Emanuela Barletta

Expression of cytokines and chemokine receptors in the cutaneous lesions of erythema multiforme and Stevens-Johnson syndrome/toxic epidermal necrolysis

Background  Erythema multiforme (EM) and Stevens–Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) are caused by a dysregulation of cellular immunity.

Association Between Genetic Polymorphisms in the XRCC1, XRCC3, XPD, GSTM1, GSTT1, MSH2, MLH1, MSH3, and MGMT Genes and Radiosensitivity in Breast Cancer Patients

PURPOSE Clinical radiosensitivity varies considerably among patients, and radiation-induced side effects developing in normal tissue can be therapy limiting. Some single nucleotide polymorphisms (SNPs) have been shown to correlate with hypersensitivity to radiotherapy. We conducted a prospective study of 87 female patients with breast cancer who received radiotherapy after breast surgery. We evaluated the association between acute skin reaction following radiotherapy and 11 genetic polymorphisms in DNA repair genes: XRCC1 (Arg399Gln and Arg194Trp), XRCC3 (Thr241Met), XPD (Asp312Asn and Lys751Gln), MSH2 (gIVS12-6T>C), MLH1 (Ile219Val), MSH3 (Ala1045Thr), MGMT (Leu84Phe), and in damage-detoxification GSTM1 and GSTT1 genes (allele deletion). METHODS AND MATERIALS Individual genetic polymorphisms were determined by polymerase chain reaction and single nucleotide primer extension for single nucleotide polymorphisms or by a multiplex polymerase chain reaction assay for deletion polymorphisms. The development of severe acute skin reaction (moist desquamation or interruption of radiotherapy due to toxicity) associated with genetic polymorphisms was modeled using Cox proportional hazards, accounting for cumulative biologically effective radiation dose. RESULTS Radiosensitivity developed in eight patients and was increased in carriers of variants XRCC3-241Met allele (hazard ratio [HR] unquantifiably high), MSH2 gIVS12-6nt-C allele (HR=53.36; 95% confidence intervals [95% CI], 3.56-798.98), and MSH3-1045Ala allele (HR unquantifiably high). Carriers of XRCC1-Arg194Trp variant allele in combination with XRCC1-Arg399Gln wild-type allele had a significant risk of radiosensitivity (HR=38.26; 95% CI, 1.19-1232.52). CONCLUSIONS To our knowledge, this is the first report to find an association between MSH2 and MSH3 genetic variants and the development of radiosensitivity in breast cancer patients. Our findings suggest the hypothesis that mismatch repair mechanisms may be involved in cellular response to radiotherapy. Genetic polymorphisms may be promising candidates for predicting acute radiosensitivity, but further studies are necessary to confirm our findings.

The CD40/CD40 ligand system is expressed in the cutaneous lesions of erythema multiforme and Stevens-Johnson syndrome/toxic epidermal necrolysis spectrum.

Background  Erythema multiforme (EM) and Stevens–Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) are determined by a dysregulation of cellular immunity.

Cell Invasion Is Affected by Differential Expression of the Urokinase Plasminogen Activator/Urokinase Plasminogen Activator Receptor System in Muscle Satellite Cells from Normal and Dystrophic Patients

The aim of this study was to evaluate the differential expression and the function in cell movement and proliferation of the urokinase plasminogen activator (u-PA) system in muscle satellite cells (MSC) of normal individuals and patients with Duchenne muscular dystrophy (DMD). By immunoenzymatic, zymographic, and radioligand binding methods and by quantitative polymerase chain reaction of the specific mRNA we have shown that both normal and DMD MSC produce u-PA and the plasminogen activator inhibitor-1 and express u-PA receptors (u-PAR). During the proliferation phase of their growth-differentiation program, MSC from DMD patients show more u-PAR than their normal counterpart, produce more plasminogen activator inhibitor-1, and release low amounts of u-PA into the culture medium. By Boyden chamber Matrigel invasion assays we have shown that normal MSC are more prone than DMD cells to spontaneous invasion but, when subjected to a chemotactic gradient of u-PA, DMD MSC sense the ligand much better and to a greater extent than normal MSC. u-PA also stimulates proliferation of MSC, but no difference is observable between normal and DMD patients. Antagonization of u-PA/u-PAR interaction with specific anti–u-PA and anti–u-PAR monoclonal antibodies and with antisense oligonucleotides inhibiting u-PAR expression indicates that u-PA/u-PAR interaction is required in spontaneous and u-PA–induced invasion, as well as in u-PA–induced proliferation.

