Mammographic breast density and characteristics of invasive breast cancer.

INTRODUCTION: Inconclusive data exist on the association between breast density and breast cancer characteristics. MATERIALS AND METHODS: We conducted a case-only study on 667 invasive breast cancers, using data from the Piedmont Cancer Registry. We applied a multivariate logistic regression model to estimate odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) of high breast density (Breast Imaging Reporting and Data System, BI-RADS 3-4) versus low (BI-RADS 1-2) in relation to histologic grade, pathological tumour size and lymph node status, histotype, estrogen and progesterone receptor, HER2 and Ki67 status. Histopathological data were assessed according to the American Joint Committee on Cancer (AJCC) Staging Manual guidelines. The model includes terms for age at diagnosis, education level, body mass index, reproductive factors, family history of breast cancer, smoking and diabetes. RESULTS: As regards histologic grade, compared to well differentiated tumours, the OR of high (versus low) breast density cases was 0.61 (95% CI 0.38-0.98) for moderately-poorly differentiated tumours. No other associations with hormonal and histopathological characteristics were observed. DISCUSSION: Our results indicate that low breast density is associated with moderately-poorly differentiated breast tumours.

Mechanical activation of Efavirenz: the effects on the dissolution and inhibitory behavior.

A poorly water-soluble drug (Efavirenz) was mechanically activated by ball-milling. The effect of the mechanical activation on the dissolution behavior and bioavailability was investigated revealing possible correlations with the grinding action, in terms of crystallinity, particle size and morphology.With proper selection of the grinding parameters the dissolution kinetics can be controlled, both in terms of dissolution velocity and as amount of dissolved drug. In vitro biological tests show that milling does not impair the ability of Efavirenz to inhibit HIV-1 infection (p value >0.05); the IC50 values of ground Efavirenz is indeed lower than values for the pristine micronized powder.

Reversal of fetal ductal constriction after maternal restriction of polyphenol-rich foods: an open clinical trial.

OBJECTIVE: To test the hypothesis that maternal restriction of polyphenol-rich foods (PRF), which, like non-steroidal anti-inflammatory drugs (NSAID), inhibit prostaglandin synthesis in the third trimester, reverse fetal ductal constriction (DC). STUDY DESIGN: An open clinical trial of 51 third trimester fetuses with DC with no history of NSAID intake was designed. All mothers were submitted to a food frequency questionnaire and were oriented to withdrawl PRF, being reassessed after 3 weeks. Doppler parameters were assessed before and after discontinuation of these substances. A control group of 26 third trimester normal fetuses, with no ductus arteriosus (DA) constriction, in which no dietary intervention was offered, was reviewed after 3 weeks. Student's t-test and Wilcoxon's test were used. RESULT: Mean gestational age was 32±3 weeks (28 to 37 weeks). After discontinuation of PRF (≥3 weeks), 48/51 fetuses (96%) showed complete reversal of DC, with decrease in mean ductal systolic velocity (1.74±0.20 m s(-1) to 1.31±0.34 m s(-1), P<0.001), mean diastolic velocity (0.33±0.09 m s(-1) to 0.21±0.07 m s(-1), P<0.001) and mean right to left ventricular dimension ratio (1.37±0.26 to 1.12±0.17, P<0.001) and increase in mean ductal pulsatility index (PI) (1.98±0.36 to 2.46±0.23, P<0.001). Median daily maternal consumption of PRF was 286 mg per day and decreased after orientation to 0 mg per day, P<0.001. In the control group, with GA of 32±4 w (29-37 w), there was no significant differences in median daily maternal consumption of PRF, mean ductal systolic velocitiy, diastolic velocity, PI and right ventricular to left ventricular diameter ratio (RV/LV) ratio. CONCLUSION: Reduction of maternal PRF intake during pregnancy, especially in the third trimester, is followed by complete reversal of DC (wide open DA), which may influence maternal dietary habits in late pregnancy.

Insertion of targeting domains into the envelope glycoprotein of Moloney murine leukemia virus (MoMLV)-based vectors modulates the route of mCAT-1-mediated viral entry.

