The program for phenotyping of genetically modified animals at AstraZeneca

Genetically modified mice offer a wide range of possibilities in preclinical drug discovery, e.g. for use in target identification, target validation and disease model generation. However, genomic modification and alteration in gene expression may cause unpredicted phenotypic alterations in the organism other than the intended ones. The aim of this study was to determine the importance of establishing the phenotype of transgenic and knockout mice models for use in pharmaceutical research.

A total number of 51 mouse models (transgenic and knockout) produced at AstraZeneca during a 4 year period were subjected to a thorough phenotyping package covering clinical as well as morphological aspects. Phenotype abnormalities were recorded in 36 (70.6%) of the mouse models. The majority of findings were considered to be minor in magnitude. Histopathological changes related to the genotype of the animals were observed in 33% of the mouse models, underlining the importance of pathology in the phenotyping program.     

Towards the phenotyping of soft tissue tumours by cell surface molecules

Summary This study is aimed at the characterization of soft tissue tumours (STT) by means of cell surface molecules. To achieve this, normal mesenchymal tissues were extensively examined for expression of leucocyte differentiation (CD) antigens and HLA molecules. The panel of antigens finally examined in STT comprised CD10, CD13, CD24, CD34, CD36, CD56, CD57, HLA-A,B,C, ₂-microglobulin, HLA-DR, -DP, and -DQ and the HLA-D-associated invariant chain (Ii). STT were determined by conventional histomorphological and immunohistochemical criteria. The immunohistological analysis was based on serial frozen sections, one of which was used to demonstrate CD53 antigen. This very broadly distributed leuco/histiocyte-restricted antigen allowed for the distinction between the background of interstitial stromal cells and the neoplastic population. In some STT, the expression pattern of the cell surface molecules corresponded to that in their non-neoplastic counterparts. The majority of STT, however, showed considerable changes in the cell surface immunophenotype compared to their cells of origin. These alterations consisted mainly in an aberrant induction/neoexpression and, to a much lesser extent, in an aberrant down-regulation/loss of cell surface antigens. Nevertheless, some immunophenotype configurations are described which, for the time being, can be considered to be useful supplements in the differential diagnosis of this complex class of tumours. The data also indicate considerable changes in cell surface antigen expression occurring in the course of neoplastic transformation of mesenchymal cells. Detailed analysis of alterations in the functional repertoire of neoplastic mesenchymal cells might provide new insights into the biology of STT, possibly leading to new concepts for therapeutic intervention.This study was supported by the Tumorzentrum Heidelberg/ Mannheim and by the Dr. Mildred-Scheel-Stiftung für Krebshilfe (W50/89/Mö2)     

Phenotypic diversity of cytomegalovirus DNA polymerase gene variants observed after antiviral therapy

BackgroundCytomegalovirus UL54 DNA polymerase mutations observed in clinical specimens are of diagnostic significance if confirmed to affect antiviral drug susceptibility.ObjectivesValidate an updated recombinant phenotyping method to determine the degree of drug resistance conferred by previously uncharacterized UL54 sequence variants, in comparison with known resistance-related mutations.Study designBacterial artificial chromosome clones of viral DNA were mutagenized by recombination, transfected to produce live virus and phenotyped by standardized reporter-based yield reduction assays.ResultsSixteen recombinant viruses were constructed, representing baseline sequences, known resistance-related mutations and amino acid changes of unproven significance from clinical specimens. Phenotypes of baseline strains and known mutants were comparable to results from prior methods and helped to resolve some published inconsistencies. Mutations F412L, F412S, L545W were newly confirmed to confer ganciclovir and cidofovir resistance, while Q578H conferred ganciclovir and foscarnet resistance with borderline cidofovir resistance. Some foscarnet-resistant mutants were appreciably growth-retarded.ConclusionsResults add to known exonuclease domain mutations that confer ganciclovir-cidofovir cross-resistance, polymerase domain mutations that confer foscarnet resistance with variably decreased ganciclovir/cidofovir susceptibility, and increase the list of sequence variants with no measurable impact on drug susceptibility. The phenotypic diversity of similar UL54 genotypic variants complicates the interpretation of genotypic resistance testing. Technical improvements are facilitating the phenotyping of remaining unknown sequence variants.     

