Laura Annovazzi

High levels of desmosines in urine and plasma of patients with pseudoxanthoma elasticum

Background  Pseudoxanthoma elasticum (PXE), a rare heritable disorder caused by mutations of the ABCC6 gene, is characterized by fragmentation and mineralization of elastic fibres. We determined the extent of degradation of elastin by measuring and comparing the amount of desmosines in plasma and urine of PXE patients, healthy carriers and normal subjects.

Solid Lipid Nanoparticles for Potential Doxorubicin Delivery in Glioblastoma Treatment: Preliminary In Vitro Studies

The major obstacle to glioblastoma pharmacological therapy is the overcoming of the blood-brain barrier (BBB). In literature, several strategies have been proposed to overcome the BBB: in this experimental work, solid lipid nanoparticles (SLN), prepared according to fatty acid coacervation technique, are proposed as the vehicle for doxorubicin (Dox), to enhance its permeation through an artificial model of BBB. The in vitro cytotoxicity of Dox-loaded SLN has been measured on three different commercial and patient-derived glioma cell lines. Dox was entrapped within SLN thanks to hydrophobic ion pairing with negatively charged surfactants, used as counterions. Results indicate that Dox entrapped in SLN maintains its cytotoxic activity toward glioma cell lines; moreover, its permeation through hCMEC/D3 cell monolayer, assumed as a model of the BBB, was increased when the drug was entrapped in SLN. In conclusion, SLN proved to be a promising vehicle for the delivery of Dox to the brain in glioblastoma treatment.

Temozolomide downregulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/glycogen synthase-3 kinase/β-catenin pathway

BACKGROUND Glioblastoma multiforme stem cells display a highly chemoresistant phenotype, whose molecular basis is poorly known. We aim to clarify this issue and to investigate the effects of temozolomide on chemoresistant stem cells. METHODS A panel of human glioblastoma cultures, grown as stem cells (neurospheres) and adherent cells, was used. RESULTS Neurospheres had a multidrug resistant phenotype compared with adherent cells. Such chemoresistance was overcome by apparently noncytotoxic doses of temozolomide, which chemosensitized glioblastoma cells to doxorubicin, vinblastine, and etoposide. This effect was selective for P-glycoprotein (Pgp) substrates and for stem cells, leading to an investigation of whether there was a correlation between the expression of Pgp and the activity of typical stemness pathways. We found that Wnt3a and ABCB1, which encodes for Pgp, were both highly expressed in glioblastoma stem cells and reduced by temozolomide. Temozolomide-treated cells had increased methylation of the cytosine-phosphate-guanine islands in the Wnt3a gene promoter, decreased expression of Wnt3a, disrupted glycogen synthase-3 kinase/β-catenin axis, reduced transcriptional activation of ABCB1, and a lower amount and activity of Pgp. Wnt3a overexpression was sufficient to transform adherent cells into neurospheres and to simultaneously increase proliferation and ABCB1 expression. On the contrary, glioblastoma stem cells silenced for Wnt3a lost the ability to form neurospheres and reduced at the same time the proliferation rate and ABCB1 levels. CONCLUSIONS Our work suggests that Wnt3a is an autocrine mediator of stemness, proliferation, and chemoresistance in human glioblastoma and that temozolomide may chemosensitize the stem cell population by downregulating Wnt3a signaling.

Stem Cell Niches in Glioblastoma: A Neuropathological View

Glioblastoma (GBM) stem cells (GSCs), responsible for tumor growth, recurrence, and resistance to therapies, are considered the real therapeutic target, if they had no molecular mechanisms of resistance, in comparison with the mass of more differentiated cells which are insensitive to therapies just because of being differentiated and nonproliferating. GSCs occur in tumor niches where both stemness status and angiogenesis are conditioned by the microenvironment. In both perivascular and perinecrotic niches, hypoxia plays a fundamental role. Fifteen glioblastomas have been studied by immunohistochemistry and immunofluorescence for stemness and differentiation antigens. It has been found that circumscribed necroses develop inside hyperproliferating areas that are characterized by high expression of stemness antigens. Necrosis developed inside them because of the imbalance between the proliferation of tumor cells and endothelial cells; it reduces the number of GSCs to a thin ring around the former hyperproliferating area. The perinecrotic GSCs are nothing else that the survivors remnants of those populating hyperproliferating areas. In the tumor, GSCs coincide with malignant areas so that the need to detect where they are located is not so urgent.

Therapeutic apheresis exchange in two patients with prolidase deficiency

Summary BackgroundProlidase deficiency is a rare genetic disorder for which a cure has not yet been found.

Short-term variability of biomarkers of proteinase activity in patients with emphysema associated with type Z alpha-1-antitrypsin deficiency.

