Elena Bonanno

Modulation of different clusterin isoforms in human colon tumorigenesis

Clusterin is a ubiquitous secretory heterodimeric disulfide-linked glycoprotein, which is implicated in several physiological processes, including immune regulation, cell adhesion and morphological transformation, lipid transportation, tissue remodelling, membrane recycling and cell–cell interactions. A large number of studies have focused their interest on clusterin gene products as mediators of cell cycle progression and cell death induction, although data on the different isoforms and their role in the different cell processes are still obscure. Recently, an increased clusterin expression in breast cancer has been reported. In order to elucidate the role of clusterin in tumor progression and whether one of its isoforms is preferentially expressed in tumorigenesis, we examined its presence throughout the different steps of human colon carcinoma, one of the best-characterized models of human tumor progression. The immunohistochemical observation of 30 bioptic and surgical colon specimens demonstrated a cell compartment clusterin translocation from the nucleus to the cytoplasm directly related to tumor progression. In fact, a nuclear localization found in healthy colonic mucosa is consistent with the involvement of the proapoptotic nuclear form in the regulation of cell cycle progression and in cell death induction. The progression towards high-grade and metastatic carcinoma leads to cytoplasmic clusterin distribution. Protein extracts from freshly isolated cells of the same patients confirm in high-grade carcinomas with metastatic nodes the complete loss of the proapoptotic nuclear form and a cytoplasmic overexpression of the highly glycosylated form. Data obtained from in vitro experiments confirm that this form is released in the extracellular space and corresponded to the fully glycosylated one. These data suggest that the controversial data on clusterin function in tumors may be related to the pattern shift of its isoform production. As the secreted form of clusterin is correlated to cell matrix formation, cell membrane remodeling and cell–cell adhesion, the overexpression of this form in highly aggressive tumors and metastatic nodes could be a potential new prognostic and predictive marker for colon carcinoma aggressiveness.

The Inhibition of the Highly Expressed Mir-221 and Mir-222 Impairs the Growth of Prostate Carcinoma Xenografts in Mice

Background MiR-221 and miR-222 are two highly homologous microRNAs whose upregulation has been recently described in several types of human tumors, for some of which their oncogenic role was explained by the discovery of their target p27, a key cell cycle regulator. We previously showed this regulatory relationship in prostate carcinoma cell lines in vitro, underlying the role of miR-221/222 as inducers of proliferation and tumorigenicity. Methodology/Principal Findings Here we describe a number of in vivo approaches confirming our previous data. The ectopic overexpression of miR-221 is able, per se, to confer a high growth advantage to LNCaP-derived tumors in SCID mice. Consistently, the anti-miR-221/222 antagomir treatment of established subcutaneous tumors derived from the highly aggressive PC3 cell line, naturally expressing high levels of miR-221/222, reduces tumor growth by increasing intratumoral p27 amount; this effect is long lasting, as it is detectable as long as 25 days after the treatment. Furthermore, we provide evidence in favour of a clinical relevance of the role of miR-221/222 in prostate carcinoma, by showing their general upregulation in patient-derived primary cell lines, where we find a significant inverse correlation with p27 expression. Conclusions/Significance These findings suggest that modulating miR-221/222 levels may have a therapeutic potential in prostate carcinoma.

Role of Inflammation in Atherosclerosis

Inflammation plays a major role in all phases of atherosclerosis. Stable plaques are characterized by a chronic inflammatory infiltrate, whereas vulnerable and ruptured plaques are characterized by an “active” inflammation involved in the thinning of the fibrous cap, predisposing the plaque to rupture. Although a single vulnerable atherosclerotic plaque rupture may cause the event, there are many other types of plaques, several of which are vulnerable. The existence of multiple types of vulnerable plaques suggests that atherosclerosis is a diffuse inflammatory process. A current challenge is to identify morphologic and molecular markers able to discriminate stable plaques from vulnerable ones, allowing the stratification of patients at high risk for acute cardiovascular and cerebrovascular events before clinical syndromes develop. With that aim in mind, this article summarizes the natural history of atherosclerotic plaques, focusing on molecular mechanisms affecting plaque progression and serum markers correlated with plaque inflammation.