Multifaceted roles of BDNF and FGF2 in human striatal primordium development. An in vitro study.

Grafting fetal striatal cells into the brain of Huntingtons disease (HD) patients has raised certain expectations in the past decade as an effective cell-based-therapy for this devastating condition. We argue that the first requirement for successful transplantation is defining the window of plasticity for the striatum during development when the progenitor cells, isolated from their environment, are able to maintain regional-specific-identity and to respond appropriately to cues. The primary cell culture from human fetal striatal primordium described here consists of a mixed population of neural stem cells, neuronal-restricted progenitors and striatal neurons. These cells express trophic factors, such as BDNF and FGF2. We show that these neurotrophins maintain cell plasticity, inducing the expression of neuronal precursor markers and cell adhesion molecules, as well as promoting neurogenesis, migration and survival. We propose that BDNF and FGF2 play an important autocrine-paracrine role during early striatum development in vivo and that their release by fetal striatal grafts may be relevant in the setting of HD cell therapy.

Interaction of tumor cells with vascular endothelia: role of platelet-activating factor.

We investigated whether tumor cell/endothelia interaction can be influenced by platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a lipid mediator that promotes adhesiveness and extravasation of leukocytes in the inflammatory reaction. We found that the PAF receptor antagonist WEB 2086 prevents adhesion of melanoma Hs294T and colon carcinoma LS180 lines to IL-1-stimulated endothelial cells. Moreover, PAF stimulated the adhesiveness of Hs294T and LS180 cells to VCAM-1 and E- selectin, respectively, in an artificial model consisting of recombinant adhesive proteins bound to protein A-coated substrata. Thus, tumoral and not endothelial cell surface seems to be involved in the PAF-mediated enhancement of tumor cell adhesiveness to IL-1-activated endothelia. This observation is supported by the finding that Hs294T and LS180 cells express high affinity and functionally active receptors for PAF. By using specific inhibitors, we found that PAF-induced enhancement of cell adhesiveness was mediated by G-protein activation and protein tyrosine phosphorylation. In addition, protein tyrosine phosphorylation was observed in Hs294T and LS180 cells stimulated by PAF. In conclusion, we demonstrated that PAF-mediated activation of tumor cells enhances their adhesiveness to IL-1-stimulated vascular endothelia.

Lipid characteristics of RSV-transformed Balb/c 3T3 cell lines with different spontaneous metastatic potentials

To determine whether a metastatic phenotype may be corelated with a characteristic lipid pattern, we compared the lipid composition of low metastasizing Balb/c 3T3 cells transformed by the B77 strain of Rous sarcoma virus (B77-3T3 cells) with that of a subclone isolated by growth in 0.6% agar, the B77-AA6 cells, which exhibit a high capacity for spontaneous metastasis. B77-3T3 cells revealed characteristics in their lipid composition common to other systems of transformed cells, i.e., an accumulation of ether-linked lipids, a reduction of the more complex gangliosides, an increase of oleic acid (18∶1) and a decrease of arachidonic (20∶4) and C22 polyunsaturated fatty acids in phospholipids. High metastatic B77-AA6 cells showed: a) an even more marked decrease of complex gangliosides; b) a more pronounced increase of 18∶1 and decrease of 20∶4 and 22 polyunsaturated fatty acids in certain phospholipid classes; and c) a higher percentage of alkyl-acyl subfractions in both phosphatidylcholine and phosphatidylethanolamine than B77-3T3 cells.Comparing the data for other systems of metastatic cells with those of lipid studies of spontaneously metastasizing B77-AA6 cell system leads us to conclude that the metastatic phenotype is characterized by a change in ether-linked lipids, rather than in fatty acids.

The role of apoptosis in the pathogenesis of dermatitis herpetiformis.

