Gene electrotransfer of IL-2 and IL-12 plasmids effectively eradicated murine B16.F10 melanoma.

Gene therapy has become an important approach for treating cancer, and electroporation represents a technology for introducing therapeutic genes into a cell. An example of cancer gene therapy relying on gene electrotransfer is the use of immunomodulatory cytokines, such as interleukin 2 (IL-2) and 12 (IL-12), which directly stimulate immune cells at the tumour site. The aim of our study was to determine the effects of gene electrotransfer with two plasmids encoding IL-2 and IL-12 in vitro and in vivo. Two different pulse protocols, known as EP1 (600 V/cm, 5 ms, 1 Hz, 8 pulses) and EP2 (1300 V/cm, 100 µs, 1 Hz, 8 pulses), were assessed in vitro for application in subsequent in vivo experiments. In the in vivo experiment, gene electrotransfer of pIL-2 and pIL-12 using the EP1 protocol was performed in B16.F10 murine melanoma. Combined treatment of tumours using pIL2 and pIL12 induced significant tumour growth delay and 71% complete tumour regression. Furthermore, in tumours coexpressing IL-2 and IL-12, increased accumulation of dendritic cells and M1 macrophages was obtained along with the activation of proinflammatory signals, resulting in CD4 + and CD8 + T-lymphocyte recruitment and immune memory development in the mice. In conclusion, we demonstrated high antitumour efficacy of combined IL-2 and IL-12 gene electrotransfer protocols in low-immunogenicity murine B16.F10 melanoma.

Tissue damage modeling in gene electrotransfer: the role of pH.

Optimal gene electrotransfer (GET) requires a compromise between maximum transgene expression and minimal tissue damage. GET in skeletal muscle can be improved by pretreatment with hyaluronidase which contributes to maximize transgene uptake and expression. Nevertheless, tissue damage remains severe close to the electrodes, with a concomitant loss of GET efficiency. Here we analyze the role of pH in tissue damage in GET protocols through in vivo modeling using a transparent chamber implanted into the dorsal skinfold of a mouse (DSC) and intravital microscopy, and in silico modeling using the Poisson-Nernst-Planck equations for ion transport. DSC intravital microscopy reveals the existence of pH fronts emerging from both electrodes and that these fronts are immediate and substantial thus giving rise to tissue necrosis. Theoretical modeling confirms experimental measurements and shows that in GET protocols whether with or without hyaluronidase pretreatment, pH fronts are the principal cause of muscle damage near the electrodes. It also predicts that an optimal efficiency in GET protocols, that is a compromise between obtaining maximum electroporated area and minimal tissue damage, is achieved when the electric field applied is near 183 V/cm in a GET protocol and 158 V/cm in a hyaluronidase+GET protocol.

BCR/ABL1 fusion transcripts generated from alternative splicing: implications for future targeted therapies in Ph+ leukaemias.

Philadelphia (Ph+) positive leukaemias are an example of haematological malignant diseases where different chromosomal rearrangements involving both BCR and ABL1 genes generate a variety of chimeric proteins (BCR/ABL1 p210, p190 and p230) which are considered pathological "biomarkers". In addition to these three, there is a variety of fusion transcripts whose origin may depend either on diverse genetic rearrangement or on alternative/atypical splicing of the main mRNAs or on the occurrence of single-point mutations. Although the therapy of Ph+ leukaemias based on Imatinib represents a triumph of medicine, not all patients benefit from such drug and may show resistance and intolerance. Furthermore, interruption of Imatinib administration is often followed by clinical relapse, suggesting a failure in the eradication of residual leukaemic stem cells. Therefore, while the targeted therapy is searching for new and implemented pharmacological inhibitors covering all the possible mutations in the kinase domain, there is urge to identify alternative molecular targets to develop other specific and effective therapeutic approaches. In this review we discuss the importance of recent advances based on the discovery of novel BCR/ABL1 variants and their potential role as new targets/biomarkers of Ph+ leukaemias in the light of the current therapeutic trends. The limits of the pharmacological inhibitors used for treating the disease can be overcome by considering other targets than the kinase enzyme. Our evaluations highlight the potential of alternative perspectives in the therapy of Ph+ leukaemias.

[Identification of candidate genes and expression profiles, as doping biomarkers]. / Individuazione di geni candidati e profili d'espressione, come indicatori molecolari di assunzione di sostanze dopanti.

Administration of prohibited substances to enhance athletic performance represents an emerging medical, social, ethical and legal issue. Traditional controls are based on direct detection of substances or their catabolites. However out-of-competition doping may not be easily revealed by standard analytical methods. Alternative indirect control strategies are based on the evaluation of mid- and long-term effects of doping in tissues. Drug-induced long-lasting changes of gene expression may be taken as effective indicators of doping exposure. To validate this approach, we used real-time PCR to monitor the expression pattern of selected genes in human haematopoietic cells exposed to nandrolone, insulin-like growth factor I (IGF-I) or growth hormone (GH). Some candidate genes were found significantly and consistently modulated by treatments. Nandrolone up-regulated AR, ESR2 and PGR in K562 cells, and SRD5A1, PPARA and JAK2 in Jurkat cells; IGF-I up-regulated EPOR and PGR in HL60 cells, and SRD5A1 in Jurkat; GH up-regulated SRD5A1 and GHR in K562. GATA1 expression was down-regulated in IGF-1-treated HL60, ESR2 was down-regulated in nandrolone-treated Jurkat, and AR and PGR were down-regulated in GH-treated Jurkat. This pilot study shows the potential of molecular biology-based strategies in anti-doping controls.