Multicentric inflammation in epicardial coronary arteries of patients dying of acute myocardial infarction

OBJECTIVES We sought to test the hypothesis of whether inflammatory cell infiltration in patients dying of an acute myocardial infarction (MI) is a multifocal event involving multiple coronary branches. BACKGROUND Coronary instability is thought to reflect local disruption of a single vulnerable plaque. However, previous postmortem studies have not addressed the question of whether activation of inflammatory cells, particularly T lymphocytes, is limited to the culprit lesion only or rather diffuse in the coronary circulation. METHODS We performed a systematic flow cytometric study in three groups of autopsied patients (group 1 = acute MI; group 2 = old MI; group 3 = no ischemic heart disease). Cell suspensions of enzymatically digested coronary arteries were stained for flow cytometry with CD3, CD68, alpha-smooth muscle actin, and human leukocyte antigen (HLA)-DR antibodies. RESULTS The coronary plaques showed: 1) a higher proportion of inflammatory cells in groups 1 and 2 than in group 3; 2) a higher percentage of T lymphocytes in group 1 than in group 2 (11.67 +/- 0.70% vs. 5.67 +/- 0.74%, p = 0.001) and in group 2 than in group 3 (p = 0.008); and 3) diffuse cell activation in the whole coronary tree of group 1, but not of group 2 subjects. CONCLUSIONS Our study suggests that lymphocytes may play a key role in coronary instability by determining activation of various cellular types throughout the coronary circulation. Activated T lymphocytes and their products may well represent a new target in both the treatment and prevention of acute coronary syndromes.

Widespread Myocardial Inflammation and Infarct-Related Artery Patency

Background—Diffuse coronary vascular inflammation is associated with acute coronary syndromes. However, it is unknown whether inflammation also occurs within the myocardium. Therefore, this study was aimed at assessing the presence of activated cells in unaffected remote myocardium of patients with acute myocardial infarction (AMI), in comparison to the peri-infarct region from the same cases, and in comparison to myocardial specimens from control hearts. Methods and Results—Sixteen patients dying 1 to 12 weeks after AMI and 16 control subjects were selected at autopsy. Myocardial specimens were taken at remote unaffected viable regions and at peri-infarct regions in cases with AMI. Confocal microscopy was performed to measure the number of activated cells (DR+), T-lymphocytes (CD3+), and activated T-lymphocytes (CD3+/DR+). Activated cells and activated T-lymphocytes were found in remote unaffected regions in 11 of 16 cases (69%), in peri-infarct zone in all cases (100%), and in none of the control hearts (0%, P <0.001 versus others). A greater myocardial inflammatory burden in remote regions but not in peri-infarct regions was associated with persistent infarct-related artery occlusion (P <0.05). Conclusions—This study for the first time shows the presence of activated T-lymphocytes in remote unaffected myocardial regions in approximately two thirds of patients with recent AMI. Because these cells are associated with persistent infarct-related artery occlusion, our data may suggest that an antigenic stimulus present also in the myocardium triggers an immune response that may be critical to precipitate artery occlusion.

Protein unlocking procedures of formalin-fixed paraffin-embedded tissues: application to MALDI-TOF imaging MS investigations.

Archival formalin‐fixed paraffin‐embedded (FFPE) tissues are a powerful tool for examining the clinical course of diseases. These specimens represent an incredible mine of valuable clinical and biological information for proteomic investigation. MALDI‐TOF imaging MS (MALDI‐IMS) is a protein profiling technique which enables the direct sampling of histological section; however, the quality of molecular data are strongly influenced by the tissue preparation condition. In fact, in previous years most of the studies employing such a technological platform have been conducted using cryo‐preserved tissues. We have developed an in vitro approach using “tissue surrogate” samples in order to explore different protein unlocking procedures which might enable a suitable recovery of polypeptides for MS analysis. The developed protocols have been compared both by MALDI‐TOF MS and nLC‐MSE analysis either on surrogate samples or on FFPE specimen from human breast cancer. The collected evidence has been applied for the preparation of FFPE tissue sections following MALDI‐IMS analysis. Our results outline the possibility to obtain valuable peptide mass spectra profiles form FFPE preparations by applying a combined two steps procedure of heat induced antigen retrieval (HIAR) in presence of EDTA and on target trypsin hydrolysis. A multivariate statistical evaluation is presented and discussed according to molecular spatial distributions and tissue morphology.

