Characterization of an immune-evading doxycycline-inducible lentiviral vector for gene therapy in the spinal cord.

Gene therapy is a powerful approach to promote spinal cord regeneration. For a clinical application it is important to restrict therapeutic gene expression to the appropriate time window to limit unwanted side effects. The doxycycline (dox)-inducible system is a widely used regulatable gene expression platform, however, this system depends on a bacterial-derived immunogenic transactivator. The foreign origin of this transactivator prevents reliable regulation of therapeutic gene expression and currently limits clinical translation. The glycine-alanine repeat (GAR) of Epstein-Barr virus nuclear antigen-1 protein inhibits its presentation to cytotoxic T cells, allowing virus-infected cells to evade the host immune system. We developed a chimeric transactivator (GARrtTA) and show that GARrtTA has an immune-evading advantage over "classical" rtTA in vivo. Direct comparison of lentiviral vectors expressing rtTA and GARrtTA in the rat spinal cord shows that the GARrtTA system is inducible for 6 doxycycline-cycles over a 47 week period, whereas with the rtTA-based system luciferase reporter expression declines during the 3rd cycle and is no longer re-inducible, indicating that GARrtTA provides an immune-advantage over rtTA. Immunohistochemistry revealed that GARrtTA expressing cells in the spinal cord appear healthier and survive better than rtTA expressing cells. Characterization of the immune response shows that expression of GARrtTA, in contrast to rtTA, does not recruit cytotoxic T-cells to the transduced spinal cord. This study demonstrates that fusion of the GAR domain to rtTA results in a functional doxycycline-inducible transactivator with a clear immune-advantage over the classical rtTA in vivo.

Manipulating the extracellular matrix and its role in brain and spinal cord plasticity and repair.

Brain and spinal cord injury can result in permanent cognitive, motor, sensory and autonomic deficits. The central nervous system (CNS) has a poor intrinsic capacity for regeneration, although some functional recovery does occur. This is mainly in the form of sprouting, dendritic remodelling and changes in neuronal coding, firing and synaptic properties; elements collectively known as plasticity. An important approach to repair the injured CNS is therefore to harness, promote and refine plasticity. In the adult, this is partly limited by the extracellular matrix (ECM). While the ECM typically provides a supportive framework to CNS neurones, its role is not only structural; the ECM is homeostatic, actively regulatory and of great signalling importance, both directly via receptor or coreceptor-mediated action and via spatially and temporally relevant localization of other signalling molecules. In an injury or disease state, the ECM represents a key environment to support a healing and/or regenerative response. However, there are aspects of its composition which prove suboptimal for recovery: some molecules present in the ECM restrict plasticity and limit repair. An important therapeutic concept is therefore to render the ECM environment more permissive by manipulating key components, such as inhibitory chondroitin sulphate proteoglycans. In this review we discuss the major components of the ECM and the role they play during development and following brain or spinal cord injury and we consider a number of experimental strategies which involve manipulations of the ECM, with the aim of promoting functional recovery to the injured brain and spinal cord.

Using multidimensional mutual information to prioritize mammographic features for breast cancer diagnosis.

The goal of this study was to demonstrate that information theory could be used to prioritize mammographic features to efficiently stratify the risk of breast cancer. We compared two approaches, Single-dimensional Mutual Information (SMI), which ranks features based on mutual information of features with outcomes without considering dependency of other features, and Multidimensional Mutual Information (MMI), which ranks features by considering dependency. To evaluate these approaches, we calculated area under the ROC curve for Bayesian networks trained and tested on features ranked by each approach. We found that both approaches were able to stratify mammograms by risk, but MMI required fewer features (ten vs. thirteen). MMI-based rankings may have greater clinical utility; a smaller set of features allows radiologists to focus on those findings with the highest yield and in the future may help improve mammography workflow.

Adaptive immune response, survival, and somatic cell score between postpartum Holstein and Norwegian Red × Holstein first-calf heifers.

