Fiorella Calabrese

Tumor-Induced Tolerance and Immune Suppression Depend on the C/EBPβ Transcription Factor

Tumor growth is associated with a profound alteration in myelopoiesis, leading to recruitment of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). We showed that among factors produced by various experimental tumors, the cytokines GM-CSF, G-CSF, and IL-6 allowed a rapid generation of MDSCs from precursors present in mouse and human bone marrow (BM). BM-MDSCs induced by GM-CSF+IL-6 possessed the highest tolerogenic activity, as revealed by the ability to impair the priming of CD8(+) T cells and allow long term acceptance of pancreatic islet allografts. Cytokines inducing MDSCs acted on a common molecular pathway and the immunoregulatory activity of both tumor-induced and BM-derived MDSCs was entirely dependent on the C/EBPbeta transcription factor. Adoptive transfer of tumor antigen-specific CD8(+) T lymphocytes resulted in therapy of established tumors only in mice lacking C/EBPbeta in the myeloid compartment, suggesting that C/EBPbeta is a critical regulator of the immunosuppressive environment created by growing cancers.

Immunosuppressive Therapy for Active Lymphocytic Myocarditis Virological and Immunologic Profile of Responders Versus Nonresponders

Background—The beneficial effect of immunosuppressive treatment on myocarditis is still controversial, possibly because the immunologic and virological profile of potential candidates is largely unknown. Methods and Results—Out of 652 biopsied patients, 112 had a histological diagnosis of active lymphocytic myocarditis; 41 of these 112 patients were characterized by progressive heart failure despite conventional therapy and were treated with prednisone and azathioprine for 6 months. All were resubmitted to cardiac catheterization, angiography, and endomyocardial biopsy at 1 and 6 months and followed-up for 1 year. A total of 21 patients responded with prompt improvement in left ventricular ejection fraction from 25.7±4.1% to 47.1±4.4% and showed evidence of healed myocarditis at control biopsy. Conversely, 20 patients failed to respond and showed a histological evolution toward dilated cardiomyopathy: 12 remained stationary, 3 underwent cardiac transplantation, and 5 died. We retrospectively performed a polymerase chain reaction on frozen endomyocardial tissue for the most common cardiotropic viruses and assessed circulating serum cardiac autoantibodies. Viral genomes were present in biopsy specimens of 17 nonresponders (85%), including enterovirus (n=5), Epstein-Barr virus (n=5) adenovirus (n=4), both adenovirus and enterovirus (n=1), influenza A virus (n=1), parvovirus-B19 (n=1), and in 3 responders, who were all positive for hepatitis C virus. Cardiac autoantibodies were present in 19 responders (90%) and in none of the nonresponders. Conclusions—In patients with active lymphocytic myocarditis, those with circulating cardiac autoantibodies and no viral genome in the myocardium are the most likely to benefit from immunosuppression. The beneficial effect of immunosuppression in hepatitis C virus myocarditis suggests a relevant immunomediated component of damage.

Neutrophil restraint by green tea: Inhibition of inflammation, associated angiogenesis, and pulmonary fibrosis

Neutrophils play an essential role in host defense and inflammation, but the latter may trigger and sustain the pathogenesis of a range of acute and chronic diseases. Green tea has been claimed to exert anti-inflammatory properties through unknown molecular mechanisms. We have previously shown that the most abundant catechin of green tea, (−)epigallocatechin-3-gallate (EGCG), strongly inhibits neutrophil elastase. Here we show that 1) micromolar EGCG represses reactive oxygen species activity and inhibits apoptosis of activated neutrophils, and 2) dramatically inhibits chemokine-induced neutrophil chemotaxis in vitro; 3) both oral EGCG and green tea extract block neutrophil-mediated angiogenesis in vivo in an inflammatory angiogenesis model, and 4) oral administration of green tea extract enhances resolution in a pulmonary inflammation model, significantly reducing consequent fibrosis. These results provide molecular and cellular insights into the claimed beneficial properties of green tea and indicate that EGCG is a potent anti-inflammatory compound with therapeutic potential.

Arrhythmogenic Right Ventricular Cardiomyopathy Causing Sudden Cardiac Death in Boxer Dogs A New Animal Model of Human Disease

