Expression of miR-487b and miR-410 encoded by 14q32.31 locus is a prognostic marker in neuroblastoma.

BACKGROUND: Combination of age at diagnosis, stage and MYCN amplification stratifies neuroblastoma into low-risk and high-risk. We aimed to establish whether a microRNA (miRNA) signature could be associated with prognosis in both groups. METHODS: Microarray expression profiling of human miRNAs and quantitative reverse-transcriptase PCR of selected miRNAs were performed on a preliminary cohort of 13 patients. Results were validated on an independent cohort of 214 patients. The relationship between miRNA expression and the overall or disease-free survival was analysed on the total cohort of 227 patients using the log-rank test and the multivariable Cox proportional hazard model. RESULTS: A total of 15 of 17 miRNAs that discriminated high-risk from low-risk neuroblastoma belonged to the imprinted human 14q32.31 miRNA cluster and two, miR-487b and miR-410, were significantly downregulated in the high-risk group. Multivariable analyses showed miR-487b expression as associated with overall survival and disease-free survival in the whole cohort, independently of clinical covariates. Moreover, miR-487b and miR-410 expression was significantly associated with disease-free survival of the non-MYCN-amplified favourable neuroblastoma: localised (stage 1, 2 and 3) and stage 4 of infant <18 months. CONCLUSION: Expression of miR-487b and miR-410 shows predictive value beyond the classical high-/low-risk stratification and is a biomarker of relapse in favourable neuroblastoma.

Intratumoral vs systemic administration of meta-tetrahydroxyphenylchlorin for photodynamic therapy of malignant gliomas: assessment of uptake and spatial distribution in C6 rat glioma model.

Malignant gliomas, with an incidence of 5 cases per 100,000 population per year, represent the most common primary brain tumour. They have an overall survival length of less than 2 years. Many different adjuvant therapies have been developed. Among them, Photodynamic Therapy (PDT), that is based on photochemical reactions between light and tumoral tissue selectively labelled with exogenous photosensitizing agents. Among photosensitizers, m-THPC (Temoporfin), seems to be the most promising one for the treatment of brain tumors, but, unfortunately, it causes problems of high skin photosensitivity. To by-pass this problem, we devised an intratumoral route of administration of this photosensitizer. The aim of this study is to investigate and compare the uptake of m-THPC in brain tumor and normal tissue after systemic and intratumoral administration of the drug. 30 female Wistar rats received m-THPC 12 days after C6 tumor implantation. Temoporfin was administered intratumorally in 24 rats at two different concentrations. 6 rats constituted the control group and received m-THPC by means of an intraperitoneal injection. The brains were extracted at 4 h, 24 h and 96 h after Temoporfin injection. The samples were examined with a confocal laser scanning microscope. All samples showed high fluorescence emission exclusively in the tumour area, without appreciable differences between the samples taken at the different times of sacrifice and the two routes of administration. No fluorescence whatsoever was detected among normal brain tissue surrounding the tumour. The intratumoral route appears to give comparable results to the systemic one, regarding intracellular uptake efficiency and tumour--normal tissue ratio, with the advantage of a much shorter time needed to reach optimal intratumoural concentration--that is just four hours from m-THPC injection.

Extensive modulation of a set of microRNAs in primary glioblastoma.

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles in animals and plants by repressing translation or cleaving RNA transcripts. The specific modulation of several microRNAs has been recently associated to some forms of human cancer, suggesting that these short molecules may represent a new class of genes involved in oncogenesis. In our study, we examined by microarray the global expression levels of 245 microRNAs in glioblastoma multiforme, the most frequent and malignant of primary brain tumors. The analysis of both glioblastoma tissues and glioblastoma cell lines allowed us to identify a group of microRNAs whose expression is significantly altered in this tumor. The most interesting results came from miR-221, strongly up-regulated in glioblastoma and from a set of brain-enriched miRNAs, miR-128, miR-181a, miR-181b, and miR-181c, which are down-regulated in glioblastoma.

A tricistronic retroviral vector expressing natural antiangiogenic factors inhibits angiogenesis in vitro, but is not able to block tumor progression in vivo.

