Marcia Martins Reis

Prognostic Values of Stromal Proportion and PCNA, Ki-67, and p53 Proteins in Patients with Resected Adenocarcinoma of the Lung

Data from 64 patients who underwent surgical resection of lung adenocarcinomas were studied to identify clinicopathologic markers that might provide prognostic information on the clinical behavior of this neoplasia. Patient staging was performed in accordance with the tumor-node-metastasis system as follows: Stage I (n = 29), Stage II (n = 11), Stage IIIA (n = 21), and Stage IIIB (n = 3). Overall follow-up time corresponded to the follow-up time for patients who were alive and to the survival time for patients who had died, all of them expressed in months. Data included age, staging, histologic type, morphometric assessment of histologic features related to tumor (stroma and vascularization), and immunohistochemical detection of proliferation cell markers (Ki-67 protein and proliferating cell nuclear antigen) and p53 protein. The morphometric assessment was made by the point-counting procedure. Data analysis included Life Tables for Survival and Cox Regression models. Overall follow-up analysis showed that significant univariate predictors (P <.05) were T stage; N stage; tumor stromal proportion; and immunohistochemical indexes of proliferating cell nuclear antigen, Ki-67, and p53 proteins. Variables that presented independent predictive value for overall follow-up with the multivariate model (P <.05) were sex, T stage, N stage, tumor stromal proportion, and immunohistochemical detection of p53 protein. We conclude that tumor stromal proportion and immunohistochemical detection of p53 protein, controlled for sex, T stage, and N stage, may be of critical value in the evaluation of recurrence of lung adenocarcinoma, serving as indicators for a more accurate prognosis.

Great amount of C.pneumoniae in ruptured plaque vessel segments at autopsy. A comparative study with stable plaques

A possible relationship between C.pneumoniae (CP) infection, atherosclerosis and acute myocardial infarction is a debated matter. Now we performed the search of CP in histological segments of fatal ruptured plaques and of stable plaques by histochemistry (Macchiavello stain), immunohistochemistry and in situ hybridization techniques. Electron microscopy and confocal laser microscopy techniques were used in two additional cases. The semi-quantification of CP + cells (0-4+) and quantification of lymphocytes demonstrated greater amount of CP + cells and more inflammation in the adventitia of vulnerable plaque vessel segments than of stable ones, larger amount of CP + cells in adventitia than in the plaque and high frequency of CP + cells in all groups studied. This preliminary study strongly suggests a direct pathogenetic involvement of adventitial CP in the rupture of the atheromatous plaque, development of acute myocardial infarction and also in the development of atherosclerosis.

Growth factors in the myocardium of patients with chronic chagasic cardiomyopathy

In this work we quantified various growth factors in the myocardium of 19 patients with chronic chagasic cardiomyopathy and heart failure, through the immunoperoxidase technique. We looked for T. cruzi antigens, growth factors (GM-CSF, TGF-beta1, PDGF-A and PDGF-B) and inflammatory cells (CD4+, CD8+, CD20+ and CD68+). The mean ratio of CD4+/CD8+ T lymphocytes was 0.6 +/- 0.3. The mean number of positive interstitial cells was 5.9 +/- 3.1 for CD68+ (macrophages); 7.5 +/- 4.3 for PDGF-A+; 2.9 +/- 2.7 for PDGF-B+, 2.2 +/- 1.9 for TGF-beta1+ and 2.3 +/- 1.9 for GM-CSF+. The immunoreaction for PDGF-A was intense, occurring also in the endothelium, smooth muscle cells and the sarcolemma; there was no correlation between the number of positive interstitial cells and the semiquantitation of the same growth factors in the other cells. TGF-beta1 presented low expression in 100% of the cases. In conclusion, PDGF-A and B are probably the growth factors most related to the proliferative lesions and fibrosis present in chronic chagasic cardiomyopathy. GM-CSF and TGF-beta1 are present in low levels. There was no statistical correlation between growth factors and the quantity of the parasitic antigens.