Background—Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary familial heart muscle disease associated with substantial cardiovascular morbidity and risk of sudden death. Efforts to discern relevant pathophysiological mechanisms have been impaired by lack of a suitable animal model. Methods and Results—ARVC was diagnosed in 23 boxer dogs (12 male; 9.1±2.3 years old). Clinical events alone or in combination included sudden death (n=9; 39%), ventricular arrhythmias of suspected right ventricular (RV) origin (n=19; 83%), syncope (n=12, 52%), and heart failure (n=3; 13%). Right ventricular enlargement or aneurysms occurred in 10 (43%). Striking histopathological abnormalities were present in each boxer dog but not in controls, including severe RV myocyte loss with replacement by fatty (n=15, 65%) or fibrofatty (n=8, 35%) tissue. Focal fibrofatty lesions were also present in both atria (n=8) and the left ventricle (LV) (n=11). Fatty replacement occupied substantially greater RV wall area in ARVC dogs than controls (40.4±18.8% versus 13.8±3.4%, respectively) (P <0.001); residual myocardium was correspondingly reduced (56.6±19.2% versus 84.8±3.8% in controls) (P <0.001). MRI demonstrated bright anterolateral and/or infundibular RV myocardial signals, confirmed as fat by histopathology. Myocarditis appeared in the RV (n=14, 61%) and LV (n=16, 70%) and in each dog with sudden death, but not in controls. Familial transmission was evident in 10 of the 23. Conclusions—We describe a novel, spontaneous, and genetically transmitted animal model of ARVC associated with sudden death in the boxer dog, closely resembling the human disease. This model may aid in understanding the pathogenic mechanisms of ARVC.

CXCR3 and Its Ligand CXCL10 Are Expressed by Inflammatory Cells Infiltrating Lung Allografts and Mediate Chemotaxis of T Cells at Sites of Rejection

The attraction of T lymphocytes into the pulmonary parenchyma represents an essential step in mechanisms ultimately leading to lung allograft rejection. In this study we evaluated whether IP-10 (CXCL10), a chemokine that is induced by interferon-gamma and stimulates the directional migration of activated T cells, plays a role in regulating the trafficking of effector T cells during lung allograft rejection episodes. Immunohistochemical examination showed that areas characterized by acute cellular rejection (grades 1 to 4) and active obliterative bronchiolitis (chronic rejection, Ca) were infiltrated by T cells expressing CXCR3, i.e., the specific receptor for CXCL10. In parallel, T cells accumulating in the bronchoalveolar lavage of lung transplant recipients with rejection episodes were CXCR3+ and exhibited a strong in vitro migratory capability in response to CXCL10. In lung biopsies, CXCL10 was abundantly expressed by graft-infiltrating macrophages and occasionally by epithelial cells. Alveolar macrophages expressed and secreted definite levels of CXCL10 capable of inducing chemotaxis of the CXCR3+ T-cell line 300-19; the secretory capability of alveolar macrophages was up-regulated by preincubation with interferon-gamma. Interestingly, striking levels of CXCR3 ligands could be demonstrated in the fluid component of the bronchoalveolar lavage in individuals with rejection episodes. These data indicate the role of the CXCR3/CXCL10 interactions in the recruitment of lymphocytes at sites of lung rejection and provide a rationale for the use of agents that block the CXCR3/CXCL10 axis in the treatment of lung allograft rejection.

2011 consensus statement on endomyocardial biopsy from the Association for European Cardiovascular Pathology and the Society for Cardiovascular Pathology.

The Association for European Cardiovascular Pathology and the Society for Cardiovascular Pathology have produced this position paper concerning the current role of endomyocardial biopsy (EMB) for the diagnosis of cardiac diseases and its contribution to patient management, focusing on pathological issues, with these aims: • Determining appropriate EMB use in the context of current diagnostic strategies for cardiac diseases and providing recommendations for its rational utilization • Providing standard criteria and guidance for appropriate tissue triage and pathological analysis • Promoting a team approach to EMB use, integrating the competences of pathologists, clinicians, and imagers.

Postmortem diagnosis in sudden cardiac death victims: macroscopic, microscopic and molecular findings

Time for primary review 29 days. When sudden death (SD) occurs in adults and elderly persons, coronary atherosclerosis is the usual cause [1,2]. On the contrary, a large spectrum of cardiovascular diseases, both congenital and acquired, may account for SD in the young [3–10]. These diseases are frequently concealed and discovered with surprise only at postmortem by means of a thorough macroscopic and microscopic investigation. This review will address the spectrum of structural substrates of cardiac SD with particular emphasis given to the possible role of molecular biology techniques in identifying subtle or even merely functional disorders accounting for electrical instability. SD is defined as a natural, unexpected fatal event occurring within 1 h of the beginning of symptoms, in an apparently healthy subject or one whose disease was not so severe enough as to predict such an abrupt outcome [11]. In the USA, the annual incidence of SD in people aged 35–74 years is 191/100 000 in men and 57/100 000 in women; almost half of all SDs occur in people with known coronary artery disease [12]. In the Veneto region, Northeast of Italy, we recently calculated an overall prevalence of SD of 0.8/100 000/year in the young, based only upon autopsy reports [13]. When focusing the attention only on young athletes the prevalence was twice that in young non athletes, i.e. 1.6/100 000/year; these figures are explained by the existence of cardiovascular diseases which cause a risk of SD during effort, such as hypertrophic cardiomyopathy (HCM), arrhythmogenic right ventricular cardiomyopathy (ARVC) and congenital coronary anomalies. As far as pathophysiology is concerned, cardiac arrest may be either mechanical, when the heart and circulatory functions are suddenly impeded by mechanical factors (i.e. cardiac tamponade, pulmonary thromboembolism, etc.) or arrhythmic (mostly ventricular fibrillation) [9]. Based … * Corresponding author. Tel.: +39-49-827-2283; fax: +39-49-827-2284 cardpath{at}ux1.unipd.it