BackgroundThe burden of proteinases from inflammatory cells in the lung of subjects with type Pi ZZ of alpha-1-antitrypsin deficiency is higher than in those without the deficiency. Cross-sectional studies have shown increased levels of biomarkers of extracellular matrix degradation in vivo. Longitudinal variability of these biomarkers is unknown but desirable for clinical studies with proteinase inhibitors.MethodsWe measured three different types of biomarkers, including desmosines, elastase-formed fibrinogen fragments and heparan sulfate epitope JM403, in plasma and urine for a period of 7 weeks in a group of 12 patients who participated in a placebo-controlled study to assess the safety of a single inhalation of hyaluronic acid.ResultsEffect of study medication on any of the biomarkers was not seen. Baseline desmosines in plasma and urine correlated with baseline CO diffusion capacity (R = 0.81, p = 0.01 and R = 0.65, p = 0.05). Mean coefficient of variation within patients (CVi) for plasma and urine desmosines was 18.7 to 13.5%, respectively. Change in urinary desmosine levels correlated significantly with change in plasma desmosine levels (R = 0.84, p < 0.01). Mean CVi for fibrinogen fragments in plasma was 20.5% and for JM403 in urine was 27.8%. No correlations were found between fibrinogen fragments or JM403 epitope and desmosines.ConclusionWe found acceptable variability in our study parameters, indicating the feasibility of their use in an evaluation of biochemical efficacy of alpha-1-antitrypsin augmentation therapy in Pi Z subjects.

Temozolomide down-regulates P-glycoprotein in human blood–brain barrier cells by disrupting Wnt3 signaling

Low delivery of many anticancer drugs across the blood–brain barrier (BBB) is a limitation to the success of chemotherapy in glioblastoma. This is because of the high levels of ATP-binding cassette transporters like P-glycoprotein (Pgp/ABCB1), which effluxes drugs back to the bloodstream. Temozolomide is one of the few agents able to cross the BBB; its effects on BBB cells permeability and Pgp activity are not known. We found that temozolomide, at therapeutic concentration, increased the transport of Pgp substrates across human brain microvascular endothelial cells and decreased the expression of Pgp. By methylating the promoter of Wnt3 gene, temozolomide lowers the endogenous synthesis of Wnt3 in BBB cells, disrupts the Wnt3/glycogen synthase kinase 3/β-catenin signaling, and reduces the binding of β-catenin on the promoter of mdr1 gene, which encodes for Pgp. In co-culture models of BBB cells and human glioblastoma cells, pre-treatment with temozolomide increases the delivery, cytotoxicity, and antiproliferative effects of doxorubicin, vinblastine, and topotecan, three substrates of Pgp that are usually poorly delivered across BBB. Our work suggests that temozolomide increases the BBB permeability of drugs that are normally effluxed by Pgp back to the bloodstream. These findings may pave the way to new combinatorial chemotherapy schemes in glioblastoma.

Stat3 Expression and Its Correlation with Proliferation and Apoptosis/Autophagy in Gliomas

Signal transducer and activator of transcription-3 (Stat3) was studied along with several steps of the PI3/Akt pathway in a series of 64 gliomas that included both malignant and low-grade tumors, using quantitative immunohistochemistry, Western blotting, and molecular biology techniques. The goal of the study was to investigate whether activated Stat3 (phospho-Stat3) levels correlated with cell proliferation, apoptosis, and autophagy. Stat3 and activated Akt (phospho-Akt) expression increased with malignancy grade, but did not correlate with proliferation and survival within the category of glioblastomas. A correlation of Stat3 with Akt was found, indicating a regulation of the former by the PI3/Akt pathway, which, in turn, was in relation with EGFR amplification. Stat3 and Akt did not show any correlation with apoptosis, whereas they showed an inverse correlation with Beclin 1, a stimulator of autophagy, which was rarely positive in glioblastomas. Autophagy seems then to be inactivated in malignant gliomas.

Antigenic and Genotypic Similarity between Primary Glioblastomas and Their Derived Neurospheres

Formation of neurospheres (NS) in cultures of glioblastomas (GBMs), with self-renewal, clonogenic capacities, and tumorigenicity following transplantation into immunodeficient mice, may denounce the existence of brain tumor stem cells (BTSCs) in vivo. In sixteen cell lines from resected primary glioblastomas, NS showed the same genetic alterations as primary tumors and the expression of stemness antigens. Adherent cells (AC), after adding 10% of fetal bovine serum (FBS) to the culture, were genetically different from NS and prevailingly expressed differentiation antigens. NS developed from a highly malignant tumor phenotype with proliferation, circumscribed necrosis, and high vessel density. Beside originating from transformed neural stem cells (NSCs), BTSCs may be contained within or correspond to dedifferentiated cells after mutation accumulation, which reacquire the expression of stemness antigens.

Collagenase production in an Antarctic strain of Arthrobotrys tortor Jarowaja

This paper describes the results of a comparative screening between the nematophagous Antarctic fungus Arthrobotrys tortor and other species of that genus for the production of extracellular collagenases. The nematode species used in this study was Caenorhabditis elegans, feeding on Escherichia coli cultures. Determination of collagenase activity was made using insoluble collagen from bovine Achilles tendon and determining the amount of solubilized hydroxyproline produced. The results show that the total amount of collagenase produced by the Antarctic strain of A. tortor was about threefold higher than that observed for the other species. In the Antarctic strain, collagenase was shown to be a constitutive enzyme.