Expression and heterodimer-binding activity of Ku70 and Ku80 in human non-melanoma skin cancer.

BACKGROUND: Experimental data suggest that exposure to ultraviolet radiation may indirectly induce DNA double-strand breaks. AIM: To investigate the contribution of the non-homologous end-joining repair pathway in basal and squamous cell carcinomas. METHODS: Levels of Ku70 and Ku80 proteins were determined by immunohistochemical analysis and Ku70-Ku80 heterodimer-binding activity by electrophoretic mobility shift assay. Matched pathological normal margins and skin from healthy people were used as controls. RESULTS: A significant increase in Ku70 and Ku80 protein levels was found for both tumour types as compared with normal skin (p<0.001). Squamous cell carcinoma showed increased immunostaining as compared with basal cell tumours (p<0.02). A direct correlation was found between Ku70 and Ku80 protein levels and expression of the proliferation markers Ki-67/MIB-1 (p<0.02 and p<0.002, respectively) in basal cell carcinoma. DNA binding activity was increased in basal cell carcinoma samples as compared with matched skin histopathologically negative for cancer (p<0.006). In squamous cell carcinomas, however, the difference was significant only with normal skin (p<0.02) and not with matched pathologically normal margins. CONCLUSIONS: Overall, an up regulation of the Ku70 and Ku80 protein levels seems to correlate only with tumour proliferation rate. As non-homologous end joining is an error-prone mechanism, its up regulation may ultimately increase genomic instability, contributing to tumour progression.

Immune response at birth, long-term immune memory and 2 years follow-up after in-utero anti-HBV DNA immunization.

Infections occurring at the end of pregnancy, during birth or by breastfeeding are responsible for the high toll of death among first-week infants. In-utero DNA immunization has demonstrated the effectiveness in inducing specific immunity in newborns. A major contribution to infant immunization would be achieved if a vaccine proved able to be protective as early as at the birth, preventing the typical 'first-week infections'. To establish its potential for use in humans, in-utero DNA vaccination efficiency has to be evaluated for short- and long-term safety, protection at delivery, efficacy of boosts in adults and effective window/s for modulation of immune response during pregnancy, in an animal model suitable with human development. Here we show that a single intramuscular in-utero anti-HBV DNA immunization at two-thirds of pig gestation produces, at birth, antibody titers considered protective in humans. The boost of antibody titers in every animal following recall at 4 and 10 months demonstrates the establishment of immune memory. The safety of in-utero fetus manipulation is guaranteed by short-term (no fetus loss, lack of local alterations, at-term spontaneous delivery, breastfeeding) and long-term (2 years) monitoring. Treatment of fetuses closer to delivery results in immune ignorance without induction of tolerance. This result highlights the repercussion of selecting the appropriate time point when this approach is used to deliver therapeutic genes. All these findings illustrate the relevance of naked DNA-based vaccination technology in therapeutic efforts aimed to prevent the high toll of death among first-week infants.

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.

A somatic mutation in the 5'UTR of BRCA1 gene in sporadic breast cancer causes down-modulation of translation efficiency.

Mutations in the 5' UTR which cause increment/decrement of translation efficiency have been recently described as a novel molecular mechanism of disease. Alterations in the consensus sequence for the translation initiation may promote context-dependent leaky scanning of ribosomes and/or initiation from a downstream AUG codon. Initiation of translation from a downstream in-frame AUG codon in BRCA1 gene was recently identified in normal cells and possibly in breast cancer. Here we present further insight into BRCA1 translational pathophysiology investigating the role of the canonical structure of the initiation consensus sequence of BRCA1. We have analysed the effect of a somatic point mutation (117 G>C) in position -3 with respect to the AUG of the BRCA1 gene, identified in a highly aggressive sporadic breast cancer. We constructed chimeric genes encoding the luciferase reporter sequence downstream of the wild type or the mutated BRCA1 5'UTR. These transcripts were tested for their activity in in vitro and in vivo systems. In in vitro transcription/translation assays the estimated translation efficiency of the construct with the mutated BRCA1 5'UTR was 30-50% lower than that with the wild type BRCA1 5'UTR. The same chimeric genes were analysed for their expression in vivo by transient transfection in human cells. While the two constructs were equally transcribed, the plasmid carrying the mutated sequence produced 70% less luciferase activity compared to the wild type sequence. Finally, to obtain a direct evaluation on translational efficiency in vivo, we analysed mRNA translation on translationally active and non-active ribosomes separated from transfected cells. Mutant mRNA was partially localized in subpolysomal particles analytically confirming a polysome recruitment defect. Thus, characterization of BRCA1 5'UTR and translation efficiency seems to provide new insight into BRCA1 role in breast and ovarian cancer pathogenesis.

Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase -- increased expression with reduced muscle damage.

The efficiency of plasmid gene transfer to skeletal muscle can be significantly improved by the application of an electrical field to the muscle following injection of plasmid DNA. However, this electrotransfer is associated with significant muscle damage which may result in substantial loss of transfected muscle fibres. Reduction of the voltage used in the technique can result in a decrease in muscle damage, with a concomitant reduction in expression, but without a significant decrease in the number of transfected fibres. Pre-treatment of the muscle with a solution of bovine hyaluronidase greatly increases the efficiency of plasmid gene transfer when used in conjunction with electrotransfer, but not when used alone. This combination treatment results in greatly enhanced levels of transfected muscle fibres without the increases in muscle damage associated with the electrotransfer process.

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.

The influence of maternal passive and light active smoking on intrauterine growth and body composition of the newborn.

OBJECTIVE: To assess the influence of passive and light active smoking on the reduction of intrauterine growth of the foetus and on modifications in the body composition of the newborn. DESIGN: Random. SETTING: Full term newborn infants at the Department of the Pediatric and Gynaecological Divisions of the City Major Hospital, Chair of Paediatrics, Verona University. SUBJECTS: One hundred and twelve mothers selected after having completed a questionnaire on smoking habits during pregnancy. One hundred and twelve newborn infants were divided into three groups: Group 1: non-smoking and non-exposed mothers; Group 2: non-smoking but exposed mothers; Group 3: light smoking mothers (under 10 cigarettes/d, whether or not also exposed to passive smoking). Examination within 24 h of birth established the anthropometric measurements and estimates of body composition through indices or equations. RESULTS: Newborns of groups 2 and 3 had a statistically significant reduction of fat mass and most anthropometric measurements: fat mass according to Dauncey (P < 0.001), birth-weight (P < 0.013), crownheel length (P < 0.000), upper- and lower-arm length (P < 0.000) and circumference (P < 0.002), triceps skinfold and sum of all skinfolds (P < 0.004). Student t-test, between groups 2 and 3, did not evidence intergroup differences. CONCLUSIONS: Exposure of the foetus to passive and/or light active smoking involves a reduction of most auxiological parameters and not only weight. As regards body composition, smoking appears to reduce fat mass. The prevention of smoking during pregnancy is therefore extremely important, as intrauterine growth seems to be negatively influenced not only by active smoking, but also by passive and light active smoking.

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.

Development of a multigenic plasmid vector for HCV DNA immunization.

HCV viral nucleocapsid protein (C), non-structural protein 3 (NS3) and the envelope glycoproteins E1 and E2 are candidate immune targets for developing anti-HCV DNA vaccine. Nevertheless, the immune response elicited by these antigens often appears weak and/or transient. Different approaches have been studied for enhancing and/or modulating the immune response of the DNA vaccine. On the basis of a prototype multigenic plasmid vector constituted of two different transcription cassettes (pRC100), we have developed a plasmid vector that allows the independent and simultaneous expression of murine IL2 and of an antigenic domain of the HCV NS3 C terminus (pRC112-HCV). The highly conserved NS3 region spans from nt 4403 to nt 4829 and contains two putative B and T epitopes. The development of this multigenic plasmid vector may combine the expression and local production of an immunomodulatory molecule (mIL2) together with the possibility of addressing the host immune response to the most immunogenic and conserved epitopes, specifically tailored in the plasmid vector.

[Relation between Doppler echo-flowmetry and oncogenic markers in pelvic masses]. / Rapporto tra ecoflussometria Doppler e marker oncogeni nelle masse pelviche.

BACKGROUND: The differential diagnosis between benign and malignant pelvic masses is still difficult and is based on clinical semeiotics and echography, whereas a reliable diagnosis can only be obtained by histological analysis. METHODS: A total of 40 patients were examined using transvaginal Doppler echoflowmetry which was used to evaluate the vascular distribution and intralesional and perilesional flowmetric values. These values were then compared with oncogenic markers and with histological tests of the neoformations. RESULTS: Histological analysis revealed that a flow with high PI is a marker of benignancy, whereas a low PI value tends to show malignancy. On the contrary oncogenic markers reveal a high level of sensitivity but low specificity, especially CA 125.

[Pregnancy and enterostomy]. / Gravidanza ed enterostomia.

The authors describe the difficulties and possible complications of pregnancy in colonostomized women. In the neoplastic forms, generally appearing in advanced age, the knowledge of the basic disease discourages any possible attempt at pregnancy. In the chronic inflammatory diseases (ulcerous rectocolitis and Crohn's disease), typical of young age, pregnancy, if opportunely followed and programmed, can get an absolutely favourable course. The authors report their own experiences and some other authors' ones.