Monica Attanasio

Transient intermittent lymphocyte activation is responsible for the instability of angina.

BackgroundBlood clotting activation is an important component of the inflammatory response; the outbursts of unstable angina are usually associated with increased thrombin formation and coronary mural thrombosis. Methods and ResultsTo investigate 1) whether monocyte activation is responsible for the enhanced thrombin formation during bursts of unstable angina and 2) what mechanism(s) might be responsible for monocyte activation, we studied patients with unstable angina (n=31), stable effort angina (n=23), left endoventricular thrombosis (n=8), and control subjects (n=44), measuring plasma fibrinopeptide A (FPA) levels and the capacity of monocytes to express procoagulant activity (PCA) and of lymphocytes to modulate this expression. Patients with unstable angina and patients with endoventricular thrombosis had significantly (p<0.0001) higher FPA plasma levels than patients with effort angina and control subjects. However, only monocytes from unstable angina patients expressed significantly increased PCA characterized as tissue factor-like activity (units/105 monocytes, median and range; 120, 1.1–463.2 versus 10.8, 0.8–39.1 in control subjects; p<0.0001 versus the other groups). When 14 patients with unstable angina were restudied 8–12 weeks later, they showed neither elevated plasma FPA levels nor monocyte PCA. In unstable angina patients, there was a correlation between FPA and PCA (r=0.56, p<0.001). For expression of PCA by monocytes, both an incubation of at least 2 hours with lymphocytes and direct monocyte-lymphocyte contact were needed. In reconstitution and cross-mixing experiments, only lymphocytes from patients with active unstable angina induced the expression ofPCA by monocytes from both control and patient groups. ConclusionsThe results demonstrate that the increased thrombin formation in unstable angina patients is due to the expression of tissue factor-like activity by activated monocytes. The monocyte activation appears to be a part of a lymphocytic cell-instructed response intermittently triggered by unknown factors.

Monitoring of low-molecular-weight heparins in cardiovascular disease.

Thrombin generation is a key event in the pathophysiology of coronary syndromes and provides the rationale for treatment with anticoagulants. Unlike standard heparin, low-molecular-weight heparin (LMWH) has little effect on activated partial thromboplastin time. LMWH treatment has been monitored by measurement of anti-Factor Xa activity, but this may not accurately reflect the anticoagulant action because LMWHs also inhibit Factor II. The Heptest is a clotting assay that is sensitive to both anti-Xa and anti-IIa activity, as well as inhibition of the extrinsic pathway by LMWH-stimulated release of tissue factor pathway inhibitor. The plasma thrombin neutralization assay has also been used to measure LMWH and to detect low concentrations to which chromogenic assays are insensitive. In the clinical setting, monitoring the anti-Xa activity in patients treated with LMWH after acute deep vein thrombosis offered no advantages over a standard weight-adjusted dose. Moreover, in acute coronary syndromes there is no increase in major hemorrhage rates with weight-adjusted LMWH. Monitoring of LMWH concentrations may be advisable in the presence of comorbid conditions carrying an increased risk of hemorrhage, such as renal disease, advanced age, severe over- or underweight, or a history of previous bleeding episodes.

Interleukin-6 and acute-phase proteins in head and neck cancer

The acute-phase response is the answer of the organism to a disturbance of its homeostasis and is characterized by dramatic changes in the concentration of some plasma proteins defined as acute-phase proteins. In recent years several data have shown that interleukin-6 (IL-6) is the major inducer of acute-phase protein synthesis in human hepatocytes. Recently, we demonstrated higher IL-6 serum levels in head and neck cancer (HNC) patients than in healthy subjects. In the present study we examined the relationship between levels of IL-6 and of several acute-phase proteins, including C-reactive protein (CRP), α1-antitrypsin (AAT), αl-acid glycoprotein (AAG), haptoglobin (HPT) and fibrinogen. Eighteen patients were studied and had squamous cell carcinoma of the larynx (n = 9), oral cavity (n = 4), oropharynx (n = 3) and hypopharynx (n = 2). Proteins were measured at three time points before and three time points after surgery. Significant (P < 0.0001) relationships were found between IL-6 and CRP (r = 0.69), and fibrinogen (r = 0.51), whereas no correlation was found with AAT (r = 0.13, P = 0.56), AAG (r = 0.38; P = 0.07) and HPT (r = 0.16; P = 0.46). These data strongly suggest that IL-6 may play a key role in acute-phase protein synthesis in HNC and in regulation of the complex host response to malignancies.

