Morphology of breast cancer as a means of triage of patients for BRCA1 genetic testing

Background: Women who have germline mutations in the BRCA1 gene are at substantially increased lifetime risk of developing breast and ovarian cancer but are otherwise normal. Currently. early age of onset of cancer and a strong family history are relied upon as the chief clues as to who should be offered genetic testing. Certain morphologic and immunohistochemical features are overrepresented in BRCA1-associated breast cancers but these differences have not been incorporated into the current selection criteria for genetic testing. Design: Each of the 4 pathologists studied 30 known cases of BRCA1- and BRCA2-associated breast cancer from kConFab families. After reviewing the literature, we agreed on a semiquantitative scoring system for estimating the chances of presence of an underlying BRCA1 mutation, based on the number of the reported prototypic features present. After a time lag of 12 months, we each examined a series of 62 deidentified cases of breast cancer, inclusive of cases of BRCA1-associated breast cancer and controls. The controls included cases of BRCA2-associated breast cancer and sporadic cases. Results: Our predictions had a sensitivity of 92%, specificity of 86%, positive predictive value of 61%, and negative predictive value of 98%. For comparison the sensitivity of currently used selection criteria are in the range of 25% to 30%. Conclusion: The inclusion of morphologic and immunohistochemical features of breast cancers in algorithms to predict the likelihood of presence of germline mutations in the BRCA1 gene improves the accuracy of the selection process.

The cyclotides and related macrocyclic peptides as scaffolds in drug design

The applicability of linear peptides as drugs is potentially limited by their susceptibility to proteolytic cleavage and poor bioavailability. Cyclotides are macrocyclic cystine-knotted mini-proteins that have a broad range of bioactivities and are exceptionally stable, being resistant to chemical, thermal and enzymatic degradation. The general limitations of peptides as drugs can potentially be overcome by using the cyclotide framework as a scaffold onto which new activities may be engineered. The potential use of cyclotides and related peptide scaffolds for drug design is evaluated herein, with reference to increasing knowledge of the structures and sequence diversity of natural cyclotides and the emergence of new approaches in protein engineering.

A DHA14 drug efflux gene from Xanthomonas albilineans confers high-level albicidin antibiotic resistance in Escherichia coli

Aims: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. Methods and Results: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other Gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. Conclusions: AlbF is the first apparent single-component antibiotic-specific efflux pump from a Gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. Significance and Impact of the Study: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.

A simple method to localise pleiotropic susceptibility loci using univariate linkage analyses of correlated traits

Univariate linkage analysis is used routinely to localise genes for human complex traits. Often, many traits are analysed but the significance of linkage for each trait is not corrected for multiple trait testing, which increases the experiment-wise type-I error rate. In addition, univariate analyses do not realise the full power provided by multivariate data sets. Multivariate linkage is the ideal solution but it is computationally intensive, so genome-wide analysis and evaluation of empirical significance are often prohibitive. We describe two simple methods that efficiently alleviate these caveats by combining P-values from multiple univariate linkage analyses. The first method estimates empirical pointwise and genome-wide significance between one trait and one marker when multiple traits have been tested. It is as robust as an appropriate Bonferroni adjustment, with the advantage that no assumptions are required about the number of independent tests performed. The second method estimates the significance of linkage between multiple traits and one marker and, therefore, it can be used to localise regions that harbour pleiotropic quantitative trait loci (QTL). We show that this method has greater power than individual univariate analyses to detect a pleiotropic QTL across different situations. In addition, when traits are moderately correlated and the QTL influences all traits, it can outperform formal multivariate VC analysis. This approach is computationally feasible for any number of traits and was not affected by the residual correlation between traits. We illustrate the utility of our approach with a genome scan of three asthma traits measured in families with a twin proband.

An n-of-1 trial service in clinical practice: Testing the effectiveness of stimulants for attention-deficit/hyperactivity disorder

OBJECTIVE. We sought to describe the clinical use of n-of-1 trials for attention-deficit/hyperactivity disorder in publicly and privately funded family and specialized pediatric practice in Australia. METHODS. We used a within-patient randomized, double-blind, crossover comparison of stimulant (dexamphetamine or methylphenidate) versus placebo or alternative stimulant using 3 pairs of treatment periods. Trials were conducted from a central location using mail and telephone communication, with local supervision by the patients' clinicians. PATIENTS. Our study population included children with clinically diagnosed attention-deficit/ hyperactivity disorder who were aged 5 to 16 years and previously stabilized on an optimal dose of stimulant. They were selected because treatment effectiveness was uncertain. MAIN OUTCOME MEASURES. Our measures included number of patients recruited, number of doctors who used the service, geographic spread, completion rates, response rate, and post-n-of-1 trial decisions. RESULTS. Forty-five doctors across Australia requested 108 n-of-1 trials, of which 86 were completed. In 69 drug-versus-placebo comparisons, 29 children responded better to stimulant than placebo. Immediately posttrial, 19 of 25 drug-versus-placebo responders stayed on the same stimulant, and 13 of 24 nonresponders ceased or switched stimulants. In 40 of 63 for which data were available, posttrial management was consistent with the trial results. For all types of n-of-1 trials, management changed for 28 of 64 children for whom information was available. DISCUSSION. Attention-deficit/hyperactivity disorder n-of-1 trials can be implemented successfully by mail and telephone communication. This type of trial can be valuable in clarifying treatment effect when it is uncertain, and in this series, they had a noticeable impact on short-term management.

