María Virginia López

A Novel A33 Promoter–Based Conditionally Replicative Adenovirus Suppresses Tumor Growth and Eradicates Hepatic Metastases in Human Colon Cancer Models

Purpose: A33 antigen is a membrane-bound protein expressed in intestinal epithelium that is overexpressed in 95% of primary and metastatic colorectal carcinomas but is absent in most epithelial tissues and tumor types. We hypothesized that A33 promoter might be useful in the design of a conditionally replicative adenovirus for the treatment of colorectal cancer (CRC). Experimental Design: We cloned an A33 promoter fragment (A33Pr) that extends from −105 to +307 bp. Using luciferase activity as a reporter gene, we showed that A33Pr was active in CRC cell lines. We next constructed a conditionally replicative adenovirus named AV22EL where E1A was placed under the control of A33Pr. The tumor-specific oncolytic effect of AV22EL was investigated both in vitro and in vivo. Results: AV22EL induced specific in vitro lysis of human CRC cell lines that expressed A33 and have negligible lytic capacity on cells that lacked or had minimal A33 expression, including normal human colonic cells. In vivo, a marked reduction of tumor growth and increased long-term survival rates were observed in nude mice xenografted with s.c. CRC tumors. Combination with 5-fluorouracil induced an additive effect in vitro with no toxic effects in vivo. Remarkably, AV22EL completely eliminated established hepatic metastases in >90% of mice and restored hepatic function according to biochemical parameters. Its systemic administration induced E1A expression only in the hepatic metastasis but not in normal organs. Conclusions: These data show that AV22EL is a stringently regulated and potent oncolytic agent for the treatment of CRC.

Development and statistical validation of a guinea pig model for vaccine potency testing against Infectious Bovine Rhinothracheitis (IBR) virus

Abstract Infectious Bovine Rhinothracheitis (IBR) caused by bovine herpesvirus 1 (BoHV-1) infection is distributed worldwide. BoHV-1 either alone or in association with other respiratory cattle pathogens causes significant economic losses to the livestock industry. The aim of this work was to validate a guinea pig model as an alternative method to the current BoHV-1 vaccine potency testing in calves. Guinea pigs were immunized with two doses of vaccine, 21 days apart and sampled at 30 days post vaccination (dpv). BoHV-1 antibody (Ab) response to vaccination in guinea pigs, measured by ELISA and virus neutralization (VN), was statistically compared to the Ab response in cattle. The guinea pig model showed a dose–response relationship to the BoVH-1 antigen concentration in the vaccine and it was able to discriminate among vaccines containing 1log10 difference in its BoHV-1 concentration with very good repeatability and reproducibility (CV≤20%). A regression analysis of the Ab titers obtained in guinea pigs and bovines at 30 and 60dpv, respectively, allowed us to classify vaccines in three potency categories: “very satisfactory”, “satisfactory” and “unsatisfactory”. Bovines immunized with vaccines corresponding to each of these three categories were experimentally challenged with BoVH-1 virus, the level of protection, as measured by reduction of virus shedding and disease severity, correlated well with the vaccine category used. Data generated by 85 experiments, which included vaccination of calves and guinea pigs with 18 reference vaccines of known potency, 8 placebos and 18 commercial vaccines, was subjected to statistical analysis. Concordance analysis indicated almost perfect agreement between the model and the target species for Ab titers measured by ELISA and almost perfect to substantial agreement when Ab titers were measured by VN. Taken together these results indicate that the developed guinea pig model represents a novel and reliable tool to estimate batch-to-batch vaccine potency and to predict efficacy of killed BoHV-1 veterinary vaccines.

Expression of a suicidal gene under control of the human secreted protein acidic and rich in cysteine (SPARC) promoter in tumor or stromal cells led to the inhibition of tumor cell growth

The successful use of transcriptional targeting for cancer therapy depends on the activity of a given promoter inside the malignant cell. Because solid human tumors evolve as a “cross-talk” between the different cell types within the tumor, we hypothesized that targeting the entire tumor mass might have better therapeutic effect. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein overexpressed in different human cancers malignant melanomas both in the malignant cells compartment as in the stromal one (fibroblasts and endothelial cells). We have shown that expression of the herpes simplex virus–thymidine kinase (TK) gene driven by the SPARC promoter in combination with ganciclovir inhibited human melanoma cell growth in monolayer as well as in multicellular spheroids. This inhibitory effect was observed both in homotypic spheroids composed of melanoma cells alone as well as in spheroids made of melanoma cells and stromal cells. Expression of the TK gene was also efficient to inhibit the in vivo tumor growth of established melanomas when TK was expressed either by the malignant cells themselves or by coadministered endothelial cells. Our data suggest that the use of therapeutic genes driven by SPARC promoter could be a valuable strategy for cancer therapy aiming to target all the cellular components of the tumor mass. [Mol Cancer Ther 2006;5(10):2503–11]

