Anna Giuffrida

Local Allergic Rhinitis in Children: Novel Diagnostic Features and Potential Biomarkers

Background Local allergic rhinitis (LAR) is a phenotype of rhinitis that has been poorly studied in children. It is characterized by the same symptoms of allergic rhinitis but with the absence of markers of systemic atopy. Objective To identify children affected by LAR and to analyze the pathogenesis of this disease. We chose to focus our attention on interleukin (IL) and thymic stromal lymphopoietin (TSLP). Methods We enrolled 20 children affected by nonallergic rhinitis (negative skin-prick test results and serum specific immunoglobulin E [sIgE] values). Each patient underwent a nasal allergen provocation test (NAPT) with dust mite and grass pollen. Before and after NAPT, nasal lavage was performed to detect sIgE, IL-5, and TSLP; anterior active rhinomanometry was used to evaluate changes in nasal obstruction. Results Two patients were positive to a nonspecific NAPT and, thus, were excluded from the study. Of the remaining 18 children, 12 (66.7%) had positive results to at least one NAPT. Among these 12 patients, nasal sIgE levels for Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Lolium perenne increased significantly after NAPT (D. pteronyssinus, p < 0.005; D. farinae, p < 0.05; L. perenne, p < 0.05). Nasal IL-5 levels showed a significant increase after NAPT (p ≤ 0.006), and this increase was significantly higher in children who had positive NAPT results than in those patients with negative NAPT results (p ≤ 0.03). Among the 12 children who had a positive NAPT result, nasal TSLP was detected in 4 patients (33.3%) and its levels showed a relevant increase after NAPT, even though the difference did not reach statistical significance (p ≤ 0.061). Conclusion Observed results raise the importance of better refining the diagnostic protocol for LAR in children. Nasal TSLP and IL-5 levels offer new insights concerning localized allergic inflammation, although the role of nasal sIgE has still to be clarified.

Breast cancer cells respond differently to docetaxel depending on their phenotype and on survivin upregulation.

Breast cancer is characterized by molecular heterogeneity, and four major breast cancer subtypes have been identified, each characterized by significant differences in survival, prognosis, and response to therapy. We have studied the effects of docetaxel treatment on apoptosis and survivin expression in four breast cancer cell lines: MCF7 (luminal A: estrogen receptor-positive and progesterone receptor-positive, ErbB2-negative), BT474 (luminal B: estrogen receptor/progesterone receptor/ErbB2-positive), SKBR3 (HER2-like: estrogen receptor/progesterone receptor-negative, ErbB2-positive), and MDA-MB231 (basal-like: estrogen receptor/progesterone receptor/ErbB2-negative). We demonstrated that docetaxel-induced apoptosis and survivin upregulation (MCF7 p = 0.002, BT474 p = 0.001, SKBR3 p = 0.001) in luminal A/B and HER2-like cells, while it induced mainly necrosis and a lower rate of survivin upregulation (MDA-MB231 p = 0.035) in basal-like cells. Wortmannin, a p-Akt inhibitor, was able to revert surviving upregulation and, at the same time, induced an increase of docetaxel-dependent apoptosis, suggesting that reduced levels of survivin can sensitize tumor cells to apoptosis. These data show that the analyzed breast cancer cell lines respond differently to docetaxel, depending on their receptor expression profile and molecular phenotype. Yet, these data confirm that one of the pathways involved in taxane-related chemoresistance is the upregulation of survivin. Further studies on the molecular mechanisms of chemoresistance and on the different modalities of apoptosis induced by chemotherapeutic agents are requested to better understand how cancer cells evade cell death, in order to design new kind of anticancer agents and survivin could represent a future target for this kind of research.

The kinesin Eg5 inhibitor K858 induces apoptosis but also survivin-related chemoresistance in breast cancer cells

SummaryInhibitors of kinesin spindle protein Eg5 are characterized by pronounced antitumor activity. Our group has recently synthesized and screened a library of 1,3,4-thiadiazoline analogues with the pharmacophoric structure of K858, an Eg5 inhibitor. We herein report the effects of K858 on four different breast cancer cell lines: MCF7 (luminal A), BT474 (luminal B), SKBR3 (HER2 like) and MDA-MB231 (basal like). We demonstrated that K858 displayed anti-proliferative activity on every analyzed breast cancer cell line by inducing apoptosis. However, at the same time, we showed that K858 up-regulated survivin, an anti-apoptotic molecule. We then performed a negative regulation of survivin expression, with the utilization of wortmannin, an AKT inhibitor, and obtained a significant increase of K858-dependent apoptosis. These data demonstrate that K858 is a potent inhibitor of replication and induces apoptosis in breast tumor cells, independently from the tumor phenotype. This anti-proliferative response of tumor cells to K858 can be limited by the contemporaneous over-expression of survivin; consequently, the reduction of survivin levels, obtained with AKT inhibitors, can sensitize tumor cells to K858-induced apoptosis.

