Laura Uxa

Platelet production and destruction in liver cirrhosis

BACKGROUND & AIMS Thrombocytopenia is common in liver cirrhosis (LC) but the mechanisms are not fully understood. The purpose of our work was to evaluate platelet kinetics in LC with different etiologies by examining platelet production and destruction. METHODS Ninety-one consecutive LC patients (36 HCV, 49 alcoholics, 15 HBV) were enrolled. As controls, 25 subjects with idiopathic thrombocytopenic purpura, 10 subjects with aplastic anemia, and 40 healthy blood donors were studied. Plasma thrombopoietin (TPO) was measured by ELISA. Reticulated platelets (RP) were determined using the Thiazole Orange method. Plasma glycocalicin (GC) was measured using monoclonal antibodies. Platelet associated and serum antiplatelet antibodies were detected by flow cytometry. B-cell monoclonality in PBMC was assessed by immunoglobulin fingerprinting. RESULTS Serum TPO was significantly lower in LC (29.9±18.1 pg/ml) compared to controls (82.3±47.6 pg/ml). The GC levels were higher in LC (any etiology) than in healthy cases. Conversely, the absolute levels of RP were lower in LC (any etiology) than in healthy controls. The platelet-associated and serum anti-platelet antibodies were higher in HCV+ LC compared to healthy subjects (p<0.0064), alcoholic LC (p<0.018), and HBV+ LC (p<0.0001). B-cell monoclonality was found in 27% of the HCV+LC, while it was not found in HBV+ or alcoholic LC. CONCLUSIONS Patients with LC present decreased plasma TPO, accelerated platelet turnover, and reduced platelet production. This indicates that LC thrombocytopenia is a multifactorial condition involving both increased platelet clearance and impaired thrombopoiesis.

Bortezomib arrests the proliferation of hepatocellular carcinoma cells HepG2 and JHH6 by differentially affecting E2F1, p21 and p27 levels.

Despite the broad anti-tumour potential of the proteasome inhibitor bortezomib, partial information is available with regard to its effects on hepatocellular carcinoma (HCC) cells. Here we studied the effects of bortezomib on two human HCC cell lines displaying a different phenotype, hepatocyte-like for HepG2 and undifferentiated for JHH6. Bortezomib induced a dose- and time-dependent increase in cell toxicity and decrease of cell viability, with JHH6 being less sensitive than HepG2. Moreover, a differential influence on major cell cycle regulatory genes was responsible for the observed decrease of S and increase of G(2)-M phase cells. In HepG2, bortezomib induced a post-transcriptional increase of cyclin E1 together with a transcriptional-mediated decrease of the transcription factor E2F1. This in turn resulted in the reduction of the hyper-phosphorylated form of pRB and in the transcriptional down-regulation of the E2F1 targets cyclin D1, cyclin A2 and CdK2 but not cyclin E1. Up-regulation of LRH1, a liver specific cyclin E1 transcription factor, accounted for the unvaried cyclin E1 mRNA levels. Additionally, bortezomib induced both transcriptional and post-translational increase of p21(waf1/cip1) and p27(kip1). In JHH6, an overall more contained variation in cell cycle mediators was observed with the reduction of E2F1, cyclin A2, LRH1 and the increase of p21(waf1/cip1) being the most evident. In conclusion, the presented data show the mechanisms regulating cell proliferation inhibition by bortezomib in two different HCC cell lines. Despite a certain phenotype-dependent effect, the potent action exerted by bortezomib makes this drug attractive for future experimentation in animal models, possibly leading to novel treatments for HCC.

MMP-9 Microsatellite Polymorphism and Susceptibility to Carotid Arteries Atherosclerosis

Objective—The aims of this study were to compare a microsatellite polymorphism (PM) of matrix metalloproteinase (MMP)-9 in patients with carotid atherosclerosis and control population, and to assess the relationship between this PM and plaque structure. Methods and Results—One hundred fifty patients referring to vascular diagnostic centers for suspected carotid atherosclerosis (at ultrasound examination: 110 positive, 40 negative) and controls (n=110) have been genotyped for MMP-9 PM. After controlling for risk factors, allelic and genotype frequencies were significantly different among the groups, with significant prevalence of long microsatellites in patients with carotid atherosclerosis. Long microsatellites (settled as 22 to 27 repeats) were associated with carotid atherosclerosis (odds ratio [OR], 5.2; 95% confidence interval [CI], 2.9 to 9.2), compared with controls; an independent case control study on patients with coronary atherosclerosis confirmed such result. Binary logistic regression showed that hypertension, long microsatellites in MMP-9 PM and smoking habits were variables accounting for the difference between ultrasound-positive patients and controls. Long microsatellites were also associated to plaques with thin fibrous cap and echolucent core (OR, 13.1; 95% CI, 1.6 to 100). These alleles were slightly more represented in female patients (&khgr;2 test=0.019; OR, 2.7; 95% CI, 1.2 to 6) but not associated with other risk factors. Plasma MMP-9 levels were related neither to MMP-9 PM nor to plaque type, and were related to gender and extension of atherosclerosis in carotid arteries. Conclusions—The number of repeats (≥22 CA) in the microsatellite of MMP-9 promoter, but not MMP-9 plasma levels, is associated to carotid atherosclerosis and particularly to plaques with a thin fibrous cap.