Several groups have inserted targeting domains into the envelope glycoprotein (Env) of Moloney murine leukemia virus (MoMLV) in an attempt to produce targeted retroviral vectors for human gene therapy. While binding of these modified Envs to the target molecule expressed on the surface of human cells was observed, specific high-titer infection of human cells expressing the target molecule was not achieved. Here we investigate the initial steps in the entry process of targeted MoMLV vectors both in murine and human cells expressing the MoMLV receptor, the mouse cationic amino acid transporter-1 (mCAT-1). We show that insertion of a small ligand targeted to E-selectin and of a single chain antibody (scFv) targeted to folate-binding protein (FBP) into the N-terminus of MoMLV Env results in the reduction of the infectivity and the kinetics of entry of the MoMLV vectors. The use of soluble receptor-binding domain (sRBD), bafilomycin A1 (BafA1) and methyl-beta-cyclodextrin (MbetaC) increase the infectivity of the MoMLV vectors targeted to FBP (MoMLV-FBP) suggesting that the scFv targeted to FBP increases the threshold for fusion and might re-route entry of the targeted MoMLV-FBP vector towards an endocytic, non-productive pathway.

Evidence for nonspecific adsorption of targeted retrovirus vector particles to cells.

The ability to specifically target a cell-type is important for the development of vectors for in vivo gene therapy. In order to produce retrovirus vectors targeting ovarian cancer cells, which specifically overexpress alpha folate receptor (alphaFR), a single chain antibody was fused as an N-terminal extension of the ecotropic and amphotropic murine leukemia virus (MLV) envelope glycoproteins. Vector particles bearing the modified glycoproteins were produced and analysed. Although conventional FACS studies indicated that viral particles bearing the modified Env could bind to ovarian cancer cells, targeted infection was not achieved. The initial step of virus-cell interaction was further studied using an immunofluorescence technique, which allows visualisation of single retrovirus particles. Vectors bearing chimeric or wild-type glycoproteins bound equally well to cells with or without the targeted receptor, although soluble chimeric glycoproteins bound specifically to FBP. Our results indicate that the incorporation of specific ligands to the virus envelope does not necessarily result in significant enhancement of vector particle binding. A similar interaction was also observed using Env-defective virus particles, suggesting that cellular factors incorporated into the lipid envelope play a dominant role in promoting initial adsorption of virus particles to cells. Significant implications arise from these observations on the interpretation of previous reports on 'targeted' vectors, and for the development of vectors for in vivo gene therapy protocols.

Development of a suspension packaging cell line for production of high titre, serum-resistant murine leukemia virus vectors.

To date, only adherent cell lines have been used for the generation of packaging cells for the production of type C retrovirus vectors. The large-scale production of high titre retrovirus vectors could benefit from the development of packaging cells growing in suspension. Here, we describe the ability of two different lymphoid cell lines, one B- and one T-lymphoblastoid cell line (Namalwa and CEM, respectively), to produce MLV-based vectors. Upon transfection with a third generation packaging construct, the virus particle production by Namalwa cells was characterised by low RT-activity, and by CEM cells as high RT activity as previously established adherent packaging cells. An amphotropic packaging cell line (CEMFLYA) was therefore established from CEM cells. Upon introduction of a lacZ vector genome, the novel packaging cell line produced vector particles routinely in the region of 10(7) infectious units/ml. The vectors were helper-free and highly stable in fresh human serum. The potential for scaled up vector production was demonstrated by continuous culture of the new packaging cells for 14 days in a 250 ml spinner flask. These suspension packaging cells should be applicable to large bioreactor systems to bulk produce high titre, complement-resistant retrovirus vectors for gene therapy.

Progress with retroviral gene vectors.

Retroviral vectors have become a standard tool for gene transfer technology. Compared with other gene transfer systems, retroviral vectors have several advantages, including their ability to transduce a variety of cell types, to integrate efficiently into the genomic DNA of the recipient cells and to express the transduced gene at high levels. The relatively well understood biology of retroviruses has made possible the development of packaging cell lines which provide in trans all the viral proteins required for viral particle formation. The design of different types of packaging cells has evolved to reduce the possibility of helper virus production. The host range of retroviruses has been expanded by pseudotyping the vectors with heterologous viral glycoproteins and receptor-specific ligands. The development of lentivirus vectors has allowed efficient gene transfer to quiescent cells. This review describes different strategies adopted for developing vectors to be used in gene therapy applications.

Gene Therapy of Glioblastoma Multiforme with a Bicistronic Retroviral Vector Expressing Human IL-2 and HSV-tK.