Angiogenesis, the formation of new blood vessels out of pre-existing capillaries, is essential for tumor progression. Many factors have been identified that are able to inhibit angiogenesis. Here, we report the construction of a tricistronic retroviral vector encoding two inhibitors of angiogenesis expressed in mammals: the N-terminal fragment of rat prolactin (16KrPRL) and a secreted form of human platelet factor 4 (sPF4). When transduced by this retroviral vector, a rat glioblastoma cell line loses its ability of promoting endothelial cell locomotion, the initial step of angiogenesis, and the formation of an endothelial cell tube network. In spite of this encouraging in vitro result, however, the anti-angiogenic vector cannot block glioblastoma progression in animal models. These results suggest that therapeutic strategies aiming to block tumor progression through the inhibition of tumor-associated angiogenesis, should not only provide large numbers of angiogenesis inhibitors, but also target the angiogenic factors produced by tumor cells. Moreover, the data described herein may confirm recent findings from other groups which indicate that in order to successfully counteract tumor progression, drugs inhibiting new blood vessel formation should be employed in combination with traditional anti-tumor strategies, such as chemotherapy or radiotherapy.

Growth inhibition and differentiation induction in murine erythroleukemia cells by 4-hydroxynonenal.

4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation. Here we show that the exposure of murine erythroleukemia (MEL) cells to 1 microM HNE, for 10.5 h over 2 days, induces a differentiation comparable with that observed in cells exposed to DMSO for the whole experiment (7 days). The exposure of MEL cells for the same length of time demonstrates a higher degree of differentiation in HNE-treated than in DMSO-treated MEL cells. The protooncogene c-myc is down-modulated early, in HNE-induced MEL cells as well as in DMSO-treated cells. However, ornithine decarboxylase gene expression first increases and then decreases, during the lowering of the proliferation rate. These findings indicate that HNE, at a concentration physiologically found in many normal tissues and in the plasma, induces MEL cell differentiation by modulation of specific gene expression.

Antibodies elicited by naked DNA vaccination against the complementary-determining region 3 hypervariable region of immunoglobulin heavy chain idiotypic determinants of B-lymphoproliferative disorders specifically react with patients' tumor cells.

Several reports have suggested that the mechanism of protection induced by antiidiotypic vaccination against low-grade lymphoproliferative disorders is likely to be antibody mediated. Here we test the hypothesis that DNA vaccination with the short peptide encompassing the complementary-determining region 3 hypervariable region of immunoglobulin heavy chain (VH-CDR3) may elicit a specific antibody immune response able to recognize the native antigens in the form required for therapy. As a test system, we used the VH-CDR3 sequences derived from two patients with non-Hodgkin's B lymphomas (PA, AS) and one patient with hairy cell leukemia (BA) to immunize outbred Swiss mice. This experimental model could mimic a clinical setting in which different patients present distinct HLA haplotypes. Individual tumor-specific VH-CDR3 sequences were amplified by a two-step procedure and directly cloned into multigenic plasmid vectors (pRC100 and derived) with and without mouse interleukin 2 (mIL-2). Each tumor-specific sequence was characterized by sequencing. Female Swiss mice were vaccinated i.m. with plasmids expressing the tumor-specific VH-CDR3 sequence alone (pRC101-PA), mIL-2 plus the VH-CDR3 sequence (pRC111-PA), or a different unrelated antigen (NS3 of hepatitis C virus; pRC112), the sole mIL-2 (pRC110), and the empty plasmid (pRC100). Boost injections were performed at 3 and 16 weeks from the first vaccination, and sera were drawn before each vaccination and at 6, 9, and 19 weeks. Induction of anti-VH-CDR3s antibodies in the sera and their ability to recognize native antigens on patients' tumor cells were evaluated by FACS analysis. Up to 56% (n = 25) of mice vaccinated with pRC111-PA plasmid and 20% (n = 15) of mice vaccinated with pRC101-PA developed a specific immune response that was maintained throughout 19 weeks of observation in 40% of pRC111-PA-vaccinated mice. No response was detected in sera obtained from mice vaccinated with the other plasmids (n = 45). pRC111-PA injection s.c. was less effective (13%, n = 15) than i.m. injection (53%, n = 15). Indeed, we demonstrated that antibodies elicited by naked DNA vaccination against three different patient-derived VH-CDR3 peptides (pRC111-PA or BA or AS) readily reacted with binding epitopes on the idiotypic proteins expressed on the surface of tumor cells derived from each patient; 60, 40, and 40% of, respectively, PA-, BA-, and AS-vaccinated mice developed specific antibodies. No cross-reactivity was detected among the three different CDR3s against tumor cells derived from the other two patients. The outbred mouse strategy confirmed the significant matching potential of three different VH-CDR3 peptides to be efficaciously presented through different MHCs. We conclude that individual VH-CDR3 DNA vaccination can result in a potentially effective specific immune response against non-Hodgkin's B lymphoma cells by a rapid and low-cost therapeutic approach.

Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by intramuscular injection of plasmid DNA.

We report on systemic delivery and long-term biological effects of apolipoprotein E (apoE) obtained by intramuscular (i.m.) plasmid DNA injection. ApoE plays an important role in lipoprotein catabolism and apoE knock-out mice develop severe hypercholesterolemia and diffuse atherosclerosis. We have injected apoE-deficient mice with 80 microg of a plasmid vector (pCMV-E3) encoding the human apoE3 cDNA under the control of the CMV promoter-enhancer in both posterior legs. Local expression of the transgene was demonstrated throughout 16 weeks. Human apoE3 recombinant protein reached 0.6 ng/ml serum level. After i.m. injection of pCMV-E3 expression vector the mean serum cholesterol concentrations decreased from 439 +/- 57 mg/dl to 253 +/- 99 mg/dl (P < 0.05) 2 weeks after injection and persisted at a significantly reduced level throughout the 16 weeks observation period (P < 0.005). Serum cholesterol was unaffected and reached an absolute level of 636 +/- 67 mg/dl in control groups. Finally, injection of pCMV-E3 into apoE-deficient mice resulted in a redistribution of cholesterol content between lipoprotein fractions, with a marked decrease in VLDL, IDL and LDL cholesterol content and an increase in HDL cholesterol. These results demonstrate that severe hypercholesterolemia in apoE-deficient mice can be effectively reversed by i.m. DNA injection, and indicate that this approach could represent a useful tool to correct several hyperlipidemic conditions resulting in atherosclerosis.

A plasmid family containing two different expression cassettes suitable for immunomodulation and genetic immunization.

We have developed an improved eukaryotic expression vector that consists of two distinct, complete, and differentially regulated transcription units. The peculiarities of this prototype vector, named pRC110, are represented by two different strong promoter/enhancer sequences, cytomegalovirus and Rous sarcoma virus, that independently drive transcription of two recombinant cDNAs, which may be easily cloned into specific rare restriction sites. Moreover, we describe a simple way to introduce an optimal translational start site context 5' to any peptide to be cloned in our vectors, thus allowing the correct and efficient expression of even a single part of a larger gene or a short synthetic peptide lacking its own AUG and neighboring regions. We demonstrate the in vivo expression efficacy of pRC110 for use in genetic vaccination through direct intramuscular gene transfer: specific antibodies are raised against one of the encoded peptides 3 weeks after muscle injection, and efficient transcription of the other syngeneic cDNA, mouse interleukin-2, is shown. The development of a "family" of vectors directly deriving from pRC110 is also described, with the common property that one of the encoded proteins may modulate the effects of the other. We recommend the use of pRC110 for genetic immunization and immunological response studies, when the concomitant local production of an immunogenic peptide and of a syngeneic immunomodulating cytokine is required.

Stability and functional effectiveness of phosphorothioate modified duplex DNA and synthetic 'mini-genes'.

