Ferdinando Cirillo

In vitro Inhibition by Defibrotide of Monocyte Superoxide Anion Generation: A Possible Mechanism for the Antithrombotic Effect of a Polydeoxyribonucleotide-Derived Drug

In an attempt to elucidate the antithrombotic potential of defibrotide (D) we have evaluated several functions of monocytes from 7 healthy subjects before and after in vitro incubation of the cells with increasing concentrations of this drug. At concentrations as high as 40 micrograms/ml, D hardly affected the expression of both the procoagulant activity of monocytes and the formation of superoxide anion in response to 1 mg/ml zymosan (STZ). In contrast, at concentrations that may be achieved in vivo following the administration of the drug (5-20 micrograms/ml), D impaired in a dose-dependent manner (p less than 0.05) the generation of O-2 in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP, 1 microM) or calcium ionophore A23187 (10 microM). Regardless of the agonist employed, at concentrations between 1 and 5 mM, extracellular Ca2+ had little effect on the impairment of superoxide anion generation by D. In contrast, the inhibitory effect was time-dependent, the maximum impairment (greater than 30%) being observed when the cells were preincubated with the drug for 20 h. These data support the concept that the antithrombotic potential of D involves the ability of the drug to affect the generation of free radicals by leukocytes and suggest that future in vivo studies for the evaluation of the activity of D should take into account the role of monocytes in hemostasis and thrombosis.

Hemostatic variables in homozygous familial hypercholesterolemia. Effect of regular plasma cholesterol removal by low density lipoprotein apheresis.

Plasma levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) and the in vitro ability of platelets to aggregate and of monocytes to express procoagulant (tissue factor) activity (PCA) were evaluated in five patients who are homozygous for familial hypercholesterolemia (FH) before and after a single and a regular 5-month cholesterol removal by low density lipoprotein (LDL) apheresis. The biweekly procedure resulted in a 25% to 30% reduction (approximately 150 mg/dl) in total and LDL cholesterol (both were greater than 550 mg/dl at the beginning of the study). The basal levels of t-PA antigen and fibrinolytic activity before and after 10 minutes of venous stasis, basal PAI activity, and PAI-1 antigen were comparable to controls and were not affected by LDL apheresis. Likewise, regardless of the cholesterol removal, the PCA of freshly isolated monocytes and that of monocytes incubated with lipopolysaccharide did not differ from control values. Finally, the pre-apheresis sensitivity of platelets to adenosine diphosphate, arachidonic acid, and collagen was 1.5 to 2 times the normal value. This ratio was unchanged throughout the 5-month procedure. We conclude that fibrinolysis and monocyte PCA are normal in FH patients, whereas platelet aggregation is abnormally high, and none of these parameters is significantly affected by a 25% to 30% reduction in total and LDL cholesterol by LDL apheresis. Furthermore, our data suggest that removal of cholesterol from plasma by LDL apheresis is important for gaining insight into the mechanisms involved in the ischemic complications of arteriosclerosis in FH patients.

Cu/Zn superoxide dismutase in patients with non-familial Alzheimer’s disease

Chromosome 21 contains genes whose altered expression has long been associated with Down’s syndrome and whose altered structure with some cases of Alzheimer’s disease (AD). The gene for the Cu/Zn superoxide dismutase enzyme (SOD- 1), a key enzyme in the metabolism of oxygen free radicals, is located on the distal portion of chromosome 21. Due to the triplication of the SOD- 1 gene, patients with Down’s syndrome have an almost 50% increase in their SOD activity. On the other hand, almost 25% of the patients with Down’s syndrome over 40 years of age develop progressive dementia, with clinical symptoms of AD. Therefore, we decided to evaluate whether abnormalities in the production of free radicals could be detected in blood cells from AD patients, and whether they correlated with molecular variations in the Cu/Zn SOD- 1 gene. Superoxide anion production was evaluated spectrophotometrically in suspensions of monocytes from 9 sporadic AD patients, and from 9 aged- matched apparently normal controls. After stimulation with increasing concentrations of n- formyl- methionyl- leucyl- phenylalanine (fMLP) or Ca ionophore A23187, monocyte free radical generation was quantitatively and qualitatively normal. Furthermore, restriction fragment length polymorphism (RFLP) analysis of leukocyte DNA digested with a variety of enzymes, gave comparable results in patients and controls. Our data support the possibility that in addition to the generation of free radicals, other directions should be explored to elucidate the mechanisms of dementia in AD. (Aging Clin. Exp. Res. 7: 49–54, 1995)

Lack of change in insulin levels as a biological marker of PAI-1 lowering in GH-deficient adults on r-HGH replacement therapy.

