Demonstration of microRNA using hybridisation on formalin fixed paraffin wax samples using conventional oligonucleotide probes: a comparison with the use of locked nucleic acid probes.

Background: MicroRNAs (miRNAs) regulate the translation of mRNA during gene expression and investigations have highlighted their importance in pathophysiology. qRT-PCR is currently the gold standard method for detecting changes in miRNA expression. However, when used on heterogeneous samples, it cannot identify individual cell types harbouring miRNAs.

Upregulated Wnt-11 and miR-21 Expression Trigger Epithelial Mesenchymal Transition in Aggressive Prostate Cancer Cells.

Prostate cancer (PCa) is the second-leading cause of cancer-related death among men. microRNAs have been identified as having potential roles in tumorigenesis. An oncomir, miR-21, is commonly highly upregulated in many cancers, including PCa, and showed correlation with the Wnt-signaling axis to increase invasion.

In situ hybridisation: Technologies and their application to understanding disease.

In situ hybridisation (ISH) is unique amongst molecular analysis methods in providing for the precise microscopic localisation of genes, mRNA and microRNA in metaphase spreads, cell and tissue preparations. The method is well established as a tool to guide appropriate therapeutic intervention in breast, gastric and lung cancer. With the description of ultrasensitive ISH technologies for low copy mRNA demonstration and the relative ease by which microRNA can be visualised, the applications for research and diagnostic purposes is set to increase dramatically.

Antigen retrieval, blocking, detection and visualisation systems in immunohistochemistry: a review and practical evaluation of tyramide and rolling circle amplification systems.

To achieve specificity and sensitivity using immunohistochemistry it is necessary to combine the application of validated primary antibodies with optimised pre-treatment, detection and visualisation steps. The influence of these surrounding procedures is reviewed. A practical evaluation of tyramide signal amplification and rolling circle amplification detection methods is provided in which formalin fixed paraffin embedded sections of adenocarcinomas of breast, colon and lung together with squamous metaplasia of lung were immunostained with CD20 and CK19 primary antibodies.

Antibody validation of immunohistochemistry for biomarker discovery: recommendations of a consortium of academic and pharmaceutical based histopathology researchers.

As biomarker discovery takes centre-stage, the role of immunohistochemistry within that process is increasing. At the same time, the number of antibodies being produced for "research use" continues to rise and it is important that antibodies to be used as biomarkers are validated for specificity and sensitivity before use. This guideline seeks to provide a stepwise approach for the validation of an antibody for immunohistochemical assays, reflecting the views of a consortium of academic and pharmaceutical based histopathology researchers.

Conference Scene: biomarkers: discovery technologies and novel disease applications.

This conference was organized by EuroSciCon and its unifying theme was the application of technology to discover and monitor biomarkers of disease. The meeting was arranged to promote interaction of presenters, and delegates and, in addition to formal presentations, an ask the panel question and answer session was included. Presentations, given by representatives from academia and companies, highlighted the requirement for the use of techniques combining exquisite sensitivity and specificity for application at the genomic and proteomic level in solid and fluid biosamples.

Conference scene: biomarkers in the field of oncology.

This conference was organized by EuroSciCon and its main theme was the development and clinical application of oncology-related biomarkers. The meeting was arranged to promote interaction of presenters and delegates and, in addition to formal presentations, an 'ask the panel' question and answer session was included. Presentations, given by representatives from pharma, academia and diagnostic companies, highlighted the growing importance of co-development of biomarkers with drugs, technology platforms for detecting biomarkers and obtaining the right biosamples to allow their measurement.

Validation and international regulatory experience for a mycoplasma touchdown PCR assay.

A PCR assay was developed to monitor rFVIII production fermenters for mycoplasma contamination. The method uses a simple extraction procedure followed by a qualitative "touchdown" (TD) PCR protocol with primers specific to the 16S rRNA gene. The method has the capacity to detect a wide range of mycoplasma species.

Cellular expression, trafficking, and function of two isoforms of human ULBP5/RAET1G.

Background: The activating immunoreceptor NKG2D is expressed on Natural Killer (NK) cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours.

Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE).

One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments.

Application of phage display to high throughput antibody generation and characterization.

We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online.

Assessing the potential of immunohistochemistry for systematic gene expression profiling.

Immunohistochemistry (IHC) is a powerful technique for identifying sites of protein expression in tissues at the cellular and sub-cellular level. Here we have investigated the potential of using IHC for genome-wide expression screening by measuring the success rate and specificity of a panel of 35 monoclonal antibodies recognizing 5 well characterised CD antigens. Antibodies were pre-screened on acetone fixed frozen sections of spleen, tonsil and colon tissues.

Office practice of adolescent medicine.