A possible role for complement in the pathogenesis of chronic chagasic cardiomyopathy

The membrane attack complex (MAC) of complement participates in several inflammatory and proliferative processes by releasing pro‐inflammatory cytokines and growth factors from target cells. Chronic Chagasic cardiomyopathy (CCH) is a parasitic dilated cardiopathy, characterized by severe fibrosis and inflammation, which differs from idiopathic dilated cardiomyopathy (DCM). Trypanosoma cruzi, the pathogenic organism of CCH, is a strong complement activator and can also induce alternative pathway activation by mammalian cells. This study explored whether the myocardium in CCH patients has increased MAC deposition, an expression of complement activation, compared to DCM patients. MAC was semi‐quantified in endomyocardial human samples (29 CCH subjects, 18 DCM subjects, and four controls) by immunohistochemistry. MAC was present in the sarcolemma of 38% of CCH, 5.5% of DCM (p<0.02), and 0% of controls, and in interstitial inflammatory cells of CCH. No difference was observed in the expression of the complement regulatory protein CD59, indicating that increased MAC deposition is likely to be the result of complement activation rather than decreased protection. It is proposed that the increased MAC deposition found in CCH, but not in DCM or controls, may help to explain the diffuse myocardial fibrosis and inflammation characteristic of the disease. Copyright

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

BackgroundChamydophila pneumoniae (CP) and/or Mycoplasma pneumoniae (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).ResultsThe 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).ConclusionInoculation 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.

Upregulation of adhesion molecules and class I HLA in the myocardium of chronic chagasic cardiomyopathy and heart allograft rejection, but not in dilated cardiomyopathy.

The immunohistochemical expression of adhesion molecules and class I HLA in chronic chagasic cardiomyopathy were compared with heart allograft rejection and dilated cardiomyopathy, to obtain new knowledge on the occurrence of autoimmunity and inflammation in the pathogenesis of chronic chagasic cardiomyopathy. Semiquantitative immunohistochemistry was performed for CD8+ T cells, ICAM-1, VCAM-1, LFA-1, and class I HLA in frozen sections of myocardial biopsies from patients presenting chronic chagasic cardiomyopathy (group I, n = 12), heart allograft rejection (group II, n = 9) or dilated cardiomyopathy (group III, n = 9). A high mean number of CD8+ T cells/mm(2) was present in group I (18.26) and group II (28.60), but not in group III (0.83). The frequency of high expression for ICAM-1 and VCAM-1 on the endothelial and interstitial cells, and for class I HLA on the cardiomyocytes was greater in group I (100%, 33.3%, and 83.3%, respectively) and group II (100%, 66.7%, and 77.8%, respectively), compared to group III (66.7%, 0%, and 0%, respectively). ICAM-1 and VCAM-1 probably participate in the development of the lymphocytic inflammatory infiltrate present in chronic chagasic cardiomyopathy, as seen in heart allograft rejection. The overexpression of adhesion molecules and the induction of class I HLA on the cardiomyocytes are probably related to the high cytokine levels at the inflammatory sites in chronic chagasic cardiomyopathy. Although the induction of class I HLA on the cardiomyocytes is consistent with an autoimmune reaction, it should not be considered as irrefutable evidence for autoimmunity in chronic chagasic cardiomyopathy. The differential expression of adhesion molecules and class I HLA in dilated cardiomyopathy compared to chronic chagasic cardiomyopathy suggests differences in the pathogenesis of these cardiomyopathies.

Co-infection ratios versus inflammation, growth factors and progression of early atheromas.