Fibrillin-1 (FBN1) gene frameshift mutations in Marfan patients: genotype-phenotype correlation

Marfan syndrome (MFS) is a multisystemic disease associated with mutations in the fibrillin‐1 gene. Most of the reported mutations are missense substitutions mainly affecting the epidermal growth factor (EGF)‐like protein domain structure and the calcium‐binding (cb) site. The aim of our study was to investigate the correlation between fibrillin‐1 frameshift mutations and the clinical phenotype in patients affected by MFS. In 48 out of 66 Marfan patients a pathogenetic mutation was found. We detected novel mutations causing premature termination codon in exons 19, 37, 40 and 41 of four Italian patients. The first mutation in exon 19 (cbEGF #8 domain) results in a clinical phenotype involving mainly the skeletal and cardiovascular systems. Interestingly, we noticed that, while mutations in exons 37 and 41 (eight cysteine domains #4 and #5) are milder, the mutation in exon 40 (cbEGF #24 domain) is more severe and causes major cardiovascular involvement with thoracic and abdominal aortic aneurysms. It is noteworthy that the degree of the severity in the phenotype of one of our patients and another from the literature carrying a mutation in exon 41 could be explained with alterations in mRNA expression.

FBN1 mutation screening of patients with Marfan syndrome and related disorders: detection of 46 novel FBN1 mutations

Fibrillin‐1 gene (FBN1) mutations cause Marfan syndrome (MFS), an inherited connective tissue disorder with autosomal dominant transmission. Major clinical manifestations affect cardiovascular and skeletal apparatuses and ocular and central nervous systems. We analyzed FBN1 gene in 99 patients referred to our Center for Marfan Syndrome and Related Disorders (University of Florence, Florence, Italy): 85 were affected by MFS and 14 by other fibrillinopathies type I. We identified mutations in 80 patients. Among the 77 different mutational events, 46 had not been previously reported. They are represented by 49 missense (61%), 1 silent (1%), 13 nonsense (16%), 6 donor splice site mutations (8%), 8 small deletions (10%), and 3 small duplications (4%). The majority of missense mutations were within the calcium‐binding epidermal growth factor‐like domains. We found preferential associations between The Cys‐missense mutations and ectopia lentis and premature termination codon mutations and skeletal manifestations. In contrast to what reported in literature, the cardiovascular system is severely affected also in patients carrying mutations in exons 1–10 and 59–65. In conclusion, we were able to detect FBN1 mutations in 88% of patients with MFS and in 36% of patients with other fibrillinopathies type I, confirming that FBN1 mutations are good predictors of classic MFS.

Cyclooxygenase and lipoxygenase metabolite synthesis by polymorphonuclear neutrophils: in vitro effect of dipyrone

Functional activity of polymorphonuclear neutrophils (PMN) is associated with the metabolism of Arachidonic Acid (AA) released from membrane phospholipids. In this study the in vitro effect of dipyrone, a non steroidal anti-inflammatory drug, on the production of AA metabolites through cyclooxygenase (CO) and lipoxygenase (LO) pathways by stimulated PMN has been investigated. PMN isolated by counterflow centrifuge elutriator were greater than 98% pure and viable. Metabolite production was evaluated by RIA of Thromboxane A2 (TxA2), Prostaglandin E2 (PGE2), Leukotriene B2 (LTB4) and Leukotriene C4 (LTC4) after PMN stimulation with calcium ionophore A 23187 (20 microM). The levels of beta-thromboglobulin (RIA) lower than 5 ng/ml allowed us to rule out activation of residual contaminant platelets. In these experimental conditions, in the absence of dipyrone the products (ng/10(6) cells) of AA metabolism were LTB4 (3.51 +/- 0.22), LTC4 (0.81 +/- 0.08), TxB2 (0.144 +/- 0.025) and PGE2 (0.150 +/- 0.017). Incubation with dipyrone induced changes of PGE2 and TXB2 production in a dose dependent fashion (r = 0.83 and r = 0.87, p less than 0.001), obtaining already at the lowest drug concentration (5 micrograms/ml) a significant inhibition (33 and 40% for TxB2 and PGE2 p less than 0.005). No significant changes of LTB4 and LTC4 production have been observed. The results of this study indicate that dipyrone relevantly affects CO metabolite synthesis by stimulated PMN at concentrations comparable to those reached in therapeutic use. The inhibition of PGE2 synthesis which is present in inflamed tissues and actively participates in inflammatory reactions, could contribute to the therapeutic anti-inflammatory action of dipyrone.

Association of Marfan syndrome and bicuspid aortic valve: frequency and outcome.

a Cardiology Service, CMSR Veneto Medica, Altavilla Vicentina, Italy b Department of Medical and Surgical Critical Care, University of Florence, Center of the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Disease to Develop Novel Therapies, University of Florence, Italy c Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, University of Florence, Italy d S. Maria agli Ulivi Center, Fondazione Don Carlo Gnocchi, Onlus, IRCCS, Florence, Italy

Cytokine gene expression and production by human LPS-stimulated mononuclear cells are inhibited by sulfated heparin-like semi-synthetic derivatives