Several gene transfer techniques that employ 'naked DNA' molecules have recently been developed and numerous gene therapy protocols that make use of 'naked-DNA' have been proposed. We studied the possibility of enhancing the stability of 'naked DNA vectors' and thus also gene transfer and expression efficiencies, by constructing phosphorothioate (PS-) double strand DNA molecules and functional transcription units. We first synthesized short PS-double strand DNA molecules by the annealing of two complementary, 35 nt long, oligonucleotides. The accessibility of DNA modifying enzymes to this molecule was significantly decreased: T4-ligase and kinase activity were respectively reduced up to 1/2 and to 1/6, as compared to the normal phosphodiester molecule. Nucleolytic stability was increased either to purified enzymes (DNase I and Bal31) or to incubations in fresh serum, cell culture medium or in muscle protein extract. Phosphorothioate end-capped complete eukaryotic transcription units (obtained by Taq polymerase amplification with PS-primers) were not significantly protected from nucleolytic attack. On the contrary, synthetic transcription units, 'mini genes', obtained by Taq amplification with 1, 2 or 3 PS-dNTP substitutions, were resistant to DNase I and Bal31 nucleolytic activity. Transcription efficiency, driven by the T7 promoter, was 96.5, 95 and 33.5% (respectively with 1, 2 or 3 substitutions), as compared to the normal phosphodiester molecules.

Accumulation of human apolipoprotein-E in rat plasma after in vivo intramuscular injection of naked DNA.

Naked DNA was found to be incorporated and consistently expressed after in vivo direct injection into striated muscle. In addition to the local expression of muscle-related or exogenous proteins, intramuscular direct gene transfer may be a useful tool to deliver recombinant proteins into the blood stream. However, no direct demonstration of recombinant protein secretion from muscle to the circulation has been reported thus far. We have injected a naked plasmid DNA containing the human receptor-binding defective apo-E2 cDNA, under the control of CMV promoter, into the quadriceps of Yoshida rats, affected by hereditary hypercholesterolemia and altered LDL-receptor activity. Plasma accumulation of human apo-E2 was demonstrated for at least 45 days after injection. On the contrary, the expression of the normal human apo-E3, injected into the muscle of normal Wistar rats, was demonstrated only in the area of muscular injection and not in the blood plasma. Endogenous rat apo-E expression was not affected by the exogenous human apo-E2 production. Our results demonstrate the availability of intramuscular direct gene transfer as a safe and simple method for the chronic systemic delivery of recombinant proteins to the circulation, although further improvements are needed in order to enhance the efficiency and stability of expression.

Histological findings and evidence of lipid conjugated dienes and malonyldialdehyde in human fetal aortas.

Recent evidence strongly suggests that peroxidative modification of lipids may play a significant role in atherogenesis. In our present research, we investigated if the oxidative stress mediated by oxygen free radicals was a pathophysiologic condition that occurred in the early stages of human development. Thus the aim of this research was to examine lipid peroxidation in human fetal aortas. Human fetal aortas and proximal iliac arteries (n = 8) were obtained from fetuses aged 7 +/- 2 months, immediately after autopsy. Lipids from the initial fatty streak lesions (LFS) and the vessels uninvolved (LUV) were extracted by the chloroform/methanol method. Lipid peroxidation levels were measured by two different methods: determination of lipid conjugate dienes (the spectrum trend was recorded from 320 to 200 nm with a spectrophotometer) and malonyldialdehyde (MDA) content (TBA method). We observed that lipid conjugated dienes were present in LFS, but not in LUV, with a characteristic absorption peak at 233 nm. In addition, MDA levels were significantly higher when the LFS = 3.85 +/- 0.91 nmol than when the LUV = 0.41 +/- 0.12 nmol (p < 0.001 versus LUV). The presence of lipid peroxidation in our samples could be mediated by free radical production in the first stages of human development. Thus these data suggest that LFS peroxidation mediated by free radicals occurs in the vascular circulation in the early stages of human development. This could influence the progression of vascular damage and atherosclerotic disease.

[The relations between atrial natriuretic factor release and adrenergic activation in hypertrophic cardiomyopathy]. / Relazioni tra rilascio di fattore natriuretico atriale ed attivazione adrenergica nella cardiomiopatia ipertrofica.