Mycoplasma pneumoniae (MP) and Chlamydophila pneumoniae (CP) antigens are encountered in complicated atheromas and may be implicated in the diversity of atherosclerotic lesions. Mycoplasma can downregulate the immune system, altering levels of inflammation, which may favor the proliferation of other co‐infectious agents. In the present study we analyze whether initially stable human atheromas exhibit different ratios of MP/CP antigens compared to ongoing atheromatous lesions. Two groups were examined for the presence of inflammatory cells, macrophages, growth factors and infectious agents: Group I (GI), n=16, early stable atheromas, <4 CD68+ macrophages/400×field, showing a normal distribution and a fibrous cap; Group II (GII), n=14, growing atheromas, ≥4 CD68+ cells/400×field, lacking a fibrous cap, showing a non‐normal macrophage distribution. The amounts of CP (but not MP) antigens and lymphocytes in GI were significantly lower than in GII. MP/CP ratios were higher in GI. MP correlated with CP and PDGFB in GI (r=0.79 and r=0.83, p<0.001), but not in GII (r=−0.4 and r=−0.08, p=0.81). MP and CP antigens are already present in early atheromas, and a higher MP/CP ratio correlates with increased growth factors, lower inflammation and plaque stability.

Basic Science ResearchInfectious Agents, Inflammation, and Growth Factors: How Do They Interact in the Progression or Stabilization of Mild Human Atherosclerotic Lesions?

Advanced complicated atherosclerotic lesions have been related to many factors, including inflammation, infectious agents, and growth factors. Mycoplasma pneumoniae (MP) and Chlamydia pneumoniae (CP), inflammation, and growth factors have been associated with severe atherosclerotic lesions in necropsy material in recent work at our lab. The present study intends to clarify the pathogenesis of atherosclerosis, analyzing which of these elements (macrophages, MP, CP, lymphocytes, and growth factors) are associated with initial development of atherosclerotic lesions, discriminating elements related to stabilization of the plaque versus those related to subendothelial active accumulation of macrophages in living patients. Surgical ascending aorta fragments presenting mild atherosclerotic lesions from 30 coronary atherosclerotic patients were immunohistochemically quantified regarding CP, MP, T cells (CD4, CD8), B cells (CD20), macrophages (CD68), and growth factors [platelet-derived growth factor A (PDGF-A), PDGF-B, transforming growth factor-ß (TGF-ß), granulocyte-macrophage colony-stimulating factor (GM-CSF)]. Cases were grouped according to the presence or not of active accumulation of macrophages at the subendothelium that indicates atheroma in development: group I (GI) fragments with <4 CD68+ cells/×400 field, in normal distribution (mean 1.8 ± 1) representing stable atherosclerotic mild lesion, and GII fragments presenting ≥4 CD68+ cells/×400 field, in a non-normal distribution, mean (8.9 ± 4.8, atheromas in progress), which was followed by increased number of lymphocytes. The median number in GI was significantly lower than that in GII: CD4 T (2.5 vs. 7.7), CD8 T (1.0 vs. 5.5), and CD20 B (1.5 vs. 5.5) cells/×400 field, p < 0.001. Percentage area positive for CP antigens was significantly lower in GI than in GII: 1.0 vs. 9.2, p < 0.001. There was a higher percentage area occupied by MP than CP in both GI and GII (7.8 vs. 13.8). There was no difference regarding mean number of growth factor-positive cells/×400 field: PDGF-A, 1.4 vs. 3.9; PDGF-B, 3.4 vs. 5.7; TGF-ß, 0.9 vs. 2.2; and GM-CSF, 2.0 vs. 2.2. Considering all cases, a positive correlation was seen between inflammatory cells and CP+ cells (r > 0.5 and p < 0.01). Growth factors did not correlate with inflammatory cells, CP, or MP and were usually seen in smooth muscle cell and fibrotic areas. Study of initial atherosclerotic lesions showed that MP is present in both kinds of lesion: stable and active subendothelial accumulation of macrophages. Stabilization was related to proportional increase of both infectious agents, which were also related to increased amount of PDGF-A and PDGF-B. Active macrophage accumulation lesions were related to higher elevation in CP concentration at subendothelial regions, in association with B cells, but not of MP and growth factors. MP and CP, inflammation, and growth factors, which were already described in severe atherosclerotic lesions in necropsy material, are also present in mild lesions in living patients, strongly favoring a pathogenetic role for these bacteria in the pathogenesis of atherosclerosis. Predominance of CP in relation to MP may favor progression of the plaque, which is associated with increased B-cell proliferation. PDGF-A and PDGF-B are associated with plaque stability, at least in arterial segments not prone for development of complicated lesions.