The objectives of this study were to evaluate antibody (AMIR) and cell-mediated immune responses (CMIR), survival, and somatic cell score (SCS) between purebred Holstein (HO) and crossbred Norwegian Red × Holstein (NRHO) first-calf heifers postpartum. Additionally, immune response traits observed as calves in a previous study were correlated with their immune response traits as first-calf heifers. Heifers, previously immunized as calves, were bled and reimmunized 6 to 9 d postcalving with known type 1 and type 2 antigens and human serum albumin (HSA). Seven days later, heifers were rebled, and background skinfold measurements of the tail fold were taken. Intradermal injections of PBS and type 1 antigen were administered on either side of the tail fold. On d 9 final skinfold measurements were taken and used to assess delayed-type hypersensitivity (DTH) as an indicator of CMIR. Blood samples were also collected for a final time on d 14 from heifers that received the antigen HSA. Serum was obtained from blood collected on d 0, 7, and 14 and analyzed by ELISA to assess AMIR. Data on survival and somatic cell count, which was converted to SCS, were obtained from CanWest Dairy Herd Improvement (DHI). All SCS, survival, and immune response data were analyzed using general linear models to determine significance between HO and NRHO first-calf heifers. To determine residual correlations between immune response traits observed in calves to their responses as first-calf heifers, residuals were obtained from models, and correlations between traits were determined using PROC CORR in SAS. Results showed NRHO had a greater primary IgG antibody response to HSA and greater tertiary IgG antibody response to the type 2 antigen compared with HO. Crossbreds (NRHO)also had significantly greater DTH response (P < 0.05) and, in general, greater survival from calving to 100 d in milk (dim), 100 to 305 dim, calving to 305 dim, and age at immune response testing as calf to 305 dim. No difference was observed between breeds for SCS. Results also showed most correlations between calf and first-calf heifer immune response traits were found to be positive and significant (P < 0.05). In conclusion, NRHO heifers have greater survival, which likely relates at least in part to increases in aspects of both AMIR and CMIR and could indicate that crossbred heifers have enhanced disease resistance.

Integrating machine learning and physician knowledge to improve the accuracy of breast biopsy.

In this work we show that combining physician rules and machine learned rules may improve the performance of a classifier that predicts whether a breast cancer is missed on percutaneous, image-guided breast core needle biopsy (subsequently referred to as "breast core biopsy"). Specifically, we show how advice in the form of logical rules, derived by a sub-specialty, i.e. fellowship trained breast radiologists (subsequently referred to as "our physicians") can guide the search in an inductive logic programming system, and improve the performance of a learned classifier. Our dataset of 890 consecutive benign breast core biopsy results along with corresponding mammographic findings contains 94 cases that were deemed non-definitive by a multidisciplinary panel of physicians, from which 15 were upgraded to malignant disease at surgery. Our goal is to predict upgrade prospectively and avoid surgery in women who do not have breast cancer. Our results, some of which trended toward significance, show evidence that inductive logic programming may produce better results for this task than traditional propositional algorithms with default parameters. Moreover, we show that adding knowledge from our physicians into the learning process may improve the performance of the learned classifier trained only on data.

Antibody and cell-mediated immune responses and survival between Holstein and Norwegian Red × Holstein Canadian calves.

As an extension of a former study, the objectives of this study were to evaluate purebred Holstein (HO; n=140) and crossbred Norwegian Red × Holstein (NRFX; n=142) calves for antibody (AMIR) and cell-mediated immune responses (CMIR) as well as survival. Blood was collected on d 0, 14, and 21, and calves were immunized on d 0 and 14 with type 1 (Candida albicans) and type 2 (hen egg white lysozyme) antigens, which have been shown to induce CMIR and AMIR, respectively. Day 21 background skin-fold measurements of either side of the tail-fold were taken and intradermal injections of test (type 1 antigen) and control (phosphate saline buffer) were administered. Day 23 final skin-fold measurements were taken to assess delayed type hypersensitivity as an indicator of CMIR. Survival data were obtained from CanWest Dairy Herd Improvement. Statistical Analysis System general linear models were used to analyze all immune response and survival data and to determine statistical significance between breeds. Results showed that NRFX had greater primary IgM, IgG, IgG1, and secondary IgG1 antibody response, as well as greater primary IgG1:IgG2 ratio to the type 2 antigen compared with HO. The NRFX also had greater primary IgG1 and IgG2, and secondary IgG2 antibody response as well as greater primary IgG1:IgG2 ratio to the type 1 antigen. The NRFX calves had a tendency toward greater survival from age at immune response testing to calving. No difference was observed between breeds for other secondary antibody response traits or delayed type hypersensitivity. Results indicate NRFX have greater AMIR and therefore may have enhanced defense against extracellular pathogens. This may contribute to increased survival compared with HO. Both breeds, however, likely have similar defense against intracellular pathogens, because no differences in CMIR were observed. In general, these results may suggest that crossbreeding could improve resistance to certain diseases in dairy calves, resulting in decreased input costs to producers for crossbred calves compared with purebred calves. However, more research with larger sample sizes and different breeds should be conducted to confirm these results and obtain a complete picture of the benefits of crossbreeding on immune response traits in calves.