Gene therapy of cancer includes strategies for augmentation of immunotherapeutic and chemoterapeutic approaches. These strategies mainly involve ex vivo and in vivo cytokine gene transfer, drug sensitization with genes for prodrug delivery, and the use of drug-resistance genes for protecting bone marrow from high-dose chemotherapy (1). Vector development remains the primary focus for any future research in the field. Retroviral vectors, especially those derived from Moloney murine leukemia virus (MoMLV), remain among the most widely utilized vectors in gene therapy trials.

Initial binding of murine leukemia virus particles to cells does not require specific Env-receptor interaction.

The initial step of virus-cell interaction was studied by immunofluorescence microscopy. Single particles of murine leukemia virus (MLV) vectors and human immunodeficiency virus (HIV) were visualized by immunofluorescence. Fluorescent dots representing single virions could be localized by staining of capsid proteins (CA) or surface envelope proteins (SU) after fixation of virus supernatants. This technique can be used to determine particle concentration in viral supernatants and also to study virus-cell interaction. We investigated the role of the Env-receptor interaction for the initial binding event between the cell and the viral particles. Ecotropic MLV vector particles were shown to bind to human cells which do not express the specific viral receptor. In addition, MLV particles defective for Env were shown to bind the cells similarly to infectious MLV. Time course experiments of virus-cell binding and dissociation showed identical profiles for infectious and Env-defective MLV particles and suggested that MLV Env is not involved in the early phases of attachment of virus to cells. The possible implication of cellular factors in enhancing viral binding and infectivity is discussed.

Gene therapy of glioblastoma multiforme via combined expression of suicide and cytokine genes: a pilot study in humans.

Retrovirus-mediated gene therapy is a particularly attractive approach for glioblastoma multiforme (GBM), given the poor prognosis of this tumour and its localized proliferation in post-mitotic tissue. In this study we assessed, for the first time in humans, the therapeutic potential of a newly designed bicistronic Moloney vector (pLIL-2-TK), combining the expression of a suicide gene (thymidine kinase, tk) with an immunomodulatory gene (human interleukin 2, IL-2). Evidence of transgene activity in the treated tumours is presented.

Herpes simplex virus chronically infected human T lymphocytes are susceptible to HIV-1 superinfection and support HIV-1 pseudotyping.

Peripheral blood lymphocytes (PBL) and CEM CD4+ T-cell line can be infected by herpes simplex virus-1 (HSV-1). CEM cells were characterized as a cellular model to study interactions occurring between HSV-1 and HIV-1. Virtually all cells were persistently infected by HSV-1 (CEM(HSV)) and expressed the latency associated transcripts, whereas only a fraction tested positive for HSV-antigens. CD4 and CXCR-4 expression and function were not affected in CEM(HSV) cells and no significant increase of deoxyribonucleotide pools was noticed. Superinfection of CEM(HSV) cells with HIV-1 led to a cell line chronically infected by both viruses (CEM(HSV/HSV)). Evidence was also obtained that this cell line can produce HIV-1 pseudotyped by HSV-1 envelope. These results may have important implications for a better understanding of AIDS pathogenesis.

Production and characterization of a bicistronic Moloney-based retroviral vector expressing human interleukin 2 and herpes simplex virus thymidine kinase for gene therapy of cancer.

Gene-based therapeutic strategies for cancer mainly include augmentation of immunotherapeutic and chemotherapeutic approaches. In this study we report the design and functional assay of a novel bicistronic Moloney-based retroviral vector expressing human interleukin-2 (IL-2) and herpesvirus thymidine kinase (tk) through a cap-dependent translation and an internal ribosome entry site (IRES)-regulated translation, respectively. This construct has the potential for allowing combination of cytokine and suicide gene therapy, especially in areas such as the brain, composed of post-mitotic cells refractory to transduction by type C retroviral vectors. Accordingly, human glioma cells were used as targets for gene transfer after selecting a packaging cell clone that produced a reasonable titer of recombinant virus and expressed high levels of IL-2 and tk transcripts. Although transduction efficiency was reduced in glioma cells as compared with murine NIH 3T3 cells, transgene expression was effectively achieved. Transduced glioma cells were sensitive to ganciclovir and secreted around 1000 U/ml IL-2 in the culture supernatants. Simultaneous production of IL-2 and tk in vivo by genetically treated tumor cells would hopefully potentiate the effect of gangiclovir-induced metabolic suicide, possibly by boosting the immune response associated with tumor debulking or by amplifying the bystander response.