Serum resistin levels in children with primary snoring.

Primary Snoring (PS) has been positioned at the milder end of the Sleep-Disordered Breathing severity continuum characterized by snoring and it is usually underestimated. PS is defined as snoring without apnea, frequent arousals, or gas exchange abnormalities and recent studies demonstrated that children with PS have increased blood pressure and reduced arterial distensibility. The association between adipokines and SDB has been recently investigated, though most of the studies were focused on OSAS where intermittent hypoxia characterizing the disease may lead to an inflammatory cascade and to the release of several adipokines, contributing to oxidative stress. Resistin, initially described as an adipokine increasing insulin resistance, has been recently identified as a novel important member of the cytokine family involved in the regulation of inflammation. The aim of our study was to investigate circulating resistin levels in normal weight children with PS. Sixty-five children of normal weight aged between 4 and 14 years of age were selected for habitual snoring. Children with positive polysomnography were excluded from the study. Serum resistin levels were detected in all children with PS. Thirty-three healthy non-snorer children with similar age, sex and BMI were selected as a control group. A significantly higher level of resistin was observed in patients with PS compared to the control group (4.67±1.91 ng/ml vs 3.98±1.58 ng/ml; p<0.01). Patients with inconclusive pulse oximetry showed significantly higher resistin levels than those with negative recordings (5.29±1.91 ng/ml vs 4.20±1.93 ng/ ml; p<0.008). Moreover, there was a significant increasing trend between sieric adipokine level and the frequency of snoring (p<0.006). Our results suggest that systemic inflammation and oxidative stress may also play a significant role in the pathophysiology of PS.

Assessing the Relationship between Serum Resistin and nasal Obstruction in Children with Allergic Rhinitis

Background Nasal obstruction has been reported as a “key symptom” of allergic rhinitis (AR) because it is deeply associated with impaired quality of life and it reflects more directly the allergic inflammation in the nasal mucosa. Resistin is known to be involved in inflammatory processes exerting an important role in the regulation of cytokine production even though its effective proinflammatory activity at nasal level has never been fully established. This study investigates the relationship between resistin levels and nasal obstruction assessed by an objective method such as active anterior rhinomanometry. Methods Fifty-three children between 4 and 10 years of age affected by persistent allergic rhinitis (PAR) were enrolled and subdivided in two groups. Serum resistin levels were detected in all children. The same day patients underwent rhinomanometry, which was considered negative (no nasal obstruction) when the fraction of predicted values (p.v.s) was between 71 and 100% and it was considered positive when the fraction of p.v. was ≤70%. Results The serum resistin levels were significantly higher in children with moderate–severe PAR than in patients with mild PAR (p < 0.03). Furthermore, serum resistin levels were significantly higher in children with positive rhinomanometry compared with negative rhinomanometry (p < 0.03). The fraction of p.v.s of nasal flows in patients with nasal obstruction had a significant negative correlation with serum resistin levels (p < 0.001). Conclusion This study provides evidence that resistin levels are increased in children with severe nasal obstruction measured by an objective and quantitative approach.

EXTRACELLULAR MATRIX REMODELLING IN A MURINE MAMMARY ADENOCARCINOMA TRANSFECTED WITH THE INTERFERON‐ALPHA1 GENE

The rejection of interferon alpha1 gene‐transfected mammary adenocarcinoma cells (TSA‐IFNα) injected into syngeneic BALB/c mice was accompanied by an unusual stromal reaction and marked CD8‐positive T‐lymphocyte involvement. To investigate the biological background of this reaction, the possibility was evaluated that an interaction between TSA‐IFNα and stromal cells might remodel the extracellular matrix (EM). When fibroblasts were co‐cultured with TSA‐IFNα or treated with exogenous IFNα, there was no change in their replication rate or collagen synthesis. By contrast, their fibronectin (FN) production and release were increased, resulting in enhanced fibroblast chemotaxis. These findings were mirrored by increased FN staining in the peritumoural and tumoural areas in vivo. IFNα thus determines increased FN production and hence massive local recruitment and activation of fibroblasts, with a modification of the EM. The several activities of IFNα should thus be considered prior to its employment in clinical trials.