Immune responses of Holstein and Norwegian Red x Holstein calves on Canadian dairy farms.

The objective of this study was to compare the immune response of Holstein and Norwegian Red x Holstein calves on 13 commercial Canadian dairy farms. Data were collected on 135 calves, 68 Holstein and 67 Norwegian Red x Holstein calves aged between 2 and 6 mo. The calves were immunized with hen egg white lysozyme to induce antibody-mediated immune response. Candida albicans was used as an in vivo indicator of cell-mediated immune response, with delayed-type hypersensitivity used as the indicator. Antibody response to hen egg white lysozyme (IgG, IgG1, and IgG2) was measured by ELISA. Calves of both breed groups produced a significant primary and secondary antibody-mediated immune response, as well as a delayed-type hypersensitivity reaction. The Norwegian Red x Holstein produced a greater primary IgG antibody-mediated immune response (d 14, and d 14 minus d 0) when compared with the Holstein. No differences were observed between the breeds for secondary response or antihen egg white lysozyme isotype (IgG1 or IgG2) production or the ratio of IgG1:IgG2. There was no effect of breed on delayed-type hypersensitivity. Nonetheless, high and low immune responders could be identified in both breed groups, but with no difference in the proportion of high and low responders observed for either antibody-mediated immune response or cell-mediated immune response between breed groups.

Movement of a biopsy-site marker clip after completion of stereotactic directional vacuum-assisted breast biopsy: case report.

A 51-year-old woman underwent stereotactic core biopsy of suspicious microcalcifications in the upper outer left breast with subsequent metallic clip deployment. Postprocedure mammograms demonstrated accurate placement of the clip. However, mammography 10 months later revealed movement of the clip 4 cm medially in the breast.

Child sexual abuse, poor parental care and adult depression: evidence for different mechanisms.

BACKGROUND: Child sexual abuse (CSA) and poor parental care (neglect and institutional care) are associated with depression in adult life. Little is known about possible mechanisms underlying these associations. AIMS: To examine the role of adult intimate-love relationships as differential mediators or moderators of the associations between CSA, poor parental care and adult depression. METHOD: Sampling was carried out in two phases. In the first, questionnaires were sent to women aged 25-36 years in five primary care practices. Second-phase subjects for interview (n=198) were drawn from three strata defined on the basis of childhood adversities. Recalled childhood experiences and recent adult relationships and depression were assessed and rated independently. Frequencies of predictor and response variables, effect estimates and their confidence intervals were weighted back to the general population questionnaire sample. RESULTS: The risk for depression associated with CSA was unaffected by quality of adult relationships, while the risk associated with poor parental care was substantially altered. CONCLUSIONS: There may be different pathways linking CSA and poor parental care to adult depression.

The impact of alternative practices on the cost and quality of mammographic screening in the United States.

The decentralized structure of health care in the Unites States hinders population-based analysis of breast cancer screening. Our objectives are to model mammography in the United States as a whole, to identify the variables that most profoundly affect cost and efficacy, and to develop a strategy to improve mammography screening from a population perspective. A spreadsheet model was used to represent the variables of mammography screening in the United States. The population-based national screening program in Sweden provides a framework for comparison. The outcome measures are the aggregate cost and the number of cancers detected by mammography. We used deterministic sensitivity analysis to calculate the impact of variation in practice. Aggregate costs of screening in the United States are in the range of $3-$5 billion dollars. The percentage of women screened, cost per mammogram, cancer to biopsy ratio, recall rate, and cost of recall have the most profound effect on the quality and cost of a national screening program. Variance of these high-impact variables, based on the U.S. population, modifies the aggregate cost of screening by over $2 billion. As mammography screening in the United States increases to include all women over age 40, high-impact variables should be optimized to decrease costs and improve breast cancer detection. Our model establishes which parameters are most important.

Childhood sexual abuse and affective symptoms in women: a general population study.