Atrial natriuretic factor (ANF) is a potent natriuretic and vasoactive (vasorelaxant) peptide localized in the secretory-like atrial specific granules. The main peptide in this storage granules is the 126 amino acid proatrial natriuretic peptide, but the principal circulating form in human plasma is the 28 amino acid, alpha-human natriuretic peptide. Animal and in vitro studies have suggested that ANF modulates autonomic circulatory control, probably with a dose-dependent mechanism. Moreover, recent human studies have resulted contradictory. In particular, it is still unclear if high circulating levels of ANF, which are present in congestive heart diseases constantly, may be correlated with sympathetic nervous system activity in man. Previously we have shown that in congestive diseases there is a relation between ANF and catecholamine secretion. From these basis, the aim of this study was to investigate on the pathophysiological relations between atrial natriuretic factor (ANF) release and adrenergic activation in patients with obstructive hypertrophic cardiomyopathy (n = 6) and non obstructive hypertrophic cardiomyopathy (n = 4). Sympathetic activation in physiologic way was induced by cycloergometer sub-maximal exercise. Then specimens of venous blood were achieved for plasma determination of ANF and catecholamines pre- and post-exercise. Results have shown that in obstructive hypertrophic cardiomyopathy patients basal levels of ANF and catecholamines were higher than levels of these parameters in non obstructive hypertrophic cardiomyopathy patients.(ABSTRACT TRUNCATED AT 250 WORDS)

[Angiotensin II stimulates endothelin-1 release from human endothelial cells]. / L'angiotensina II stimola il rilascio di endotelina-1 da cellule endoteliali umane.

Endothelin-1 (ET-1) is an endothelium-derived vasoconstrictor peptide isolated from the culture supernatant of porcine aortic endothelial cells. This 21 amino-acid residue peptide has potent vasoconstrictive properties in vitro and in vivo. ET-1 action involves phosphatidylinositol turnover, calcium mobilization and protein kinase C activation. Endothelial cells have distinct receptors for different operating through hydrosoluble hormones. The aim of this study was to investigate on a possible role of angiotensin II (ANG II) to modulate the release ET-1 from human endothelial cells in vitro. These data revealed a time- and a dose-dependent increase of ET-1 production in response to ANG II. This mechanism may have important pathophysiological implications in vivo. In fact, a double-mechanism of secretion of ET-1 from endothelial cells could exist: one active in a physiological condition and an other in response to a vasoconstrictor stimuli (as well as ANG II). Furthermore, these results may suggest an additional favourable effect of ACE-inhibition in human hypertension therapy.

Role of arterial hypertension in plasma secretion of prostacyclin during renal failure in man.

Cardiac function and morphology in chronic hemodialyzed patients are modified in consequence of both vascular and neurohormonal factors. In the present study we investigate on the role of prostacyclin (PGI2) vasodilator agent, during hemodialytic (HD) treatment. Twenty-four patients (13 males and 11 females; 9 hypertensive and 15 normotensive) aged 58.5 +/- 14.4 years were studied; 2.5 ml of venous blood were collected before (time 0) and 15', 120', and 240' of dialytic session. The PGI2 levels were measured in plasma, after extraction in ethyl acetate by RIA method, as levels of 6-Keto-PGF1 alpha, a stable metabolite. The results have shown as increase of PGI2 levels at 15' in hypertensive HD patients (HHD) from the begin of dialysis that increased until 240'. This phenomenon was more significant in hypertensive than in normotensive group (NHD) (p < 0.05 vs NHD). These preliminary data suggest that in HHD patients the role of PGI2 is more important than in NHD patients as regards the effects on regulation of circulatory tone. The increment of PGI2 levels could be in relation with the sympathetic activation occurred during hemodialytic treatment.

The ribosomal protein L2 in S. cerevisiae controls the level of accumulation of its own mRNA.

The expression of the yeast L2 r-protein gene is controlled at the level of mRNA accumulation. The product of the gene appears to participate in this regulation by an autogenous feedback mechanism. This control does not operate at the level of transcription but instead affects L2 mRNA accumulation. This autogenous regulation of mRNA accumulation provides an interesting analogy to the autogenous translational regulation of r-proteins in Escherichia coli.

The ABF1 factor is the transcriptional activator of the L2 ribosomal protein genes in Saccharomyces cerevisiae.

The same factor, ABF1, binds to the promoters of the two gene copies (L2A and L2B) coding for the ribosomal protein L2 in Saccharomyces cerevisiae. In vitro binding experiments and in vivo functional analysis showed that the different affinities of the L2A and L2B promoters for the ABF1 factor are responsible for the differential transcriptional activities of the two gene copies. The presence of ABF1-binding sites in front of many housekeeping genes suggests a general role for ABF1 in the regulation of gene activity.