Genetic engineering and preclinical evaluation of oncolytic herpes simplex viruses retargeted to cancer-specific receptors

Oncolytic virotherapy exploits the ability of viruses to infect and kill cells. It is suitable as treatment for tumors that are not accessible by surgery and/or respond poorly to the current therapeutic approach. HSV is a promising oncolytic agent. It has a large genome size able to accommodate large transgenes and some attenuated oncolytic HSVs (oHSV) are already in clinical trials phase I and II. The aim of this thesis was the generation of HSV-1 retargeted to tumor-specific receptors and detargeted from HSV natural receptors, HVEM and Nectin-1. The retargeting was achieved by inserting a specific single chain antibody (scFv) for the tumor receptor selected inside the HSV glycoprotein gD. In this research three tumor receptors were considered: epidermal growth factor receptor 2 (HER2) overexpressed in 25-30% of breast and ovarian cancers and gliomas, prostate specific membrane antigen (PSMA) expressed in prostate carcinomas and in neovascolature of solid tumors; and epidermal growth factor receptor variant III (EGFRvIII). In vivo studies on HER2 retargeted viruses R-LM113 and R-LM249 have demonstrated their high safety profile. For R-LM249 the antitumor efficacy has been highlighted by target-specific inhibition of the growth of human tumors in models of HER2-positive breast and ovarian cancer in nude mice. In a murine model of HER2-positive glioma in nude mice, R-LM113 was able to significantly increase the survival time of treated mice compared to control. Up to now, PSMA and EGFRvIII viruses (R-LM593 and R-LM613) are only characterized in vitro, confirming the specific retargeting to selected targets. This strategy has proved to be generally applicable to a broad spectrum of receptors for which a single chain antibody is available.

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson’s disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson’s disease.

Social stress and the reactivation of latent herpes simplex virus type 1

Psychological stress is thought to contribute to reactivation of latent herpes simplex virus (HSV). Although several animal models have been developed in an effort to reproduce different pathogenic aspects of HSV keratitis or labialis, until now, no good animal model existed in which application of a psychological laboratory stressor results in reliable reactivation of the virus. Reported herein, disruption of the social hierarchy within colonies of mice increased aggression among cohorts, activated the hypothalamic-pituitary-adrenal axis, and caused reactivation of latent HSV type 1 in greater than 40% of latently infected animals. However, activation of the hypothalamic-pituitary-adrenal axis using restraint stress did not activate the latent virus. Thus, the use of social stress in mice provides a good model in which to investigate the neuroendocrine mechanisms that underlie behaviorally mediated reactivation of latent herpesviruses.

Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Their efficacy depends on the extent of both intratumoral viral replication and induction of a host antitumor immune response. To enhance these properties while employing ample safeguards, two conditionally replicating HSV-1 vectors, termed G47Δ and R47Δ, have been constructed by deleting the α47 gene and the promoter region of US11 from γ34.5-deficient HSV-1 vectors, G207 and R3616, respectively. Because the α47 gene product is responsible for inhibiting the transporter associated with antigen presentation (TAP), its absence led to increased MHC class I expression in infected human cells. Moreover, some G47Δ-infected human melanoma cells exhibited enhanced stimulation of matched antitumor T cell activity. The deletion also places the late US11 gene under control of the immediate-early α47 promoter, which suppresses the reduced growth properties of γ34.5-deficient mutants. G47Δ and R47Δ showed enhanced viral growth in a variety of cell lines, leading to higher virus yields and enhanced cytopathic effect in tumor cells. G47Δ was significantly more efficacious in vivo than its parent G207 at inhibiting tumor growth in both immune-competent and immune-deficient animal models. Yet, when inoculated into the brains of HSV-1-sensitive A/J mice at 2 × 106 plaque forming units, G47Δ was as safe as G207. These results suggest that G47Δ may have enhanced antitumor activity in humans.

