Gigliola Leoncini

Neutral endopeptidase (3.4.24.11) in plasma and synovial fluid of patients with rheumatoid arthritis. A marker of disease activity or a regulator of pain and inflammation

SummaryIn recent years the role of the peripheral nervous system has been focused on the pathogenesis of rheumatoid arthritis (RA). In particular, substance P (SP), released by the sensory terminals, has been demonstrated to be involved in cartilage breakdown [13]. The aim of our work was to study the levels of SP and its peptidases, neutral endopeptidase (3.4.24.11) (NEP) and angiotensin-converting enzyme (ACE), in the synovial fluid and plasma of 30 patients with RA and 14 patients with osteoarthritis (OA). ACE and NEP were determined with a fluorimetric assay and SP with a radioimmunoassay (RIA) method. ACE levels were normal in the plasma of patients with RA and OA (6.1±1.9 and 6.7±1.4 pmol/ml/min, respectively); we found no differences in the values, of ACE between RA and OA synovial fluid (5.7±4.2 and 5.5±4.1 pmol/ml/min, respectively). NEP levels were significantly increased in plasma (139.3±36 pmol/ml/min) and synovial fluid (133.8±32 pmol/ml/min in synovial fluid) and healthy controls (89.7±14 pmol/ml/min in plasma). In synovial fluid, SP was significantly higher in RA patients (43.1±16.6 pg/ ml) than in OA patients (12±13.1 pg/ml), while plasma levels did not show any difference (RA: 14.4±10.2; OA: 13.6±10.6; healthy subjects: 11.3±3.9 pg/ml). The only relationship detected in controls and in OA was among plasma NEP and ESR (P<0.05) and synovial fluid NEP (P<0.001). Our data confirmed that SP could have a role in the pathogenesis of RA synovial inflammation through a control on neurogenic inflammation (SP degradation), vascular tone control (endothelin degradation) and on nociception (enkephalin degradation).

Fibroblast growth factor 23 (FGF23) gene polymorphism in children with Kawasaki syndrome (KS) and susceptibility to cardiac abnormalities

BackgroundFibroblast Growth Factor (FGF) 23 influences endothelial integrity and few reports have studied the association between FGF23 and Kawasaki syndrome (KS), a childhood vasculitis displaying a high risk of subsequent cardiac abnormalities (CaA).AimTo investigate the genetic variation in the FGF23 gene in a cohort of KS children and its association with serum FGF23 levels and eventual development of CaA, including both coronary artery dilatations and aneurysms.Patients and methods84 Italian KS children were recruited; 24/84 (28.6%) developed CaA. Each patient underwent evaluation of serum FGF23 levels and FGF23 genotype: the frequency of the c.212-37insC (rs3832879) polymorphism in intron 1 was examined and compared with sex, age at disease onset, fever duration, laboratory data, and occurrence of CaA. Univariate statistical analysis of categorical parameters was performed by the Pearson’s Chi-square test or Fisher’s exact test as appropriate. Parametric variables were assessed by Student’s t-test for unpaired data. Independent predictors of disease were studied by a logistic regression model.Results28/84 patients carried the FGF23 polymorphism (33.3%) and had higher serum FGF23 levels (p < 0.01). FGF23 polymorphism was significantly associated with CaA compared to wild type FGF23 children (respectively, p = 0.03 and p = 0.05). The comparison with demographical, clinical or laboratory data was not significant.ConclusionsThe prevalent segregation of the c.212-37insC polymorphism in children with CaA advocates a possible functional FGF23 role in the predisposition to higher serum levels of FGF23 and potential occurrence of any coronary artery abnormalities in KS.

Are Angiotensin Converting Enzyme and von Willebrand Factor Circulating Levels Useful Surrogate Parameters to Monitor Disease Activity in Kawasaki Disease

To verify if Angiotensin Converting Enzyme (ACE) and von Willebrand factor (vWF) may be used as a laboratory marker for the follow-up of endothelial derangement and therapeutic efficacy in Kawasaki disease (KD), circulating ACE, vWF routine hematological tests and cardiac involvement were assessed in 32 children with established diagnosis of KD before and up to six months after intravenous gamma-globulins (i.v.IG) treatment. I.v.IG treatment normalized progressively all the hematological parameters to levels comparable with healthy controls within 30 days. At baseline, ACE levels resulted significantly lower (1.8 +/- 1.3 pmol/ml/min), and vWF levels significantly increased (210.3 +/- 35.2%) when compared with controls (respectively 7.0 +/- 0.9 pm/ml/min and 99 +/- 17.9%). Seven days after the treatment vWF levels were decreased (188 +/- 18.4%) but still significantly higher than controls, and fully normalized after 15 days (104.8 +/- 14.3%). ACE levels were found progressively increased at 7, 15, and 30 days after the treatment (respectively 2.7 +/- 1.0, 3.7 +/- 0.4, 5.04 +/- 0.9 pm/ml/min) and reached the range of normality only after two months (7.74 +/- 2.46 pm/ml/min). The present study shows that ACE and vWF circulating levels are significantly modified during the acute phase of the disease.