Physicians have an important role to play in the health of teenagers who come to their offices. The physician can help teenagers to understand their physical and emotional changes, to talk about sensitive issues in the family,and to identify and reduce some of the risks of adolescent life. The best strategy is to screen each adolescent and offer brief interventions or advice.

Resin tissue microarrays: a universal format for immunohistochemistry.

Tissue microarray (TMA) technology allows the miniaturization and characterization of multiple tissue samples on a single slide and commonly uses formalin-fixed paraffin-embedded (FFPE) tissue or acetone-fixed frozen tissue. The former provides good morphology but can compromise antigenicity, whereas the latter provides compromised morphology with good antigenicity. Here, we report the development of TMAs in glycol methacrylate resin, which combine the advantages of both methods in one embedding format.

Tissue microarrays: fast-tracking protein expression at the cellular level.

Tissue microarrays maximize returns in cellular pathology whilst minimizing the use of cells and tissues. They are made by arraying cores of tissue taken from multiple donor blocks into a single recipient block. Accordingly, the histology and pathology of several hundred tissues can be represented in one tissue microarray that, when stained by immunohistochemistry, provides comprehensive topographic information on protein expression.

Expression profiling by high-throughput immunohistochemistry.

Immunohistochemistry (IHC) provides valuable information on expression of proteins within tissues at a cellular and subcellular level. Recent developments in the practice of IHC now make it possible to contemplate using this technique as a high-throughput expression profiling system. Advances have been made in creation and use of tissue microarrays, in automated IHC and in image capture/analysis.

Comparison of conventional viral cultures with direct fluorescent antibody stains for diagnosis of community-acquired respiratory virus infections in hospitalized children.

Objective: Because of the widespread availability of rapid viral antigen testing, many institutions never adopted a routine practice of ordering viral cultures to detect community-acquired respiratory viruses (CRVs). The ease of performing complete viral studies in our on site laboratory allowed us to assess the clinical implications of the absence of conventional culture results in previously healthy hospitalized children with CRV infections.

Methods: From June 1997 through May 2000, the results of direct immunofluorescence assay (DFA) of 1069 nasopharyngeal swab (NP) specimens were compared with simultaneously inoculated conventional tube cell cultures for detection of CRVs.

Cross-reactivity and clinical impact of the antibody response to hepatitis C virus second envelope glycoprotein (E2).

The genotype of hepatitis C virus (HCV) can profoundly affect the success of antiviral therapy for HCV infection. A possible contributing factor is a varied immune response elicited by infection with different HCV genotypes. In this study, full-length E2 proteins of HCV genotypes 1a, 1b, 2a, and 2b were used to determine the fraction of the humoral immune response to HCV E2 that is genotype specific.

High degree of interlaboratory reproducibility of human immunodeficiency virus type 1 protease and reverse transcriptase sequencing of plasma samples from heavily treated patients.

We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.

Isolation and tissue profiles of a large panel of phage antibodies binding to the human adipocyte cell surface.

Phage display is a powerful technique for the rapid selection and isolation of antibodies to any given target antigen. We have applied this technology to isolate over 100 different human antibodies that bind to antigens expressed in situ on the human adipocyte cell surface. This is a diverse panel of antibodies, as indicated by the V-region sequences.

Reproducibility of human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase sequencing of plasma samples from heavily treated HIV-1-infected individuals.

The reproducibility of population-based human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT) sequencing was assessed using replicate aliquots of cryopreserved plasma samples obtained from seven heavily treated HIV-1-infected individuals. The sequence of each sample replicate was compared with the consensus sequence for that sample and 99.4% of 35128 amino acids were found to be concordant with the sample consensus.

HIV-1 genotypic resistance patterns predict response to saquinavir-ritonavir therapy in patients in whom previous protease inhibitor therapy had failed.

Background: Tests for resistance to HIV drugs are available for clinical use; however, their predictive value has not been fully assessed.

Objectives: To determine HIV-1 genotypic predictors of a virologic response to saquinavir-ritonavir therapy in patients in whom at least one previous protease inhibitor-containing regimen had failed and to compare the predictive value of baseline genotype with that of standard clinical evaluation.

Design: Retrospective clinical cohort study.

Cytomegalovirus gB genotype distribution differs in human immunodeficiency virus-infected patients and immunocompromised allograft recipients.

Cytomegalovirus isolates can be grouped into 4 gB and 2 gH genotypes. gB genotypes were studied in patients infected with human immunodeficiency virus (HIV) and in allograft transplantation recipients. In allograft recipients, the distribution of gB 1, -2, -3, and -4 in leukocytes and urine, respectively, was 36%, 21%, 43%, and 0% and 39%, 30%, 17%, and 13%.