Generalized isotropic Lipkin-Meshkov-Glick models: ground state entanglement and quantum entropies

We introduce a new class of generalized isotropic Lipkin–Meshkov–Glick models with su(m+1) spin and long-range non-constant interactions, whose non-degenerate ground state is a Dicke state of su(m+1) type. We evaluate in closed form the reduced density matrix of a block of Lspins when the whole system is in its ground state, and study the corresponding von Neumann and Rényi entanglement entropies in the thermodynamic limit. We show that both of these entropies scale as a log L when L tends to infinity, where the coefficient a is equal to (m  −  k)/2 in the ground state phase with k vanishing magnon densities. In particular, our results show that none of these generalized Lipkin–Meshkov–Glick models are critical, since when L-->∞ their Rényi entropy R_q becomes independent of the parameter q. We have also computed the Tsallis entanglement entropy of the ground state of these generalized su(m+1) Lipkin–Meshkov–Glick models, finding that it can be made extensive by an appropriate choice of its parameter only when m-k≥3. Finally, in the su(3) case we construct in detail the phase diagram of the ground state in parameter space, showing that it is determined in a simple way by the weights of the fundamental representation of su(3). This is also true in the su(m+1) case; for instance, we prove that the region for which all the magnon densities are non-vanishing is an (m  +  1)-simplex in R^m whose vertices are the weights of the fundamental representation of su(m+1).

High prevalence of herpes simplex virus (HSV)- type 2 co-infection among HIV-positive women in Ukraine, but no increased HIV mother-to-child transmission risk.

BACKGROUND Over 3500 HIV-positive women give birth annually in Ukraine, a setting with high prevalence of sexually transmitted infections. Herpes simplex virus Type 2 (HSV-2) co-infection may increase HIV mother-to-child transmission (MTCT) risk. We explored factors associated with HSV-2 seropositivity among HIV-positive women in Ukraine, and its impact on HIV MTCT. METHODS Data on 1513 HIV-positive women enrolled in the Ukraine European Collaborative Study from 2007 to 2012 were analysed. Poisson and logistic regression models respectively were fit to investigate factors associated with HSV-2 seropositivity and HIV MTCT. RESULTS Median maternal age was 27 years (IQR 24-31), 53% (796/1513) had been diagnosed with HIV during their most recent pregnancy and 20% had a history of injecting drugs. Median antenatal CD4 count was 430 cells/mm(3) (IQR 290-580). Ninety-six percent had received antiretroviral therapy antenatally. HSV-2 seroprevalence was 68% (1026/1513). In adjusted analyses, factors associated with HSV-2 antibodies were history of pregnancy termination (APR 1.30 (95% CI 1.18-1.43) for ≥ 2 vs. 0), having an HIV-positive partner (APR 1.15 (95% CI 1.05-1.26) vs partner's HIV status unknown) and HCV seropositivity (APR 1.23 (95 % CI 1.13-1.35)). The overall HIV MTCT rate was 2.80% (95% CI 1.98-3.84); no increased HIV MTCT risk was detected among HSV-2 seropositive women after adjusting for known risk factors (AOR 1.43 (95% CI 0.54-3.77). CONCLUSION No increased risk of HIV MTCT was detected among the 68% of HIV-positive women with antibodies to HSV-2, in this population with an overall HIV MTCT rate of 2.8%. Markers of ongoing sexual risk among HIV-positive HSV-2 seronegative women indicate the importance of interventions to prevent primary HSV-2 infection during pregnancy in this high-risk group.

Characterization Of Microsatellite Markers Developed From Prosopis Rubriflora And Prosopis Ruscifolia (leguminosae - Mimosoideae), Legume Species That Are Used As Models For Genetic Diversity Studies In Chaquenian Areas Under Anthropization In South America.

Prosopis rubriflora and Prosopis ruscifolia are important species in the Chaquenian regions of Brazil. Because of the restriction and frequency of their physiognomy, they are excellent models for conservation genetics studies. The use of microsatellite markers (Simple Sequence Repeats, SSRs) has become increasingly important in recent years and has proven to be a powerful tool for both ecological and molecular studies. In this study, we present the development and characterization of 10 new markers for P. rubriflora and 13 new markers for P. ruscifolia. The genotyping was performed using 40 P. rubriflora samples and 48 P. ruscifolia samples from the Chaquenian remnants in Brazil. The polymorphism information content (PIC) of the P. rubriflora markers ranged from 0.073 to 0.791, and no null alleles or deviation from Hardy-Weinberg equilibrium (HW) were detected. The PIC values for the P. ruscifolia markers ranged from 0.289 to 0.883, but a departure from HW and null alleles were detected for certain loci; however, this departure may have resulted from anthropic activities, such as the presence of livestock, which is very common in the remnant areas. In this study, we describe novel SSR polymorphic markers that may be helpful in future genetic studies of P. rubriflora and P. ruscifolia.