The kinesin Eg5 inhibitor K858 induces apoptosis and reverses the malignant invasive phenotype in human glioblastoma cells

SummaryGlioblastoma multiforme is the most common primary malignant brain tumor and its current chemotherapeutic options are limited to temozolomide. Recently, some synthetic compounds acting as inhibitors of kinesin spindle protein Eg5 have shown pronounced antitumor activity. Our group has recently demonstrated that one of these kinesin Eg5 inhibitors, named K858, exerted important antiproliferative and apoptotic effects on breast cancer cells. Since glioblastoma cells usually express high levels of kinesin Eg5, we tested the effect of K858 on two human glioblastoma cell lines (U-251 and U-87) and found that K858 inhibited cell growth, induced apoptosis, reversed epithelial-mesenchymal transition and inhibited migration in both cell lines. We also detected that, at the same time, K858 increased the expression of survivin, an anti-apoptotic molecule, and that the forced down-regulation of survivin, obtained with the specific inhibitor YM155, boosted K858-dependent apoptosis. This indicated that the anti-tumor activity of K858 on glioblastoma cells is limited by the over-expression of survivin and that the negative regulation of this protein sensitizes tumor cells to K858. These data confirmed that kinesin Eg5 is an interesting target for new therapeutic approaches for glioblastoma. We showed that K858, specifically, was a potent inhibitor of replication, an inducer of apoptosis and a negative regulator of the invasive phenotype for glioblastoma cells.

Resistance to the mTOR inhibitor everolimus is reversed by the downregulation of survivin in breast cancer cells

Everolimus (RAD001) is an inhibitor of mammalian target of rapamycin used in combination with exemestane to treat hormone receptor-positive advanced breast cancer. However, not all patients are equally sensitive to RAD001 and certain patients develop resistance. Therefore, the present study analyzed the mechanisms involved in the resistance of breast cancer cells to RAD001 in order to identify a potential tool to overcome it. The effects of RAD001 on the inhibition of cell viability, on the induction of apoptosis and autophagy and on the regulation of survivin, an anti-apoptotic protein, were evaluated in two breast cancer cell lines: BT474 (luminal B) and MCF7 (luminal A). RAD001 was demonstrated to induce autophagy in the two cell lines at following a short period of treatment (4 h) and to induce apoptosis exclusively in BT474 cells following longer periods of treatment (48 h). RAD001 induced the downregulation of survivin in BT474 cells and its upregulation in MCF7 cells. Consequently, inhibiting survivin with YM155 resulted in the acquired resistance of MCF7 cells to RAD001 being reverted, restoring RAD001-induced apoptosis. These data demonstrated that RAD001 exerted anti-proliferative and pro-apoptotic effects on breast cancer cells, but that these effects were repressed by the simultaneous up-regulation of survivin. Finally, the results demonstrated that inhibiting the expression of survivin resulted in the restoration of the anti-neoplastic activity of RAD001.

Augmentation of mouse natural killer (NK) activity by GM-1/p, a processed form of monosialoganglioside GM-1

We describe the immunomodulatory activity of GM-1/P a processed form of GM-1 (monosialoganglioside) extracted from ox brain, purified and physically modified. We examined the effect of in vivo and in vitro treatment of GM-1/P on natural (NK) activity and its ability to induce the production of interleukin-2 (IL-2) in the mouse. In vivo treatment with GM-1/P (1 mg/Kg, i.v., day-1) resulted in a marked increase and in a change of distribution of NK activity, which was associated with lower density Percoll fractions. Marked increase was already observed at 18 hrs and then declined by day 4. In vitro treatment with GM-1/P (2 micrograms/ml) enhanced NK activity of B6 spleen cells, already after 6 hours of incubation, remaining at plateau levels within 18 hours. A role of IL-2 in this enhancement was suggested by the ability of an anti-IL-2 rabbit antiserum to abolish in vitro increased cytotoxicity. The presence of IL-2 in the supernatants of splenocytes from GM-1/P (1mg/Kg, i.v., ,day-1) treated mice stimulated with Con A or Con A plus TPA for 48 hrs was evaluated by proliferation of an IL-2 dependent CTLL cell line. GM-1/P by itself was unable to stimulate IL-2 production; however it markedly increased IL-2 production induced by Con A or Con A plus TPA.

Enhancement of lymphocyte proliferation and IL-2 receptor expression by a processed form (GM-1/P) of monosialoganglioside GM-1.

In this study we investigated the ability of GM-1/P, a calcium mediated processed form of monosialoganglioside GM-1, of in vivo augmenting mouse T and B-lymphocyte blastogenesis induced by mitogens. We have also determined its effect on IL-2 responsiveness by analyzing the induction of the expression of IL-2 receptor (IL-2r) on mouse spleen cells. Lymphocyte blastogenesis was evaluated by 3H-TdR incorporation of spleen cells from untreated or GM-1/P (1mg/Kg, i.v., day-1) treated mice cultured in the presence of T (PHA, ConA) B (LPS) cell specific mitogens. The stimulatory effects appeared to be due to a direct action on T and B lymphocytes, since proliferative response was not abolished by removal of macrophages. Splenocytes from GM-1/P treated mice showed increased proliferation in response to various concentrations of HrIL-2; moreover under these conditions an increased generation of LAK activity was found. A direct evidence for enhanced expression of IL-2r was obtained by immunofluorescence and FACS analysis using a monoclonal antibody (PC.61) directed against the p55 subunit of murine IL-2r. 29% PC.61+ cells were found in IL-2 cultures from treated spleen cells.