Mycobiota from Cyclamen persicum and its interaction with Botrytis cinerea

Sixty-six fungi isolated from cyclamen phylloplanes were identified and assessed in vitro for antagonism to B. cinerea on leaves, petals, petioles and peduncles. The estimation of pathogen conidial production was used as indicator of biocontrol ability of each of the strains. They were classified by cluster analysis resulting in four categories according to their behavior in the different organs. The most promising category included 34 isolates that significantly reduced pathogen inoculum in all the organs. Correspondence analysis showed association among leaf isolations, strains of Clonostachys rosea and Penicillium spp. and the best biocontrol performance. The statistical analysis was successful in dealing with this complex set of experimental data. Leaf fungal diversity was higher than those of petals and petiols, with Shannon values of 2.7, 0.9 and 0.5 respectively. Evidence for antibiosis and hyperparasitism was found for C. rosea.

Measuring University Students’ Approaches to Learning Statistics: An Invariance Study

The aim of the current study was to provide evidence that an abbreviated version of the Approaches and Study Skills Inventory for Students (ASSIST) was invariant across different languages and educational contexts in measuring university students’ learning approaches to statistics. Data were collected on samples of university students attending undergraduate introductory statistics courses in five countries (Argentina, Italy, Australia, Turkey, and Vietnam). Using factor analysis, we confirmed the three-factor (Deep, Surface, and Strategic approach) model holds across the five samples, and we provided evidence of configural and measurement invariance. The current version of the ASSIST for statistics learners is a suitable scale for researchers and teachers working in the field of statistics education and represents promising tool for multinational studies on approaches to learning statistics.

A Comparison of first year statistics units’ content and contexts in a multinational study, with a case study for the validation of ASSIST in Australia

The study of statistics has become widespread throughout many degrees around the world in many universities, as the emphasis on evidence-based decision making has gained momentum in the business world. Students’ approaches to their learning bear significant weight over the skills and understanding that students acquire during their studies. Three distinct learning approaches have been identified by researchers over the last three decades: deep, surface (British Journal of Educational Psychology 46:115–127, 1976) and strategic (Educational Research Journal 5:18–28, 1990). The discrepancy between desired learning outcomes and the aptitude and skills that students of statistics acquire (e.g. International Statistical Review 63:25–34, 1995) is well documented but the underlying reasons for choosing different learning approaches in statistics has only been investigated in limited studies and only from the perspective of a student’s demographics. It is therefore important to understand how unit and student characteristics might encourage students to utilise certain approaches, especially students who do not major in statistics. The aims of the current chapter are therefore to provide a brief review of learning approaches, a detailed description of the multinational study and validation of the Approaches and Study Skills Inventory for Students (ASSIST) as a measure of the learning approaches utilised by a cohort of Australian students of statistics.

78. Expression of the Herpes Simplex Virus Thymidine Kinase Gene by a Promoter Region of the Human SPARC Gene Inhibits Human Melanoma Cell Growth In Vitro and In Vivo