Censored Linear Regression Models For Irregularly Observed Longitudinal Data Using The Multivariate-t Distribution.

In acquired immunodeficiency syndrome (AIDS) studies it is quite common to observe viral load measurements collected irregularly over time. Moreover, these measurements can be subjected to some upper and/or lower detection limits depending on the quantification assays. A complication arises when these continuous repeated measures have a heavy-tailed behavior. For such data structures, we propose a robust structure for a censored linear model based on the multivariate Student's t-distribution. To compensate for the autocorrelation existing among irregularly observed measures, a damped exponential correlation structure is employed. An efficient expectation maximization type algorithm is developed for computing the maximum likelihood estimates, obtaining as a by-product the standard errors of the fixed effects and the log-likelihood function. The proposed algorithm uses closed-form expressions at the E-step that rely on formulas for the mean and variance of a truncated multivariate Student's t-distribution. The methodology is illustrated through an application to an Human Immunodeficiency Virus-AIDS (HIV-AIDS) study and several simulation studies.

Augmented Mixed Models For Clustered Proportion Data.

Often in biomedical research, we deal with continuous (clustered) proportion responses ranging between zero and one quantifying the disease status of the cluster units. Interestingly, the study population might also consist of relatively disease-free as well as highly diseased subjects, contributing to proportion values in the interval [0, 1]. Regression on a variety of parametric densities with support lying in (0, 1), such as beta regression, can assess important covariate effects. However, they are deemed inappropriate due to the presence of zeros and/or ones. To evade this, we introduce a class of general proportion density, and further augment the probabilities of zero and one to this general proportion density, controlling for the clustering. Our approach is Bayesian and presents a computationally convenient framework amenable to available freeware. Bayesian case-deletion influence diagnostics based on q-divergence measures are automatic from the Markov chain Monte Carlo output. The methodology is illustrated using both simulation studies and application to a real dataset from a clinical periodontology study.

Possibilidades e limites da avaliação da qualidade de vida : análise dos instrumentos WHOQOL e modelos clássicos de qualidade de vida no trabalho = Possibilities and limits of quality of life assessment : analysis of WHOQOL instruments and quality of work life classical models

Universidade Estadual de Campinas . Faculdade de Educação Física

Comparative study of dental arch width in plaster models, photocopies and digitized images

The aim of this study was to comparatively assess dental arch width, in the canine and molar regions, by means of direct measurements from plaster models, photocopies and digitized images of the models. The sample consisted of 130 pairs of plaster models, photocopies and digitized images of the models of white patients (n = 65), both genders, with Class I and Class II Division 1 malocclusions, treated by standard Edgewise mechanics and extraction of the four first premolars. Maxillary and mandibular intercanine and intermolar widths were measured by a calibrated examiner, prior to and after orthodontic treatment, using the three modes of reproduction of the dental arches. Dispersion of the data relative to pre- and posttreatment intra-arch linear measurements (mm) was represented as box plots. The three measuring methods were compared by one-way ANOVA for repeated measurements (α = 0.05). Initial / final mean values varied as follows: 33.94 to 34.29 mm / 34.49 to 34.66 mm (maxillary intercanine width); 26.23 to 26.26 mm / 26.77 to 26.84 mm (mandibular intercanine width); 49.55 to 49.66 mm / 47.28 to 47.45 mm (maxillary intermolar width) and 43.28 to 43.41 mm / 40.29 to 40.46 mm (mandibular intermolar width). There were no statistically significant differences between mean dental arch widths estimated by the three studied methods, prior to and after orthodontic treatment. It may be concluded that photocopies and digitized images of the plaster models provided reliable reproductions of the dental arches for obtaining transversal intra-arch measurements.