Summary.  Background: The K5 polysaccharide obtained from Escherichia coli strain 010:K5:H4 is a polymer of the disaccharidic unit formed by D‐glucuronic acid and N‐acetylglucosamine. This structure is akin to N‐acetylheparosan, the precursory polymer of heparin and of heparan sulfate. This structural affinity with N‐acetylated heparin and with de‐sulfated heparin makes the K5 polysaccharide extremely useful for the preparation of sulfated heparin‐like semi‐synthetic derivatives. It has been demonstrated that heparins are able to inhibit tissue factor and cytokine production and expression by human monocytes. Objective: The aim of this study was to evaluate the effects of four different heparin‐like semi‐synthetic derivatives on inflammatory cytokine production and expression by human mononuclear cells. Results: The simultaneous addition of lipopolysaccharide (LPS; 0.2 and 10 µg mL−1) and the K5 polysaccharide did not inhibit interleukin (IL)‐1β, IL‐6 or tumor necrosis factor (TNF)‐α production by stimulated mononuclear cells. IL‐1β, IL‐6 and TNF‐α concentrations in supernatants of LPS‐stimulated mononuclear cells were not influenced by the addition of N,O‐sulfated K5 polysaccharide (K5‐N, OS) and epimerized N‐sulfated K5 polysaccharide (K5 NS epi) at 5 and 10 µg mL−1, whereas the addition of epimerized N,O‐sulfated K5 polysaccharide (K5‐N, OS epi) (5 and 10 µg mL−1) and O‐sulfated K5 polysaccharide (K5‐OS) (5 and 10 µg mL−1) to LPS‐stimulated cells caused a significant dose‐dependent inhibition of IL‐1β, IL‐6 and TNF‐α. All sulfated heparin‐like semi‐synthetic derivatives did not influence the IL‐10 production by LPS‐stimulated mononuclear cells. In LPS‐stimulated cells (0.2 and 10 µg mL−1), K5‐OS or K5‐N, OS epi at 5 and 10 µg mL−1 markedly decreased TNF‐α mRNA expression. Conclusions: These results indicate that the sulfated heparin‐like semi‐synthetic derivatives K5‐OS and K5‐N, OS epi are able to inhibit both expression and production of inflammatory cytokines, whereas they do not influence the anti‐inflammatory cytokine IL‐10, suggesting a potential role for these products as modulators of inflammatory reactions.

Interleukin-1 beta and interleukin-6 release by peripheral blood monocytes in head and neck cancer.

In patients with advanced head and neck squamous cell carcinoma (HNSC), evidence of cell-mediated immunity and monocyte functional abnormalities has been reported. We studied the production of interleukin 1 beta (IL-1 beta) and interleukin 6 (IL-6) by peripheral blood monocytes from 22 patients with HNSC (12 larynx and ten oral cavity cancers) in comparison with monocyte cytokine production of age-matched healthy subjects. Pure monocytes were incubated with and without lipopolysaccharides (LPS) (10 micrograms ml-1) for 4 h at 37 degrees C and IL-1 beta and IL-6 concentrations were determined in supernatants by specific ELISA. There was no significant difference in IL-1 beta levels in monocyte supernatants from cancer in comparison to control subjects; conversely, a higher IL-6 production by unstimulated and LPS-activated cells from HNSC patients than from controls was found. No relationship was observed between cytokine production and cancer stage. The regression analysis evidenced a significant correlation between IL-1 beta and IL-6 monocyte-release in HNSC patients and in controls, so suggesting a possible autocrine control of IL-6 production by other cytokines.

Multiple primary tumors of the upper aerodigestive tract: Is there a role for constitutional mutations in the p53 gene?†

Head‐and‐neck cancer (HNC) patients have a high risk of developing second primary tumors of the upper aerodigestive tract, the main cause of death. Although the roles of tobacco and diet in multiple head‐and‐neck carcinogenesis have been thoroughly investigated, little is known about individual genetic susceptibility factors involved in this process. Genomic instability, reflecting the propensity and the susceptibility of the genome to acquire multiple alterations, could be considered a driving force behind multiple carcinogenesis. Mutation of the p53 tumor‐suppressor gene has been proposed to play an important role in this process. Therefore, we evaluated the incidence of inherited p53 germ‐line alteration(s) in a population of 24 consecutive HNC patients and their first‐degree relatives affected by multiple malignancies as well as the occurrence of p53 somatic acquired mutation(s) in 16 cancers, including first and second primaries from 5 HNCs of the same group. Mutations in exons 4–11 of the p53 gene were investigated using SSCP‐PCR analysis and DNA sequencing. Analysis was extended to the peripheral blood and cancer biopsies available from first‐degree relatives of cancer‐prone families with p53 germ‐line mutations. p53 germ‐line mutations were identified in the peripheral blood and corresponding cancers of 3 HNC patients who had multiple malignancies. The only missense mutation detected was mapped in exon 6; it is a GTG to GAG substitution with an amino acid change from Val to Glu at codon 197. The remaining 2 p53 germ‐line mutations were single‐nucleotide substitutions without amino acid change in exon 6 (codon 213, CGA to CGG) and in exon 8 (codon 295, CCT to CCC), respectively. These mutations were found in HNC patients with a family history of cancer. Abnormal expression of wild‐type p53 protein in normal and pathological tissues from patients with the same sense single‐nucleotide substitutions was detected by immuno‐histochemistry. Int. J. Cancer 82:180–186, 1999.