Determination of HPRT mutant frequency and molecular analysis of T-lymphocyte mutants derived from coke-oven workers.

We measured the frequency of mutant (MF) lymphocytes at the hprt locus in a population of 43 coke-oven workers exposed to PAH and in a group of 26 non-exposed workers. A non-significant increase in MF in the exposed group (19.0 +/- 16.3) compared to the non-exposed group (15.8 +/- 14.6) was observed. Moreover, when we considered smoking habits for the overall population, the MF values were higher, although not significantly, in smokers than in non-smokers. For some T-cell mutant clone structural alterations, splicing and coding errors were detected by PCR-based methods. We analysed 161 HPRT- clones, derived from exposed and non-exposed workers by multiplex-PCR and 56 HPRT- clones by reverse transcriptase-PCR. Overall, the percentages of the different types of gene alterations were similar in exposed and non-exposed subjects. Only the frequency of splice mutations in mutant clones derived from coke-oven workers was higher (22%) than in non-exposed donors (11%).

[Statistical results of 700 electrophysiologic tests (ERG) in patients without ophthalmologic manifestations treated with synthetic antimalarials for rheumatologic or dermatologic disease]. / Bilan statistique de 700 examens électrophysiologiques (ERG) chez des patients sans signe ophtalmologique traités par antipaludéens de synthèse pour maladie rhumatologique ou dermatologique.

737 adapto-electroretinograms are registered on patients treated by chloroquine for a rheumatoid arthritis (RA) or for a lupus. Their ophthalmological examinations are completely normal. In term of the cumulative doses, the statistical results of the electrological characteristics of the different waves of the ERG issued from white, red and blue stimulations show: a great stability of the latencies of the "a" and "b" waves; a gradual decrease of the "b" wave amplitudes for the RA; a gradual increase of the "b" wave amplitudes till a level dose reached between 800 and 900 g and then a decrease of the "b" wave amplitudes, for the lupus. We discuss the electrophysiological criterions for a beginning intoxication. As a standard, for the R.A., it seems that a certain amount of "b" wave amplitude decrease could be admitted but the "a" and "b" wave latencies must be very constant. For the lupus, the hyper normality or at least stability of the "b" wave amplitude can be admitted till a dose around 800 g, associated to a good stability of the "a" and "b" wave latencies. Beyond these statistically established limits a beginning intoxication should be suspected.

[Visual electrophysiology in 101 children with encephalopathy]. / Etude de l'électrophysiologie visuelle chez 101 enfants encéphalopathes.

The electrophysiological recordings (E.R.G. associated with C.V.E.P.) performed on 101 children suffering from encephalopathy of different origins demonstrate a trend towards retinal integrity for subjects with encephalopathy and no suspicion of acid-base disorders. However a functional macular deficiency is frequently observed in encephalopathy with suspicion of acid-base disorders (encephalopathy due to intracranial hypertension or cardio-vascular disorders or metabolic deficiencies with acid-base disturbance). In these cases, a common pathophysiological substratum with disturbance of the acute or chronic acid-base equilibrium has been assumed. Over a period of time, in fixed encephalopathy, the electrophysiological results reflect a real but slow maturation of the visual pathways.

Data processing software for electrophysiological visual exploration.

The software we have developed originated after careful study of a routine functional visual processing session. It has three objectives: (i) to have available a large library of data processing programs; (ii) to add to these electrophysiological results, automatically transferred into a file, a clinical record card, creating a personalized file for study of visual characteristics of each patient; (iii) to allow programs providing statistical studies, creation of groups based on the same clinical features and various comparisons between files or groups. The database now contains 5000 different files and the results of its use are very encouraging. Some results are presented, especially those of a study on multiple sclerosis. The goal of the operation is the creation, in the future, of an expert system.

[Retinal protection using glasses filtering short wave lengths in patients with hereditary degenerative diseases. First electrophysiologic results]. / Protection rétinienne par des verres filtrant les courtes longueurs d'ondes chez des patients atteints de maladies hérédo-dégénératives. Premiers résultats électrophysiologiques.

The electrophysiological action of the ORMA RT glasses filtering the short and middle wavelengths are exposed through the first results of this work by retinitis pigmentosa patients. In 90% of the tested patients A.E.R.G. and V.E.P. are improved.