Relational machine learning for electronic health record-driven phenotyping

ObjectiveElectronic health records (EHR) offer medical and pharmacogenomics research unprecedented opportunities to identify and classify patients at risk. EHRs are collections of highly inter-dependent records that include biological, anatomical, physiological, and behavioral observations. They comprise a patient’s clinical phenome, where each patient has thousands of date-stamped records distributed across many relational tables. Development of EHR computer-based phenotyping algorithms require time and medical insight from clinical experts, who most often can only review a small patient subset representative of the total EHR records, to identify phenotype features. In this research we evaluate whether relational machine learning (ML) using inductive logic programming (ILP) can contribute to addressing these issues as a viable approach for EHR-based phenotyping.MethodsTwo relational learning ILP approaches and three well-known WEKA (Waikato Environment for Knowledge Analysis) implementations of non-relational approaches (PART, J48, and JRIP) were used to develop models for nine phenotypes. International Classification of Diseases, Ninth Revision (ICD-9) coded EHR data were used to select training cohorts for the development of each phenotypic model. Accuracy, precision, recall, F-Measure, and Area Under the Receiver Operating Characteristic (AUROC) curve statistics were measured for each phenotypic model based on independent manually verified test cohorts. A two-sided binomial distribution test (sign test) compared the five ML approaches across phenotypes for statistical significance.ResultsWe developed an approach to automatically label training examples using ICD-9 diagnosis codes for the ML approaches being evaluated. Nine phenotypic models for each ML approach were evaluated, resulting in better overall model performance in AUROC using ILP when compared to PART (p = 0.039), J48 (p = 0.003) and JRIP (p = 0.003).DiscussionILP has the potential to improve phenotyping by independently delivering clinically expert interpretable rules for phenotype definitions, or intuitive phenotypes to assist experts.ConclusionRelational learning using ILP offers a viable approach to EHR-driven phenotyping.     

Phenotyping of malignant hematopoietic cells

Summary 1255 cases of leukemia-lymphoma were tested between 1972 and 1984 by multiple marker analysis. Routine leukemia phenotyping was performed using standard morphological and cytochemical techniques in combination with clinical and histo-pathological information; the main emphasis was put on immunological surface marker analysis using erythrocyte rosette assays, TdT and a large panel of poly- and monoclonal antibody tests. The 1255 cases were divided into these major types and subtypes: 349 cases of ALL and related immature T- and Burkitt-lymphomas (cALL, pre B-ALL, B-ALL and Burkitt-lymphomas, T-ALL and immature, mostly leukemic T-lymphomas, Null-ALL), 454 cases of mature T- and B-cell malignancies (T-CLL, mycosis fungoides, Sezary-syndrome, T-lymphomas, B-CLL, hairy cell leukemia, multiple myeloma, B-lymphomas), 263 cases of acute myeloid leukemias (AML, AMMoL/AMoL), 182 cases of chronic myeloid leukemias (CML in chronic phase, CMoL, CML in blast crisis), 6 cases of erythroleukemia and 1 case of megakaryoblastic leukemia. A simplified classification scheme which has been used in our laboratories is presented. Phenotyping is of diagnostic, prognostic and therapeutic relevance, most evidently for patients with ALL. Routine leukemia phenotyping should be performed with highly standardized techniques and reagents and by combining information from several fields in the multiple marker analysis. New areas of leukemia research might become very useful for the routine procedure of phenotyping.Abbreviations ALL acute lymphoblastic leukemia - AML acute myeloblastic leukemia - AMMoL acute myelomonoblastic leukemia - AMoL acute monoblastic leukemia - cALL common ALL - CLL chronic lymphocytic leukemia - CML chronic myelocytic leukemia - CML-BC CML in blastic crisis - CMoL chronic monocytic leukemia     

The 2013 EAU Guidelines on Chronic Pelvic Pain: Is Management of Chronic Pelvic Pain a Habit,a Philosophy,or a Science? 10 Years of Development

Context

Progress in the science of pain has led pain specialists to move away from an organ-centred understanding of pain located in the pelvis to an understanding based on the mechanism of pain and integrating, as far as possible, psychological, social, and sexual dimensions of the problem. This change is reflected in all areas, from taxonomy through treatment. However, deciding what is adequate investigation to rule out treatable disease before moving to this way of engaging with the patient experiencing pain is a complex process, informed by pain expertise as much as by organ-based medical knowledge.

Objective

To summarise the evolving changes in the management of patients with chronic pelvic pain by referring to the 2012 version of the European Association of Urology (EAU) guidelines on chronic pelvic pain.

Evidence acquisition

The working panel highlights some of the most important aspects of the management of patients with chronic pelvic pain emerging in recent years in the context of the EAU guidelines on chronic pelvic pain. The guidelines were completely updated in 2012 based on a systematic review of the literature from online databases from 1995 to 2011. According to this review, levels of evidence and grades of recommendation were added to the text. A full version of the guidelines is available at the EAU office or Web site (www.uroweb.org).