Prevalence of drug-resistance mutations and non-subtype B strains among HIV-infected infants from New York State

Summary: Prevalence studies indicate that transmission of drug-resistant HIV has been rising in the adult population, but data from the perinatally infected pediatric population are limited. In this retrospective study, we sequenced the pol region of HIV from perinatally infected infants diagnosed in New York State in 2001-2002. Analyses of drug resistance, subtype diversity, and perinatal antiretroviral exposure were conducted, and the results were compared with those from a previous study of HIV-infected infants identified in 1998-1999. Eight of 42 infants (19.1%) had provirus carrying at least 1 drug-resistance mutation, an increase of 58% over the 1998-1999 results. Mutations conferring resistance to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 7.1%, 11.9%, and 2.4% of specimens, respectively. Consistent with previous results, perinatal antiretroviral exposure was not associated with drug resistance (P = 0.70). Phylogenetic analysis indicated that 16.7% of infants were infected with a non-subtype B strain of HIV. It seems that drug-resistant and non-subtype B strains of HIV are becoming increasingly common in the perinatally infected population. Our results highlight the value of resistance testing for all HIV-infected infants upon diagnosis and the need to consider subtype diversity in diagnostic and treatment strategies.

Alginates as therapeutic and drug delivery systems

Alginate is widely used as a viscosity enhancer in many different pharmaceutical formulations. The aim of this thesis is to quantitatively describe the functions of this polyelectrolyte in pharmaceutical systems. To do this the techniques used were Viscometry, Light Scattering, Continuous and Oscillatory Shear Rheometry, Numerical Analysis and Diffusion. Molecular characterization of the Alginate was carried out using Viscometry and Light Scattering to determine the molecular weight, the radius of gyration, the second virial coefficient and the Kuhn statistical segment length. The results showed good agreement with similar parameters obtained in previous studies. By blending Alginate with other polyelectrolytes, Xanthan Gum and 'Carbopol', in various proportions and with various methods of low and high shear preparation, a very wide range of dynamic rheological properties was found. Using oscillatory testing, the parameters often varied over several decades of magnitude. It was shown that the determination of the viscous and elastic components is particularly useful in describing the rheological 'profiles' of suspending agent blends and provides a step towards the non-empirical formulation of pharmaceutical disperse systems. Using numerical analysis of equations describing planar diffusion, it was shown that the analysis of drug release profiles alone does not provide unambiguous information about the mechanism of rate control. These principles were applied to the diffusion of Ibuprofen in Calcium Alginate gels. For diffusion in such non-Newtonian systems, emphasis was placed on the use of the elastic as well as the viscous component of viscoelasticity. It was found that the diffusion coefficients were relatively unaffected by increases in polymer concentration up to 5 per cent, yet the elasticities measured by oscillatory shear rheometry were increased. This was interpreted in the light of several theories of diffusion in gels.

Compressed orally disintegrating tablets:excipients evolution and formulation strategies

Introduction: Orally disintegrating tablets (ODTs) have emerged as one of the novel solid oral dosage forms with a potential to deliver a wide range of drug candidates to both paediatric and geriatric patient populations. Of the plethora of available technologies, compression of excipients offers a cost-effective and translatable methodology for the manufacture of ODTs. Areas covered: The review is a modest endeavour from the authors to assemble literature published over the last couple of decades on formulation development of compressed ODT. It describes the main ODT excipients used since the introduction of this dosage form in the 1990s and explores the switch from cellulose-based excipients towards sugar/polyols. Furthermore, it unfolds the key properties of ODT fillers, binders and disintegrants with an emphasis on their advantages and drawbacks. The review also provides a critical assessment of the various strategies employed for performance enhancement of compressed ODT with a focus on the underlying mechanisms for fast disintegration and acceptable mechanical strength. Expert opinion: Recent increase in the total number of compression-based technologies for ODT development promises to reduce the manufacturing cost of this dosage form in the future. However, some of the developed methods may affect the stability of tablets due to susceptibility to moisture, collapse of pores or the generation of less stable polymorphs which require rigorous testing prior to commercialization. © 2013 Informa UK, Ltd.