MMP-9 microsatellite polymorphism and susceptibility to exudative form of age-related macular degeneration.

Purpose: To assess if a polymorphism (PM) of the microsatellite (CA13–27) in the promoter region of Matrix Metalloproteinase 9 (MMP-9) was associated with the exudative form of age-related macular degeneration (AMD) and to its risk factors.Methods: In 107 patients with AMD (AMD Group) and 223 age- and gender-matched controls (Control Group) with cataract, demographic, clinical data, and MMP-9 PM have been compared.Results: The comparison of allelic frequencies showed a different pattern of CA repeats between AMD and Control Group (P < 0.00005), in particular the prevalence of longer microsatellites (≥ 22 CA repeats) was higher in AMD than in Control Group (O.R. 2.49, 95% CI 1.71 - 3.37, P < 0.001). Analyses of genetic frequencies gave similar results. Logistic regression confirmed that 22 or more CA repeats are associated to AMD. The only association between MMP-9 PM and other risk factors for AMD was with BMI (Spearman’s R = 0.298, P < 0.00005): all patients with both microsatellites ≥ 22 CA repeats were overweight or obese (χ2 test P < 0.0005, compared to other genotypes).Conclusions: Longer microsatellites in the promoter of MMP-9 are associated to the exudative form of AMD and to body mass index, a well-known risk factor for the disease.

Serum response factor depletion affects the proliferation of the hepatocellular carcinoma cells HepG2 and JHH6

For hepatocellular carcinoma (HCC), a leading cause of cancer death world-wide, there is no effective therapy especially for the advanced stage of the disease. Thus, we started the investigations about a novel anti HCC approach based on the depletion of the transcription factor serum response factor (SRF) in HCC cell lines; SRF choice was based on its recently proposed contribution to HCC tissue development and on its important role in cell proliferation. SRF depletion, obtained by a siRNA (siSRF797), was studied in two HCC cell lines, i.e. HepG2 and JHH6 assigned to high and low hepatocytic differentiation grade on the base of the capacity to synthesize albumin. In the HCC cell lines examined, siSRF797 reduced both the mRNA and protein levels of SRF without inducing unspecific interferon response or cytotoxicity. Moreover, SRF depletion induced the reduction of S-phase cells and a decrease in cell number and vitality. Particularly in HepG2, cell growth impairment was paralleled by the decrease of the levels of the transcription factor E2F1 together with some of its regulated genes. In HepG2 but not in JHH6, SRF depletion was associated with apoptosis. Finally, in both HepG2 and JHH6, the combined administration of siSRF797 and bortezomib, a proteasome inhibitor whose therapeutic potential for HCC is considered attractive, further reduced cell viability compared to either siSRF797 or bortezomib treatment alone. In conclusion, SRF depletion affects the expansion of the high and low differentiation grade HCC cells HepG2 and JHH6. These results can pave the way to understand the role of SRF in HCC development and possibly to identify novel anti HCC therapeutic strategies.

Relationship Between Human Leucocyte Antigen Class I and Class II and Chronic Idiopathic Urticaria Associated With Aspirin and/or NSAIDs Hypersensitivity

Background. HLA genes play a role in the predisposition of several diseases. The aim was to analyze the prevalence of HLA class I phenotypes and HLA-DRB1* genotype in patients with CIU associated with ASA and NSAIDs hypersensitivity (AICU). Methods. 69 patients with AICU, and 200 healthy subjects. Results. Subjects with HLA-B44 and HLA-Cw5 antigens were more represented in patients with AICU than in control group. Subjects with HLA-A11, HLA-B13, HLACw4, and HLA-Cw7 antigen were more represented in control group than in patients with AICU. Multiple logistic regression demonstrated an association of HLA-Cw4 and HLA-Cw7 with a lower risk of AICU, whereas carriers of HLA-B44 phenotype had a higher risk of AICU. No differences were found between patients and controls as regards to HLA-DRB1* genotype. Conclusions. We observed an association between some HLA class-I antigens and AICU. To the best of our knowledge this is the first description of such association.