Establishment and Characterization of a Human Small Cell Osteosarcoma Cancer Stem Cell Line: A New Possible In Vitro Model for Discovering Small Cell Osteosarcoma Biology

Osteosarcoma (OSA) is the most common primary malignant bone tumor, usually arising in the long bones of children and young adults. There are different subtypes of OSA, among which we find the conventional OS (also called medullary or central osteosarcoma) which has a high grade of malignancy and an incidence of 80%. There are different subtypes of high grade OS like chondroblastic, fibroblastic, osteoblastic, telangiectatic, and the small cell osteosarcoma (SCO). In this study, for the first time, we have isolated, established, and characterized a cell line of cancer stem cells (CSCs) from a human SCO. First of all, we have established a primary finite cell line of SCO, from which we have isolated the CSCs by the sphere formation assay. We have proved their in vitro mesenchymal and embryonic stem phenotype. Additionally, we have showed their neoplastic phenotype, since the original tumor bulk is a high grade osteosarcoma. This research demonstrates the existence of CSCs also in human primary SCO and highlights the establishment of this particular stabilized cancer stem cell line. This will represent a first step into the study of the biology of these cells to discover new molecular targets molecules for new incisive therapeutic strategies against this highly aggressive OSA.

Painful vein overdistension in migraine patients: no relationship with serotoninergic parameters

Overdistension of the hand-forearm veins after a period of ischaemia-induced stasis causes local pain in a high percentage of migraineurs, but never in healthy subjects. To investigate the mechanism of such pain, we compared 5-hydroxytryptamine (5HT) whole blood levels and hand vein 5HT reactivity of migraine subjects who did experience pain during venous overdistension to those who did not. No differences were found in whole blood 5HT levels or in the venoconstrictor activity of 5HT between subjects experiencing pain and those who did not. No correlation was found between whole blood 5HT levels and the degree of 5HT-induced venoconstriction. Our results suggest that, if platelets are considered as a model of central antinociceptive 5HT neurons, pain appearance is not due to reduced 5HT at a central level and, therefore, to increased perception of peripheral nociceptive stimuli. Moreover, the similar 5HT venoconstrictive effect (indirect marker of venous tone and, therefore, of venous distensibility) seems to indicate that a mechanical factor is not involved in pain appearance during the HAVD test.

OP0276 Fibroblast growth factor (FGF23) gene polymorphism in kawasaki disease: A risk of coronary damage

Background Kawasaki disease may be complicated by coronary artery disease (CAD): genetic predisposition might play a role in the susceptibility both to KD and CAD. FGF23, a novel phosphaturic factor that influences phosphate renal reabsorption acts through FGF-receptor1 on the vasculature and heart. A study of our group (submitted work) has detected high serum FGF23 levels in all patienta with KD mainly in those with coronary artery involvement. Objectives To investigate FGF23 gene looking for possible mutations or polymorphisms responsible of abnormal serum FGF23 levels and evaluate a potential predisposition to CAD. Methods Genomic DNA extracted from peripheral blood and the 3 FGF23 exons, including the intron-exon boundary regions, were PCR-amplified and analyzed on ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA). The obtained sequences were compared to the wild type reference sequence of the FGF23 gene published on Genbank Database (NT-009759).Intact FGF23 was measured using an ELISA method (Immunotopics Inc. San Clemente, CA, USA). One hundred and eighteen KD Caucasian pts, 75M, 43F, median age 32.5 months were studied. Twenty eight out of 118 developed CAD. All underwent 2D-echocardiogram at admission, 15 days, 2, 6, 12 months. Pts with CAD were more closely controlled. Seventy-six sex-age-matched healthy children were used as controls, after clinical and laboratory exclusion of rheumatic, endocrinological and chronic renal diseases. Ethical Committee’s approval and informed consents by relatives were obtained. Pearson’s chi-square (χ2) analysis was performed to evaluate the distribution of FGF23 gene polymorphisms in the population. Analysis of covariance (ANCOVA), followed by LSD protected least significant difference, was performed to evaluate the correlation between FGF23 polymorphisms and serum FGF23 values. Results were expressed as means ±SEM. Results DNA analysis has shown a new C insertion in the intronic region between -36 and -37 nucleotide (rs3832879:NM_020638.2:c.212-37_212-36insC). Subjects without FGF23 polymorphism were indicated as WT, homozygous as pattern1 and heterozygous as pattern 2. To verify the frequency of the rs3832879 variant and evaluate the presence of polymorphic changes, DNA analysis of 100 Caucasian voluntaries confirmed this change. The χ2 analysis showed that 36.5% of the total population carried out the polymorphic site, and 63.5% not. Only 13.56% of KD pts without CAD had the FGF23 polymorphism, while it was present in 85.19% of pts with CAD (χ2: 41.2; df=1, p=0.001). ANCOVA analysis showed a statistically significant correlation between the presence of polymorphism and serum FGF23 levels. Pts WT had lower levels of serum FGF23 than heterozygotes and homozygotes (54.4±23 SD vs 51,9±28 and 135,3±35). All pts with FGF23 polymorphysm had higher FGF23 serum levels than those without (120±40 vs 38.2±5). Conclusions From our preliminary data the segregation of FGF23 genotype with the CD advocates a possible functional role of this new polymorphism in the predisposition to CAD in pts with KD. Disclosure of Interest None Declared