Patients with growth hormone (GH) deficiency (GHD) have decreased life expectancy and increased risk of cardiovascular events [3,22,25]. An impaired fibrinolysis is a major determinant of cardiovascular risk [4,27]. PAI-1, the main physiologic inhibitor of tPA, regulates the fibrinolytic system. Raised plasma levels of PAI-1 have been documented in ischemic heart disease and in subjects who subsequently develop myocardial infarction [7]. Abnormally high circulating levels of PAI-1 are associated with high risk for stroke and myocardial ischemia [4,27]. Prospectively, raised levels of PAI-1 is a strong determinant of vascular ischemic events [11,18,20,21]. It is also known [see 16,17 for a review], that PAI-1 inhibition rises with the increase in t-PA antigen, so that high levels of either factor reflect an impaired fibrinolysis. This information is based on a series of data: a) concentrations of t-PA antigen above normal ranges are present in subjects with high plasma PAI-1 levels; b) increases in t-PA antigen reflect the inhibitory effect of PAI-1 on t-PA

Identification of the first dominant mutation of LAMA5 gene causing a complex multisystem syndrome due to dysfunction of the extracellular matrix

Background The laminin alpha 5 gene (LAMA5) plays a master role in the maintenance and function of the extracellular matrix (ECM) in mammalian tissues, which is critical in developmental patterning, stem cell niches, cancer and genetic diseases. Its mutations have never been reported in human disease so far. The aim of this study was to associate the first mutation in LAMA5 gene to a novel multisystem syndrome. Methods A detailed characterisation of a three-generation family, including clinical, biochemical, instrumental and morphological analysis, together with genetics and expression (WES and RNAseq) studies, was performed. Results The heterozygous LAMA5 mutation c.9418G>A (p.V3140M) was associated with skin anomalies, impaired scarring, night blindness, muscle weakness, osteoarthritis, joint and internal organs ligaments laxity, malabsorption syndrome and hypothyroidism. We demonstrated that the mutation alters the amount of LAMA5 peptides likely derived from protein cleavage and perturbs the activation of the epithelial-mesenchymal signalling, producing an unbalanced expression of Sonic hedgehog and GLI1, which are upregulated in cells from affected individuals, and of ECM proteins (COL1A1, MMP1 and MMP3), which are strongly inhibited. Studies carried out using human skin biopsies showed alteration of dermal papilla with a reduction of the germinative layer and an early arrest of hair follicle downgrowth. The knock-in mouse model, generated in our laboratory, shows similar changes in the tissues studied so far. Conclusions This is the first report of a disease phenotype associated with LAMA5 mutation in humans.

Changes in insulin levels following 6-month treatment with recombinant human growth hormone in growth hormone-deficient adults

Introduction: Eighty-six adult patients with GH deficiency (GHD) of adult or childhood onset were treated for 6 months, with recombinant human growth hormone (r-hGH) at a low (LD) or conventional dose (CD). The treatment effect on insulin levels was investigated. Methods: This manuscript refers to the Italian addendum to an International Study (B9R-EW-GDED) in which patients with GHD were randomized to receive r-hGH replacement therapy at a dose of either 3 μg/kg/day or 6 μg/kg/day for the 3 months. The dose was then doubled for the next 3 months. Results: After 6 months of r-hGH treatment, insulin levels increased with both GH dosages, with a greater increase achieved in the low-dose subgroup. Insulin levels also increased significantly in the childhood-onset, while even decreased in the adult-onset subgroup. On the whole, in more than 50% of patients, insulin values rose by >13%. Moreover, mean levels of IGF-I increased 2–3 fold (p<0.001 vs baseline) in both the LD and CD groups. Significant and similar increases in IGF binding protein-3 levels were seen in both the LD and CD groups over the treatment period, regardless the time of onset of GHD. Conclusion: Insulin increased with both GH dosages and more than half of patients presented an important increase in insulin plasma levels. It would be of interest to assess if there is a correlation between the changes in insulin levels and other cardiovascular risk factors such as hemostatic parameters.

The effect of LDL-apheresis on some hemostatic parameters in homozygous familial hypercholesterolemia

Some selected parameters of hemostasis that are currently thought to play a major role in thrombogenesis were evaluated in 5 patients homozygous for familial hypercholesterolemia (FH) before and 7 and 14 days after a single LDL-apheresis. The sensitivity of platelets to threshold concentrations of ADP was normal and it was not affected by LDL-apheresis. On the other hand, the sensitivity to arachidonic acid or collagen was abnormally high and this enhanced sensitivity was unchanged 7 and 14 days after the apheresis. Monocyte pro-coagulant activity (PCA) was normal before the apheresis and its values were little affected by the procedure. Likewise, the levels of plasminogen activator inhibitor (PAI) as well as the release by vessel walls of tissue plasminogen activator (tPA) the major phisiological activator of fibrinolysis were functionally and antigenicaliv normal before as well as 7 and 14 days after the apheresis. We conclude that the ability of LDL-apheresis to reduce the tendency to thrombosis of these patients is not associated with changes in these major parameters of hemostasis.