Linear dimensional changes in plaster die models using different elastomeric materials

Dental impression is an important step in the preparation of prostheses since it provides the reproduction of anatomic and surface details of teeth and adjacent structures. The objective of this study was to evaluate the linear dimensional alterations in gypsum dies obtained with different elastomeric materials, using a resin coping impression technique with individual shells. A master cast made of stainless steel with fixed prosthesis characteristics with two prepared abutment teeth was used to obtain the impressions. References points (A, B, C, D, E and F) were recorded on the occlusal and buccal surfaces of abutments to register the distances. The impressions were obtained using the following materials: polyether, mercaptan-polysulfide, addition silicone, and condensation silicone. The transfer impressions were made with custom trays and an irreversible hydrocolloid material and were poured with type IV gypsum. The distances between identified points in gypsum dies were measured using an optical microscope and the results were statistically analyzed by ANOVA (p < 0.05) and Tukey's test. The mean of the distances were registered as follows: addition silicone (AB = 13.6 µm, CD=15.0 µm, EF = 14.6 µm, GH=15.2 µm), mercaptan-polysulfide (AB = 36.0 µm, CD = 36.0 µm, EF = 39.6 µm, GH = 40.6 µm), polyether (AB = 35.2 µm, CD = 35.6 µm, EF = 39.4 µm, GH = 41.4 µm) and condensation silicone (AB = 69.2 µm, CD = 71.0 µm, EF = 80.6 µm, GH = 81.2 µm). All of the measurements found in gypsum dies were compared to those of a master cast. The results demonstrated that the addition silicone provides the best stability of the compounds tested, followed by polyether, polysulfide and condensation silicone. No statistical differences were obtained between polyether and mercaptan-polysulfide materials.

Allometric models for estimating the phytomass of a secondary Atlantic Forest area of southeastern Brazil

The purpose of this study was to develop and validate equations to estimate the aboveground phytomass of a 30 years old plot of Atlantic Forest. In two plots of 100 m², a total of 82 trees were cut down at ground level. For each tree, height and diameter were measured. Leaves and woody material were separated in order to determine their fresh weights in field conditions. Samples of each fraction were oven dried at 80 °C to constant weight to determine their dry weight. Tree data were divided into two random samples. One sample was used for the development of the regression equations, and the other for validation. The models were developed using single linear regression analysis, where the dependent variable was the dry mass, and the independent variables were height (h), diameter (d) and d²h. The validation was carried out using Pearson correlation coefficient, paired t-Student test and standard error of estimation. The best equations to estimate aboveground phytomass were: lnDW = -3.068+2.522lnd (r² = 0.91; s y/x = 0.67) and lnDW = -3.676+0.951ln d²h (r² = 0.94; s y/x = 0.56).

Descriptor-and fragment-based QSAR models for a series of Schistosoma mansoni purine nucleoside inhibitors

The enzyme purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive molecular target for the treatment of major parasitic infectious diseases, with special emphasis on its role in the discovery of new drugs against schistosomiasis, a tropical disease that affects millions of people worldwide. In the present work, we have determined the inhibitory potency and developed descriptor- and fragment-based quantitative structure-activity relationships (QSAR) for a series of 9-deazaguanine analogs as inhibitors of SmPNP. Significant statistical parameters (descriptor-based model: r² = 0.79, q² = 0.62, r²pred = 0.52; and fragment-based model: r² = 0.95, q² = 0.81, r²pred = 0.80) were obtained, indicating the potential of the models for untested compounds. The fragment-based model was then used to predict the inhibitory potency of a test set of compounds, and the predicted values are in good agreement with the experimental results

Comparison of some theoretical models for fittings of the temperature dependence of the fundamental energy gap in GaAs

In this work we report on a comparison of some theoretical models usually used to fit the dependence on temperature of the fundamental energy gap of semiconductor materials. We used in our investigations the theoretical models of Viña, Pässler-p and Pässler-ρ to fit several sets of experimental data, available in the literature for the energy gap of GaAs in the temperature range from 12 to 974 K. Performing several fittings for different values of the upper limit of the analyzed temperature range (Tmax), we were able to follow in a systematic way the evolution of the fitting parameters up to the limit of high temperatures and make a comparison between the zero-point values obtained from the different models by extrapolating the linear dependence of the gaps at high T to T = 0 K and that determined by the dependence of the gap on isotope mass. Using experimental data measured by absorption spectroscopy, we observed the non-linear behavior of Eg(T) of GaAs for T > ΘD.