BACKGROUND: Child sexual abuse (CSA) is associated, after controlling for other adversities, with a range of non-psychotic disorders in adult life. There is a need to clarify whether CSA contributes to risk of disorder in the absence of such adversities, and given that associations may be accounted for by genetic mechanisms, whether they are seen where the perpetrator of CSA is not a biological relative, and where there has been only one incident. METHODS: A questionnaire-based study of a socio-economically representative sample of women age 25-36 (N = 862) was carried out. Parental care and control were assessed using the Parental Bonding Instrument, and CSA using a previously validated questionnaire. Current affective symptoms were assessed from the depression scale of the GHQ-28. RESULTS: Low maternal and paternal care were associated with risk of abuse by a biologically unrelated perpetrator before the age of 11, but not during early adolescence. Low maternal care and CSA each made independent contributions to the prediction of affective symptoms. CSA by a non-relative was strongly associated with GHQ depression, as was CSA by a non-relative that had occurred only once. CONCLUSIONS: Quality of parental care probably influences risk of CSA by unrelated abusers in younger children, while there may be a combination of genetic and parental influences on the risk of abuse by a relative. The strong association of affective symptoms in adult life with CSA by a non-relative suggests an environmental effect. Studies, utilizing genetic designs, of the role of childhood trauma in relation to adult affective symptoms are needed.

A Bayesian network for mammography.

The interpretation of a mammogram and decisions based on it involve reasoning and management of uncertainty. The wide variation of training and practice among radiologists results in significant variability in screening performance with attendant cost and efficacy consequences. We have created a Bayesian belief network to integrate the findings on a mammogram, based on the standardized lexicon developed for mammography, the Breast Imaging Reporting And Data System (BI-RADS). Our goal in creating this network is to explore the probabilistic underpinnings of this lexicon as well as standardize mammographic decision-making to the level of expert knowledge.

Genetic evaluation for herd life in Canada.

Methods were developed for the national genetic evaluation of herd life for Canadian Holstein sires. The genetic evaluations incorporate information from survival (direct herd life) and information from conformation traits that are related to herd life (indirect herd life) after adjustment for production in first lactation to remove the effect of culling for production. Direct genetic evaluations for herd life were based on survival in each of the first three lactations, which was analyzed using a multiple-trait animal model. Sire evaluations thus obtained for survival in each of the first three lactations were combined based on their economic weights into an overall sire evaluation for direct herd life. Sire evaluations for indirect herd life were based on an index of sire evaluations for mammary system, feet and legs, rump, and capacity. A multiple-trait sire model based on multiple-trait across country evaluation methodology was used to combine direct and indirect genetic evaluations for herd life into an overall genetic evaluation for herd life. Sire evaluations for herd life were expressed in estimated transmitting ability as the number of lactations and represent expected differences among daughters in functional herd life (number of lactations); the average functional herd life was set equal to three lactations. Estimated transmitting abilities were normally distributed and ranged from 2.31 to 3.43 lactations.

Merrill C. Sosman Lecture. Surviving managed care.

Managed care has arrived, especially on the West Coast. This tsunami is moving eastward at a rapid rate. Perhaps the experience with killer bees will be repeated with managed care, with decreased virulence as this wave moves across the country. Institutions would be wise to prepare for these changes with improved efficiency and cost-effectiveness. Radiologists are particularly at risk and need to aggressively adapt to managed care by becoming informed, developing a clear strategy for coping, and then executing their plan. Evolution will demonstrate once again the survival of the fittest.

Optimal size of progeny groups for progeny-testing programs by artificial insemination firms.

A semi-stochastic model for the simulation of genetic improvement in a dairy cattle population was used to evaluate and optimize progeny-testing programs for AI firms that operate in a competitive market for semen from progeny-tested bulls with regard to number of bulls sampled and size of progeny groups. The population was serviced by four firms. The competition for market share and semen sales was determined by the relative rank of progeny-tested bulls from a firm based on EBV for a trait with a heritability of 25%. For a fixed total number of daughters from young bulls for an AI program (test capacity), optimal size of the progeny groups was highly dependent on the objective to be maximized. The rate of genetic gain was maximized with a progeny group of 57 to 61 daughters per bull, but was relatively robust to changes in size of progeny groups. The number of marketable bulls was maximized with progeny groups between 20 and 40 daughters, depending on the test capacity. However, when a relationship between price per dose of semen and EBV of marketable bulls was considered, returns from semen sales were maximized at 49 and 82 daughters per bull, respectively, for linear and quadratic functions for semen price. The critical objective, net returns from semen sales, subtracting costs of sampling bulls, was maximized for progeny groups of between 95 and 105 daughters. Optimal size of progeny groups was robust to changes in economic parameters and the breeding programs of competitors. For economic parameters that were typical for Canadian AI firms, net returns per annual cohort of young bulls were 40% higher for the optimal size of the progeny groups than for sampling with 60 daughters per bull.