Transcriptional targeting utilizes tumor-associated gene (TAG) promoters to direct the expression of therapeutic genes specifically to the malignant tumor cells. However, solid human tumors are highly heterogeneous and TAGs are currently expressed in only a percentage of malignant cells. In addition, tumor progression evolves as a cross talk between the different cell types within the tumor and the surrounding stroma, endothelial cells and fibroblasts. As a first approach to co-target the different components of a tumor mass we investigated the properties of the human SPARC promoter. SPARC expression is highest during embryo development in bone and cartilage areas while in adults its expression is restricted to tissues undergoing extensive cellular turnover. Different studies have shown that SPARC is overexpressed in a variety of cancer types not only in malignant cells but also in tumor-associated fibroblasts and endothelium. First, we compared the transcriptional activity of different constructs encompassing the human SPARC promoter region to drive luciferase expression in human malignant cells from different origins as well as in fibroblasts and endothelial cells. We analyzed the activity of a fragment corresponding to the SPARC promoter (hSPPr) that extends from -1176 bp to +71 bp and includes the untranslated exon 1. hSPPr activity was higher in malignant cells, fibroblasts and endothelial cells overexpressing SPARC compared to cells with lower levels or none SPARC expression. A SPARC promoter version with a 10 bp deletion between two GGA boxes hSPPr(|[Auml]|10) that appears to be inhibitory, increased the promoter activity 4 to 7 fold in SPARC-expressing tumor cells and 3 to 5 fold in SPARC non-expressing cells. We investigated the properties of human SPARC promoter-based gene therapy. We prepared plasmid-based vectors carrying the thymidine kinase gene (TK) driven by the different forms of SPARC promoter and evaluated its efficacy in the presence of gancyclovir (GCV) in vitro, in cell culture and spheroids, and in vivo after xenograft transplantation. SPARC-expressing melanoma cells stably producing TK driven by hSPPr (MEL-SPPr-TK) were killed in the presence of GCV when grown as monolayers and spheroids even when the latter were made of malignant cells and fibroblasts. The sensitization to GCV was retained in vivo. Indeed, almost complete tumor regression was observed in nude mice injected with MEL-SPPr-TK cells and treated daily with GCV starting from day 10 after tumor cells injection. Thus, suicide gene therapy driven by SPARC gene promoter appears as a useful strategy for conditional targeting in cancer gene therapy.

THE ANALYSIS OF THE TWO-PERIOD REPEATED MEASUREMENTS CROSSOVER DESIGN WITH APPLICATION TO A FORESTRY PROBLEM

The two-period repeated measurements crossover design is not often used in agricultural studies. It is, however, an attractive model, involving the confluence of two powerful statistical ideas, treatment crossover and repeated measurements on the same experimental unit. This paper presents one approach for the statistical analysis of such design based on the work of Wallenstein and Fisher (1977). It is shown how the data may be transformed so that it can be analyzed under the framework of a completely randomized repeated measurements design. We formalize the analysis in the context of a forestry experiment conducted on poplar trees (Populus SP,), to compare the efficacy of two treatments to prevent damage by the coleopteran insect Platypus sulcatus (ambrosia beetle). Two insecticides were applied in a crossover fashion to two groups of 8 poplar trees each. Each tree was treated with one insecticide and evaluated on three occasions during the first year, received no treatment during the following one-year washout phase, and then (in the third year) received the other treatment and was evaluated on three occasions. One of the parameters analyzed to test for treatment differences was the number of tree lesions attributed to the insect. We present the results of our work and discuss the potential usefulness as well as the limitations of this interesting design.

SEQUENTIAL ANALYSIS OF AGRICULTURAL EXPERIMENTS

Interim monitoring of accumulating data has been widely used in clinical trials, but it has not received the same attention in agricultural experimentation. The methodology, however, can be a useful tool in agronomic trials designed to find better production techniques or optimal animal treatments at low cost, plus the possible economic advantages resulting from correct early decisions. These sequential procedures for testing hypothesis with available data in successive periods of time dictate termination of the experiment when a significant difference is detected, or otherwise continuation of the experiment to the end of the stipulated time or until all the planned sample size is realized. The statistical cost of repeated testing of part of the same data is a reduction in the significance levels a to the time-related significance levels a j (aj<a). We apply three methods for this type of analysis, which we illustrate with two examples involving respectively, comparisons of two proportions and two means from normally distributed random variables with unknown variances. The examples show the usefulness and limitations of the proposed methods and also that there can be no absolute rule for choosing the best method of analysis in a particular case. The optimal strategy depends on the specifics of the trial and the investigators criterion to choose the ajO

TWO-FACTOR AGRICULTURAL EXPERIMENT WITH REPEATED MEASURES ON ONE FACTOR IN A COMPLETE RANDOMIZED DESIGN

A typical agricultural experiment involves comparisons of several treatments at different points in time. The ensuing lack of independence between observations of the same experimental unit may then impair the attainment of statistical significance by the standard analysis of variance, and calls for the application of more powerful methods. This paper addresses one such method, the so-called two-factor experiment with repeated measures on one factor. We discuss the adequacy of this model in the context of three concrete examples drawn from agricultural experimentation.