Enhancement of a novel gene therapy approach for Sandhoff disease through complimentary drug therapy

GM2 gangliosidoses is a family of severe, neurodegenerative disorders resulting from a deficiency in the β-hexosaminidase A (Hex A) enzyme. This disorder is typically caused by a mutation to either the HEXA gene, causing Tay Sachs disease, or a mutation to the HEXB gene, causing Sandhoff disease. The HEXA and HEXB genes are required to produce the α and β subunits of the Hex A enzyme respectively. Using a Sandhoff disease (SD) mouse model (Hexb-/-) we tested the potential of a low dose of systemically delivered single stranded adeno-associated virus 9 (ssAAV9) expressing human HEXB and human HEXA cDNA under the control of a single promoter through the use of a bicistronic vector design with a P2A linker to correct the neurological phenotype. Neonatal mice were injected with either this ssAAV9-HexB-P2A-HexA vector (HexB-HexA) or a vehicle solution via the superficial temporal vein. HexB-HexA treatment alone conferred an increase in survival of 56% compared to vehicle-injected controls and biochemical analysis of the brain tissue and serum revealed an increase in HexA activity and a decrease in brain GM2 ganglioside buildup. Additionally, treatments with the non-steroidal anti-inflammatory drug indomethacin (Indo), the histone deactylase inhibitor ITF2357 (ITF) and the pharmacological chaperone pyrimethamine (Pyr) were tested. The anti-inflammatory treatments of Indo and ITF conferred an increase in survival of 12% and 8% respectively while causing no alteration in the HexA activity or GM2 ganglioside buildup. Pyr had no observable effect on disease progression. Lastly HexB-HexA treatment was tested in conjunction with Indo, ITF and Pyr individually. Additive increases in survival and behavioural testing results were observed with Indo and ITF treatments while no additional benefit to HexA activity or GM2 ganglioside levels in the brain tissue was observed. This indicates the two treatments slowed the progression of the disease through a different mechanism than the reduction of the GM2 ganglioside substrate. Pyr treatment was shown to have no effect when combined with HexB-HexA treatment. This study demonstrates the potential amelioration of SD with a novel AAV9 gene therapy approach as well as helped to identify the additive potential of anti-inflammatory treatments in gene therapy of GM2 gangliosidoses.

Specific humoral response of hosts with variable schistosomiasis susceptibility

The schistosome blood flukes are some of the largest global causes of parasitic morbidity. Further study of the specific antibody response during schistosomiasis may yield the vaccines and diagnostics needed to combat this disease. Therefore, for the purposes of antigen discovery, sera and antibody-secreting cell (ASC) probes from semi-permissive rats and sera from susceptible mice were used to screen a schistosome protein microarray. Following Schistosoma japonicum infection, rats had reduced pathology, increased antibody responses and broader antigen recognition profiles compared with mice. With successive infections, rat global serological reactivity and the number of recognized antigens increased. The local antibody response in rat skin and lung, measured with ASC probes, increased after parasite migration and contributed antigen-specific antibodies to the multivalent serological response. In addition, the temporal variation of anti-parasite serum antibodies after infection and reinfection followed patterns that appear related to the antigen driving the response. Among the 29 antigens differentially recognized by the infected hosts were numerous known vaccine candidates, drug targets and several S. japonicum homologs of human schistosomiasis resistance markers-the tegument allergen-like proteins. From this set, we prioritized eight proteins that may prove to be novel schistosome vaccine and diagnostic antigens.

Multi-purpose utility of circulating plasma DNA testing in patients with advanced cancers.

Tumor genomic instability and selective treatment pressures result in clonal disease evolution; molecular stratification for molecularly targeted drug administration requires repeated access to tumor DNA. We hypothesized that circulating plasma DNA (cpDNA) in advanced cancer patients is largely derived from tumor, has prognostic utility, and can be utilized for multiplex tumor mutation sequencing when repeat biopsy is not feasible. We utilized the Sequenom MassArray System and OncoCarta panel for somatic mutation profiling. Matched samples, acquired from the same patient but at different time points were evaluated; these comprised formalin-fixed paraffin-embedded (FFPE) archival tumor tissue (primary and/or metastatic) and cpDNA. The feasibility, sensitivity, and specificity of this high-throughput, multiplex mutation detection approach was tested utilizing specimens acquired from 105 patients with solid tumors referred for participation in Phase I trials of molecularly targeted drugs. The median cpDNA concentration was 17 ng/ml (range: 0.5-1600); this was 3-fold higher than in healthy volunteers. Moreover, higher cpDNA concentrations associated with worse overall survival; there was an overall survival (OS) hazard ratio of 2.4 (95% CI 1.4, 4.2) for each 10-fold increase in cpDNA concentration and in multivariate analyses, cpDNA concentration, albumin, and performance status remained independent predictors of OS. These data suggest that plasma DNA in these cancer patients is largely derived from tumor. We also observed high detection concordance for critical 'hot-spot' mutations (KRAS, BRAF, PIK3CA) in matched cpDNA and archival tumor tissue, and important differences between archival tumor and cpDNA. This multiplex sequencing assay can be utilized to detect somatic mutations from plasma in advanced cancer patients, when safe repeat tumor biopsy is not feasible and genomic analysis of archival tumor is deemed insufficient. Overall, circulating nucleic acid biomarker studies have clinically important multi-purpose utility in advanced cancer patients and further studies to pursue their incorporation into the standard of care are warranted.