Evidence synthesis

The previously mentioned issues are explored in this paper, which refers throughout to dilemmas for the physician and treatment team as well as to the need to inform and engage the patient in a collaborative empirical approach to pain relief and rehabilitation. These issues are exemplified in two case histories.

Conclusions

Chronic pelvic pain persisting after appropriate treatment requires a different approach focussing on pain. This approach integrates the medical, psychosocial, and sexual elements of care to engage the patient in a collaborative journey towards self-management.     

La psychiatrie connectée,une psychiatrie augmentée ?

The use of digital technologies in medicine is constantly growing. The adoption of digital technologies in the clinical practice in psychiatry seems to be a question of when, not if. How can we use those tools effectively? To address those issues, this short review discusses three of the major contributions of digital psychiatry: 1/the assistance and improvement of current care; 2/the development of new treatments; 3/the production of scientific and medical knowledge.     

<Emphasis Type="Italic">CYP2C9</Emphasis> genetic variants and losartan oxidation in a Turkish population

Objective Cytochrome P₄₅₀ 2C9 (CYP2C9) is a polymorphic enzyme catalysing the metabolism of several important drugs. Losartan has recently been suggested as a selective probe for CYP2C9 metabolic activity. The aim of the study was to determine the activity of CYP2C9, using losartan as a probe drug, in relation to CYP2C9 genotype in healthy Turkish subjects.Methods A single oral dose of 25 mg losartan was given to 85 Turkish unrelated subjects. Concentrations of losartan and its carboxylic acid metabolite, E3174, were analysed by means of high-performance liquid chromatography in urine collected for 8 h. The CYP2C9 genotypes were determined in 85 subjects using polymerase chain reaction-based endonuclease digestion methods specific for CYP2C9*2 and *3. Losartan oxidation was also studied in vitro, using human CYP2C8 and CYP2C9 enzymes expressed in yeast.Results The frequencies of the allelic variants CYP2C9*2 and CYP2C9*3 were 0.100 and 0.088, respectively. The urinary losartan/E3174 ratio was significantly higher in subjects with CYP2C9*1/*3 genotype (median 2.35, n=12) than in subjects with CYP2C9*1/*1 (0.71, n=58) and *1/*2 (0.85, n=10) genotypes (P<0.05). In contrast to CYP2C9, no E3174 was formed by CYP2C8 in vitro.Conclusion The urinary losartan to E3174 metabolic ratio after a 25-mg losartan dose was found to be a safe and useful phenotyping assay for CYP2C9 activity in vivo. CYP2C9*3 variant allele is a major determinant of the enzyme activity, and it decreases losartan metabolism significantly, while CYP2C9*2 allele has less impact on enzyme function.     

不动杆菌属在NC21板上耐药表型分类研究

目的 研究不动杆菌属在NC21板上耐药表型及比率.方法 对临床分离的275株不动杆菌属耐药表型进行分类.结果 β-内酰胺类抗菌药物耐药表型Ⅰ～Ⅴ分别为67.3%、1.8%、1.8%、19.6%和0.7%,8.8%未能分类;庆大霉素、阿米卡星和妥布霉素均敏感的菌株占74.2%,庆大霉素耐药表型7.3%,庆大霉素-阿米卡星耐药表型1.1%,庆大霉素、阿米卡星、妥布霉素耐药表型10.9%,未能分类6.5%;Ⅳ型和庆大霉素、阿米卡星、妥布霉素耐药表型同时存在菌株占8.7%,均对亚胺培南敏感,氨苄西林/舒巴坦对50.0%菌株敏感.结论 表型分类有利于合理使用抗菌药物和对多药耐药菌株监测,亚胺培南、氨苄西林/舒巴坦有较高的抗菌活性,氨曲南不适合不动杆菌属感染的治疗.     

Abnormalities of B cell subsets in patients with systemic lupus erythematosus

The prototypic autoimmune disease, SLE, is known to be associated with polyclonal B cell hyperreactivity. Developing an understanding of the complex nature of human B cell differentiation, largely through the application of multiparameter flow cytometry to an analysis of circulating B cells has permitted an assessment of whether specific stages of B cell maturation are affected by the tendency for polyclonal B cell activation. Moreover, the analysis of perturbations of the specific stages of B cell maturation has generated new information on whether abnormalities in B cell differentiation are primarily involved in autoimmune disease immunopathology or, rather, are secondary to the inflammatory environment characteristic of subjects with this autoimmune disease. Multivariant analysis has begun to document abnormalities in B cell maturation that are primarily associated with lupus, or, alternatively related to disease duration, disease activity and concomitant medication. Together, these analyses have provided new insights on the role of B cell over-reactivity in SLE.