Nonadditive genetic effects and inbreeding depression for somatic cell counts of Holstein cattle.

A total of 65,491 lactation means of log2-transformed SCC measures were analyzed from first lactation Holstein cows in Ontario. Effects of inbreeding on SCC were estimated by a nonadditive sire and dam model that included additive, dominance, and additive by additive genetic effects and regression of lactation somatic cell score on inbreeding coefficients of the cows. Variance components were estimated using the tildehat approximation to REML. Solutions were by iteration on data. Estimates of heritability for lactation somatic cell score in the narrow sense were .165 and in the broad sense were .203. The additive by additive component (2.5% of the total phenotypic variance) was almost twice as large as the dominance component (1.3%). The regression coefficient of lactation somatic cell score per 1% increase of inbreeding was .012. The average increase of the population mean of lactation somatic cell score caused by a 10% increase of inbreeding coefficient was estimated to be 10.5% of the original phenotypic standard deviation of 1.153. The inbreeding depression was thus relatively low, but, on average, inbred animals tended to have higher lactation somatic cell score. This study provides preliminary evidence that inbreeding is related to disease prevalence in large purebred dairy populations.

Production traits of Holstein cattle: estimation of nonadditive genetic variance components and inbreeding depression.

Additive, dominance, and additive by additive components of genetic variance and inbreeding depression were estimated for production traits from a group of daughters of young sires from the Canadian Holstein population. First lactations of 92,838 cows were analyzed. Three sire and dam models (additive, additive plus dominance, additive plus dominance plus additive by additive genetic effects), all including regression of the trait on inbreeding coefficient of the cow, were used to estimate the effect of inbreeding on production traits. For all production traits, heritability in the narrow sense was overestimated with the simplest model, in which only the additive effect was fitted. Estimates of dominance variance were low for all traits, .9 to 3%. Additive by additive components were low for milk, 2.8%, and fat yield, 2.8%, but higher for protein yield, 6.8%, and for fat, 9%, and protein percentages, 8.9%. Estimates of inbreeding depression for the five traits were similar across all models (-25, -.9, and -.8 kg; .05% and .05% per 1% increase in inbreeding for milk, fat, and protein production and fat and protein percentages, respectively). More accurate estimates of additive effects might be obtained with the inclusion of nonadditive effects for genetic evaluation. If the estimation of inbreeding depression is the only objective, simple models and small random samples of the population may be adequate.

Genetic response and inbreeding with different selection methods and mating designs for nucleus breeding programs of dairy cattle.

Stochastic simulation was used to study the effect of selection and mating strategy on rates of genetic response and inbreeding with a closed nucleus breeding program for juvenile and adult schemes with 8 males and 64 females selected to produce 1024 progeny (512 females). Selection strategies considered using all available information or only individual and sibling records. Selection of sires was either unrestricted or restricted to between full-sib families. The effect of avoidance of mating of relatives to limit inbreeding was also evaluated. Four mating designs were examined: each dam was mated to 1, 2, 4, or all sires. Mating designs involving one sire per dam and more than one dam per sire were referred to as hierarchical. Use of several mates per dam resulted in a factorial mating design. Selected parents were mated either randomly, best to best, or best to worst. An index based on relative inbreeding to response ratio was used to describe the effectiveness of strategies for reducing inbreeding relative to changes in rates of genetic response. Strategies that lower index values were preferred and include selection on BLUP or approximations of BLUP and factorial mating designs that involve the random mating of dams to several sires. Factorial mating designs were effective for a range of heritabilities. Avoidance of matings of full sibs and restriction of selection of sires to between full-sib families enabled appreciable reductions in the index. Nucleus breeding programs based entirely on the selection of juveniles were not indicated because they had higher index values than adult schemes.