Small Breast Cancers: In Vivo Percutaneous US-guided Radiofrequency Ablation with Dedicated Cool-Tip Radiofrequency System

PURPOSE To evaluate in vivo the efficacy of a newly developed breast radiofrequency (RF) ablation system in human small invasive breast carcinomas in terms of induction of complete tumor necrosis, reproducibility of ablation lesion size and shape, and cosmetic outcome. MATERIALS AND METHODS This study had institutional review board approval, and written informed consent was obtained. Thirty-four postmenopausal women (mean age, 53 years +/- 5 [standard deviation]; range, 49-62 years) with small (< or = 2 cm) biopsy-proved invasive ductal breast carcinomas were enrolled. RF energy was delivered through a 25-mm 15-gauge monopolar cool-tip needle electrode by using the temperature-controlled mode. Patients were divided into three groups according to their breast pattern as assessed at mammography. The volumetric size and geometry of the coagulation zone, together with ablation time, were determined. Histopathologic data were compared with postprocedural 3.0-T contrast material-enhanced magnetic resonance (MR) images. Cosmesis after RF ablation was assessed. Four weeks after RF ablation, patients underwent definitive surgery. RESULTS All ablation procedures were performed successfully. For 97% of the procedures, nicotinamide adenine dinucleotide in its reduced form-diaphorase staining showed no evidence of viable cells. The mean induced ablation volume, as assessed with histologic analysis, was 12.50 cm(3) +/- 0.8. Tumor ablation volume on the postablation MR images showed good correlation with results of histopathologic analysis (r = 0.823, P < .005). No differences were observed in terms of duration of the procedure or ablation volume with respect to the glandular pattern of the breast (P > .05 for both). The general shape of the induced necrosis was close to a sphere in all cases. Cosmesis was excellent in 28 patients. CONCLUSION A dedicated breast cool-tip RF ablation system can induce complete tumor necrosis and reproducible ablation volumes independently of breast glandular pattern, providing excellent cosmesis. Postablation MR images are a reliable tool in predicting histologic findings.

99mTc-interleukin-2 scintigraphy for the in vivo imaging of vulnerable atherosclerotic plaques

PurposeSeveral histopathological studies have demonstrated that vulnerable plaques are enriched in inflammatory cells. The aims of this study were: (1a) to test the ability of 99mTc-labelled interleukin-2 (99mTc-IL2) to bind to IL2R-positive (IL2R+) cells in carotid plaques and (1b) to correlate the plaque uptake of 99mTc-IL2, measured in vivo, with the number of IL2R+ cells within the plaque, measured ex vivo by histology (transversal study, TS), and (2) to evaluate changes in 99mTc-IL2 uptake in plaques, before and after treatment with a statin or a hypocholesterolaemic diet (longitudinal study, LS).MethodsUltrasound scan was performed for plaque characterisation and localisation. Fourteen patients (16 plaques) eligible for endoarterectomy were recruited for the TS and underwent 99mTc-IL2 scintigraphy before surgery. Nine patients (13 plaques) were recruited for the LS; these patients received atorvastatin or a standard hypocholesterolaemic diet and 99mTc-IL2 scintigraphy was performed before and after 3 months of treatment.ResultsThe degree of 99mTc-IL2 uptake was expressed as the plaque/background (T/B) ratio. In patients from TS, T/B ratios correlated with the percentage of IL2R+ cells at histology (r=0.707; p=0.002) and the number of IL2R+ cells at flow cytometry (r=0.711; p=0.006). No correlations were observed between ultrasound scores and either scintigraphic or histological findings. In patients from the LS, the mean 99mTc-IL2 uptake decreased in statin-treated patients (1.75±0.50 vs 2.16±0.44; p=0.012), while it was unchanged in the patients on the hypocholesterolaemic diet (2.33±0.45 vs 2.34±0.5).Conclusion99mTc-IL2 accumulates in vulnerable carotid plaques; this accumulation is correlated with the amount of IL2R+ cells and is influenced by lipid-lowering treatment with a statin.

Sphingosine 1-phosphate induces antimicrobial activity both in vitro and in vivo

Sphingosine 1-phosphate (S1P), a polar sphingolipid metabolite, is involved in a wide spectrum of biological processes, including Ca(++) mobilization, cell growth, differentiation, motility, and cytoskeleton organization. Here, we show a novel role of S1P in the induction of antimicrobial activity in human macrophages that leads to the intracellular killing of nonpathogenic Mycobacterium smegmatis and pathogenic M. tuberculosis. Such activity is mediated by host phospholipase D, which favors the acidification of mycobacteria-containing phagosomes. Moreover, when it was intravenously injected in mycobacteria-infected mice, S1P reduced mycobacterial growth and pulmonary tissue damage. These results identify S1P as a novel regulator of the host antimicrobial effector pathways.

miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration.

A differentiation-promoting micro-RNA regulates actin cable dynamics, intercellular adhesion, and cell migration in human and mouse epidermis.