Sarcoidosis is a Th1/Th17 multisystem disorder

Background and aims Sarcoidosis is characterised by a compartmentalisation of CD4+ T helper 1 (Th1) lymphocytes and activated macrophages in involved organs, including the lung. Recently, Th17 effector CD4+ T cells have been claimed to be involved in the pathogenesis of granuloma formation. The objective of this study was to investigate the involvement of Th17 cells in the pathogenesis of sarcoidosis. Methods Peripheral and pulmonary Th17 cells were evaluated by flow cytometry, real-time PCR, immunohistochemistry analyses and functional assays in patients with sarcoidosis in different phases of the disease and in control subjects. Results Th17 cells were detected both in the peripheral blood (4.72±2.27% of CD4+ T cells) and in the bronchoalveolar lavage (BAL) (8.81±2.25% of CD4+ T lymphocytes) of patients with sarcoidosis and T cell alveolitis. Immunohistochemical analysis of lung and lymph node specimens showed that interleukin 17 (IL-17)+/CD4+ T cells infiltrate sarcoid tissues surrounding the central core of the granuloma. IL-17 was expressed by macrophages infiltrating sarcoid tissue and/or forming the granuloma core (7.88±2.40% of alveolar macrophages). Analysis of some lung specimens highlighted the persistence of IL-17+/CD4+ T cells in relapsed patients and their absence in the recovered cases. Migratory assays demonstrated the ability of the Th17 cell to respond to the chemotactic stimulus CCL20—that is, the CCR6 ligand (74.8±8.5 vs 7.6±2.8 migrating BAL lymphocytes/high-powered field, with and without CCL20, respectively). Conclusions Th17 cells participate in the alveolitic/granuloma phase and also to the progression towards the fibrotic phase of the disease. The recruitment of this cell subset may be driven by CCL20 chemokine.

IL-32, a novel proinflammatory cytokine in chronic obstructive pulmonary disease.

RATIONALE Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disorder of the lung, yet the mechanisms that regulate this immune-inflammatory response are not fully understood. OBJECTIVES We investigated whether IL-32, a newly discovered cytokine, was related to markers of inflammation and clinical progression in COPD. METHODS Using immunohistochemistry, expression of IL-32 was examined in surgically resected specimens from 40 smokers with COPD (FEV(1) = 39 +/- 4% predicted), 11 smokers with normal lung function, and 9 nonsmoking control subjects. IL-32 was quantified in alveolar macrophages, alveolar walls, bronchioles, and arterioles, and confirmed by molecular analysis. The levels of IL-32 were correlated with the cellular infiltrates, markers of inflammation, and clinical data. MEASUREMENTS AND MAIN RESULTS Macrophage staining for IL-32 was increased in smokers with COPD compared with control smokers and nonsmokers (P = 0.0014 and P < 0.0001, respectively), and similar differences were observed in alveolar walls (P = 0.0004 and P = 0.0005) and bronchiolar epithelium (P = 0.004 and P = 0.0009). This increase was also detected at the mRNA level (P = 0.007 vs. control smokers and P = 0.029 vs. nonsmokers) and was mainly due to non-alpha isoforms. Moreover, IL-32 expression was positively correlated with tumor necrosis factor-alpha (P = 0.004, r(s)=0.70), CD8(+)cells (P = 0.02, r(s)=0.46), phospho p38MAPK (P < 0.01, r(s)=0.60) and negatively with FEV(1) values (P = 0.004, r(s)= -0.53). CONCLUSIONS This is the first study to demonstrate increased expression of IL-32 in lung tissue of patients with COPD, where it was colocalized with tumor necrosis factor-alpha and correlated with the degree of airflow obstruction. These results suggest that IL-32 is implicated in the characteristic immune response of COPD, with a possible impact on disease progression.

Myocarditis and inflammatory cardiomyopathy: microbiological and molecular biological aspects.

Myocarditis is an inflammatory disease of the myocardium associated with cardiac dysfunction. The natural history of myocarditis is frequently characterised by the evolution in dilated cardiomyopathy. Due to its variable clinical manifestation from latent to very severe clinical forms, such as acute congestive heart failure and sudden death, its prevalence is still unknown and probably underestimated. In spite of the development of various diagnostic modalities, early and definite diagnosis of myocarditis still depends on the detection of inflammatory infiltrates in endomyocardial biopsy specimens according to the Dallas criteria. Routine application of immunohistochemistry, used for identification and characterisation of inflammatory cell populations, has now significantly increased the sensistivity of the diagnosis of inflammatory cardiomyopathy. Various molecular techniques, such as PCR, gene sequencing and real-time PCR, often applied on the same endomyocardial specimen, have become an essential part of the diagnostic armamentarium for rapid, specific and sensitive identification of infective agents. The correct application of molecular techniques will allow increasingly more information to be obtained: new epidemiology, new patient risk stratification and overall more appropriate medical treatment.