Previous exercise training increases levels of PPAR-α in long-term post-myocardial infarction in rats, which is correlated with better inflammatory response.

OBJECTIVE: Exercise is a protective factor for cardiovascular morbidity and mortality, with unclear mechanisms. Changing the myocardial metabolism causes harmful consequences for heart function and exercise contributes to metabolic adjustment modulation. Peroxisome proliferator-activated receptors (PPARs) are also myocardium metabolism regulators capable of decreasing the inflammatory response. We hypothesized that PPAR-α is involved in the beneficial effects of previous exercise on myocardial infarction (MI) and cardiac function, changing the expression of metabolic and inflammatory response regulators and reducing myocardial apoptosis, which partially explains the better outcome. METHODS AND RESULTS: Exercised rats engaged in swimming sessions for 60 min/day, 5 days/week, for 8 weeks. Both the exercised rats and sedentary rats were randomized to MI surgery and followed for 1 week (EI1 or SI1) or 4 weeks (EI4 or SI4) of healing or to sham groups. Echocardiography was employed to detect left ventricular function and the infarct size. Additionally, the TUNEL technique was used to assess apoptosis and immunohistochemistry was used to quantitatively analyze the PPAR-α, TNF-α and NF-κB antigens in the infarcted and non-infarcted myocardium. MI-related mortality was higher in SI4 than in EI4 (25% vs 12%), without a difference in MI size. SI4 exhibited a lower shortening fraction than EI4 did (24% vs 35%) and a higher apoptosis/area rate (3.97±0.61 vs 1.90±1.82) in infarcted areas (both p=0.001). Immunohistochemistry also revealed higher TNF-α levels in SI1 than in EI1 (9.59 vs 4.09, p<0.001) in infarcted areas. In non-infarcted areas, EI4 showed higher levels of TNF-α and positive correlations between PPAR-α and NF-κB (r=0.75, p=0.02), in contrast to SI4 (r=0.05, p=0.87). CONCLUSION: Previously exercised animals had better long-term ventricular function post-MI, in addition to lower levels of local inflammatory markers and less myocardial apoptosis, which seemed to be related to the presence of PPAR-α.

Do Archaea and bacteria co-infection have a role in the pathogenesis of chronic chagasic cardiopathy?

UNLABELLED Chronic cardiopathy (CC) in Chagas disease is a fibrotic myocarditis with C5b-9 complement deposition. Mycoplasma and Chlamydia may interfere with the complement response. Proteolytic enzymes and archaeal genes that have been described in Trypanosoma cruzi may increase its virulence. Here we tested the hypothesis that different ratios of Mycoplasma, Chlamydia and archaeal organisms, which are frequent symbionts, may be associated with chagasic clinical forms. MATERIALS AND METHODS eight indeterminate form (IF) and 20 CC chagasic endomyocardial biopsies were submitted to in situ hybridization, electron and immunoelectron microscopy and PCR techniques for detection of Mycoplasma pneumoniae (MP), Chlamydia pneumoniae(CP), C5b-9 and archaeal-like bodies. RESULTS MP and CP-DNA were always present at lower levels in CC than in IF (p < 0.001) and were correlated with each other only in CC. Electron microscopy revealed Mycoplasma, Chlamydia and two types of archaeal-like bodies. One had electron dense lipid content (EDL) and was mainly present in IF. The other had electron lucent content (ELC) and was mainly present in CC. In this group, ELC correlated negatively with the other microbes and EDL and positively with C5b-9. The CC group was positive for Archaea and T. cruzi DNA. In conclusion, different amounts of Mycoplasma, Chlamydia and archaeal organisms may be implicated in complement activation and may have a role in Chagas disease outcome.