Design of experiment in drug development: optimal allocations in multi-arm clinical trials and design space for multi-step processes

In this thesis, we deal with the design of experiments in the drug development process, focusing on the design of clinical trials for treatment comparisons (Part I) and the design of preclinical laboratory experiments for proteins development and manufacturing (Part II). In Part I we propose a multi-purpose design methodology for sequential clinical trials. We derived optimal allocations of patients to treatments for testing the efficacy of several experimental groups by also taking into account ethical considerations. We first consider exponential responses for survival trials and we then present a unified framework for heteroscedastic experimental groups that encompasses the general ANOVA set-up. The very good performance of the suggested optimal allocations, in terms of both inferential and ethical characteristics, are illustrated analytically and through several numerical examples, also performing comparisons with other designs proposed in the literature. Part II concerns the planning of experiments for processes composed of multiple steps in the context of preclinical drug development and manufacturing. Following the Quality by Design paradigm, the objective of the multi-step design strategy is the definition of the manufacturing design space of the whole process and, as we consider the interactions among the subsequent steps, our proposal ensures the quality and the safety of the final product, by enabling more flexibility and process robustness in the manufacturing.

Iis--integrated Interactome System: A Web-based Platform For The Annotation, Analysis And Visualization Of Protein-metabolite-gene-drug Interactions By Integrating A Variety Of Data Sources And Tools.

High-throughput screening of physical, genetic and chemical-genetic interactions brings important perspectives in the Systems Biology field, as the analysis of these interactions provides new insights into protein/gene function, cellular metabolic variations and the validation of therapeutic targets and drug design. However, such analysis depends on a pipeline connecting different tools that can automatically integrate data from diverse sources and result in a more comprehensive dataset that can be properly interpreted. We describe here the Integrated Interactome System (IIS), an integrative platform with a web-based interface for the annotation, analysis and visualization of the interaction profiles of proteins/genes, metabolites and drugs of interest. IIS works in four connected modules: (i) Submission module, which receives raw data derived from Sanger sequencing (e.g. two-hybrid system); (ii) Search module, which enables the user to search for the processed reads to be assembled into contigs/singlets, or for lists of proteins/genes, metabolites and drugs of interest, and add them to the project; (iii) Annotation module, which assigns annotations from several databases for the contigs/singlets or lists of proteins/genes, generating tables with automatic annotation that can be manually curated; and (iv) Interactome module, which maps the contigs/singlets or the uploaded lists to entries in our integrated database, building networks that gather novel identified interactions, protein and metabolite expression/concentration levels, subcellular localization and computed topological metrics, GO biological processes and KEGG pathways enrichment. This module generates a XGMML file that can be imported into Cytoscape or be visualized directly on the web. We have developed IIS by the integration of diverse databases following the need of appropriate tools for a systematic analysis of physical, genetic and chemical-genetic interactions. IIS was validated with yeast two-hybrid, proteomics and metabolomics datasets, but it is also extendable to other datasets. IIS is freely available online at: http://www.lge.ibi.unicamp.br/lnbio/IIS/.

Study Of The Homogeneity Of Drug Loaded In Polymeric Films Using Near-infrared Chemical Imaging And Split-plot Design.

Split-plot design (SPD) and near-infrared chemical imaging were used to study the homogeneity of the drug paracetamol loaded in films and prepared from mixtures of the biocompatible polymers hydroxypropyl methylcellulose, polyvinylpyrrolidone, and polyethyleneglycol. The study was split into two parts: a partial least-squares (PLS) model was developed for a pixel-to-pixel quantification of the drug loaded into films. Afterwards, a SPD was developed to study the influence of the polymeric composition of films and the two process conditions related to their preparation (percentage of the drug in the formulations and curing temperature) on the homogeneity of the drug dispersed in the polymeric matrix. Chemical images of each formulation of the SPD were obtained by pixel-to-pixel predictions of the drug using the PLS model of the first part, and macropixel analyses were performed for each image to obtain the y-responses (homogeneity parameter). The design was modeled using PLS regression, allowing only the most relevant factors to remain in the final model. The interpretation of the SPD was enhanced by utilizing the orthogonal PLS algorithm, where the y-orthogonal variations in the design were separated from the y-correlated variation.