerous exosomes, possibly removing and degrading abnormal AMZ1 collagenase, are associated with indeterminate form. Archaeal microvesicles and their exosomes, possibly associated with release of archaeal AMZ1 in heart failure, are future candidates of heart failure biomarkers if confirmed in larger series, and the therapeutic focus in the treatment of Chagas disease

Comparison of the Protective Effects of Individual Components of Particulated trans-Sialidase (PTCTS), PTC and TS, against High Cholesterol Diet-Induced Atherosclerosis in Rabbits

Previous studies showed the presence of Mycoplasma pneumoniae (M. pneumoniae) and membrane-shed microparticles (MPs) in vulnerable atherosclerotic plaques. H&S Science and Biotechnology developed PTCTS, composed by natural particles from medicinal plants (PTC) combined with trans-Sialidase (TS), to combat MPs and Mycoplasma pneumoniae. Our aim was to determine the effects of the different components of PTCTS in a rabbit model of atherosclerosis. Rabbits were fed with high cholesterol diet for 12 weeks and treated during the last 6 weeks with either vehicle, PTC, TS, or PTCTS. Lipid profile and quantification of MPs positive for Mycoplasma pneumoniae and oxidized LDL antigens were carried out. Aortas and organs were then histologically analyzed. PTCTS reduced circulating MPs positive for Mycoplasma pneumoniae and oxidized LDL antigens, reduced the plaque area in the abdominal aorta, and caused positive remodeling of the ascendant aorta. PTC caused positive remodeling and reduced plaque area in the abdominal aorta; however, TS had a lipid lowering effect. PTCTS components combined were more effective against atherosclerosis than individual components. Our data reinforce the infectious theory of atherosclerosis and underscore the potential role of circulating MPs. Therefore, the removal of Mycoplasma-derived MPs could be a new therapeutic approach in the treatment of atherosclerosis