Apoptosis is a form of cell death that is claimed to be involved in a number of chronic inflammatory and malignant skin diseases. The aim of this study was to investigate whether apoptosis may contribute to the pathogenesis of epidermal changes in dermatitis herpetiformis (DH) and, in particular, whether certain apoptosis-related markers such as Bax, Bcl-2, Fas and Fas ligand (FasL) take part in this process. For the detection of apoptotic nuclei, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling technique (TUNEL) was employed on cryostat sections. Skin lesions from six and perilesional skin from four DH patients were stained with monoclonal antibodies to Bax, Bcl-2, Fas and FasL. The same evaluation was also performed on three patients affected by bullous pemphigoid (BP) and in two healthy donors. Using TUNEL technique, a remarkable increase in the apoptotic rate within the epidermal compartment was observed in DH and BP patients in comparison with normal controls. In our immunohistochemical analysis, Bax/Bcl-2 ratio was almost the same in the epidermis of perilesional/lesional DH, BP and healthy skin specimens. In DH and BP specimens both Bax and Bcl-2 proteins were increased in the dermal perivascular compartment. Fas showed a prevalently epidermal staining, both in DH and BP lesions, while FasL was distributed in perivascular and subjunctional dermis; some FasL+ cells infiltrated the DEJ and the basal layer of epidermis. This study allowed us to highlight conspicuous apoptotic phenomena in basal and suprabasal keratinocytes within lesional and perilesional skin of DH. We conclude that in DH, as well as in BP, apoptosis plays a role in the pathogenesis of cutaneous lesions in concert with other pathogenetic mechanisms.

Low doses of 3-aminobenzamide, a poly(ADP-ribose) polymerase inhibitor, stimulate angiogenesis by regulating expression of urokinase type plasminogen activator and matrix metalloprotease 2

BackgroundPoly(ADP-Ribose) polymerase (PARP) activity has been demonstrated fundamental in many cellular processes, including DNA repair, cell proliferation and differentiation. In particular, PARP activity has been recently found to affect proliferation, migration, and tube formation of human umbilical vein endothelial cells. In recent times, PARP inhibitors have entered in clinical trials to potentiate cancer treatments by preventing DNA repair, but little is known about the effects performed by different drug concentrations on neoangiogenesis, an essential step in tumor growth.MethodsHuman umbilical vein endothelial cells were treated with 3 aminobenzamide (3ABA), a PARP inhibitor, and tested for several different cellular parameters.ResultsHere we present in vitro evidence that a low concentration of 3ABA (50 μM), stimulates angiogenesis by decreasing fibrinolytic activity, carried out by urokinase-type plasminogen activator (uPA), and by enhancing matrix metalloprotease-2 (MMP-2) gelatinolytic activity, in fibroblast growth factor-2-stimulated endothelial cells. These unbalanced pathways modify in vitro angiogenic steps, inhibiting chemoinvasion and stimulating tubulogenic activity.ConclusionsOur results suggest that the proangiogenic effect of low concentrations of 3ABA alerts on the efficacy of PARP inhibitors to potentiate anticancer therapy. Moreover, they indicate that endothelial chemoinvasion and tubulogenesis depend on distinct proteolytic pathways.

Platelet Activating Factor Inhibits the Expression of Matrix Metalloproteinases and Affects Invasiveness and Differentiation in a System of Human Neuroblastoma Clones

Abstract Platelet Activating Factor (PAF), an inflammatory bioactive lipid, has been shown to be involved in the regulation of the activity of matrix metalloproteinases (MMPs). In view of the role played by MMPs in tumor cell invasiveness, we investigated whether PAF influences MMP activity in a system of neuroblastoma clones, the AA5 and AE12 cells, isolated from the human LaN1 neuroblastoma cell line. These clones were characterized by an inverse relationship between invasiveness and differentiative capacity and by the expression of specific cell surface PAF receptors. We found that the levels of mRNAs specific for MMP-2 and for MT1-MMP, the MMP-2 activator, were reduced in both clones treated with 300 nM PAF. These changes are consistent with the reduced secretion and activation of MMP-2 found in the neuroblastoma clones exposed to PAF. These effects were accompanied by an inhibition of invasiveness through Matrigel and by a promotion of differentiation, as revealed by an increased percentage of cells with neurites. The finding that both neuroblastoma clones exposed to the metalloproteinase inhibitors, BB3103 and 1,10-phenanthroline, increased their differentiative capacity and reduced their invasiveness through Matrigel, represents a further indication that PAF modulates differentiation and invasiveness by affecting the activity of MMPs.