Industrial-scale, genomics-based drug design and discovery.

The demands on drug discovery organizations have increased dramatically in recent years, partly because of the need to identify novel targets that are both relevant to disease and chemically tractable. This is leading to an industrial approach to traditional biology and chemistry, inspired in part by the revolution in genomics. The purpose of this article is to highlight the flow of investigation from gene sequence of potential therapeutic targets, through mRNA and protein expression, to protein structure and drug design. To deal with this scale of activity, many commercial and public organizations have been established and some of the key players will be listed in this article.

An AP-1 site in the promoter of the human IL-5R alpha gene is necessary for promoter activity in eosinophilic HL60 cells.

Interleukin-5 (IL-5) plays a crucial role in the proliferation, differentiation and activation of eosinophils. The IL-5 receptor is composed of an IL-5-specific alpha subunit, which is expressed by eosinophils and basophils, and a beta c-subunit shared with the receptors for IL-3 and GM-CSF. We identified an AP-1 element which is important for IL-5R alpha promoter activity in eosinophilic HL60 cells. The AP-1 site and the previously identified EOS1 site cooperate, since single mutation of either of the sites decreased promoter activity. We show that the AP-1 site of the IL-5R alpha promoter binds multiple proteins, including cJun, CREB, and CREM.

Methods and approaches in the analysis of gene expression data.

The application of high-density DNA array technology to monitor gene transcription has been responsible for a real paradigm shift in biology. The majority of research groups now have the ability to measure the expression of a significant proportion of the human genome in a single experiment, resulting in an unprecedented volume of data being made available to the scientific community. As a consequence of this, the storage, analysis and interpretation of this information present a major challenge. In the field of immunology the analysis of gene expression profiles has opened new areas of investigation. The study of cellular responses has revealed that cells respond to an activation signal with waves of co-ordinated gene expression profiles and that the components of these responses are the key to understanding the specific mechanisms which lead to phenotypic differentiation. The discovery of 'cell type specific' gene expression signatures have also helped the interpretation of the mechanisms leading to disease progression. Here we review the principles behind the most commonly used data analysis methods and discuss the approaches that have been employed in immunological research.

A micromethod for the induction and assay of specific in vitro antibody responses by human lymphocytes.

Human lymphocytes obtained from peripheral blood or spleen were cultured with influenza virus in a microculture system to produce strain specific antibody which was detected by the enzyme linked immunosorbent assay (ELISA). Nanogram amounts of specific antibody could be detected in cultures of 4 x 10(5) cells.

Analysis of immune system gene expression in small rheumatoid arthritis biopsies using a combination of subtractive hybridization and high-density cDNA arrays.

Subtractive hybridization of cDNAs generated from synovial RNA which had been isolated from patients with rheumatoid arthritis (RA) or normal controls was used in conjunction with high-density array hybridization to identify genes of immunological interest. The method was designed to detect gene expression in small needle biopsy specimens by means of a prior amplification of nanogram amounts of total RNA to full-length cDNA using PCR. The latter was cut with Rsa I, ligated with adapters, hybridized with unmodified driver cDNA, and subjected to suppression subtraction PCR. Differentially expressed products were cloned into E. coli and picked into 384 well plates. Inserts were obtained by PCR across the multiple cloning site, and the products arrayed at high density on nylon filters. The subtracted cDNAs were also labelled by random priming for use as probes for library screening. The libraries chosen were the subtracted one described above and a set of 45,000 ESTs from the I.M. A.G.E consortium. Clones showing positive hybridization were identified by sequence analysis and homology searching. The results showed that the subtracted hybridization approach could identify many gene fragments expressed at different levels, the most abundant being immunoglobulins and HLA-DR. The expression profile was characteristic of macrophage, B cell and plasma cell infiltration with evidence of interferon induction. In addition, a significant number of sequences without matches in the nucleotide databases were obtained, this demonstrates the utility of the method in finding novel gene fragments for further characterisation as potential members of the immune system. Although RA was studied here, the technology is applicable to any disease process even in cases where amounts of tissue may be limited.

Gene expression analysis as an aid to the identification of drug targets.

Many drug targets are components of complex signalling pathways, and in order to understand the true biological consequences of modulating these targets it is necessary to understand the biology of the system in great detail. Genomics research can contribute some of the tools to achieve this, for example through the use of cDNA microarrays. Since activation of signalling pathways leads to mRNA expression, microarray technology can be used to provide a detailed quantitative assessment of the consequences of this activation, often providing a completely new biological perspective on well established cellular systems. This review will discuss some of the results obtained using mRNA profiling of yeast and mammalian cells to analyse signalling pathways of relevance to inflammation and cancer, and will point towards the future applications of this exciting approach to drug target evaluation.

Cambridge Healthtech Institute's 5th Annual Conference: impact of genomics on medicine.

The recent publications in Nature and Science by the Human Genome Consortium and Celera Genomics, respectively, while being landmark achievements in themselves, have also given pause for thought. A definitive catalogue of human genes is still not available but the broad picture of how humans compare with lower organisms at the genomic level is becoming clearer. The full impact of these findings on the practice of medicine is hard to predict, but research being conducted now, in the early years of the 21st century, will form the basis of future advances in the diagnosis and treatment of disease. Exactly what this will entail is the subject of intense debate, but there are some common starting points that were discussed at this meeting in Munich. The main theme to emerge was the need to move beyond the human genome sequence towards an understanding of proteins and their interactions in complex biological pathways, thereby increasing opportunities for drug discovery through the identification of new targets. The majority of the talks were therefore devoted to the description of technological advances in the analysis of gene and protein expression (and interaction) and in the use of various methods of gene deletion in order to validate individual proteins as drug targets. Perhaps it will still be a few years before it will be possible to report on the application of genomic analyses to routine medical practice at the first point of care for patients but when that happens, the research efforts described here will have been worthwhile.

Interleukin-5 receptor alpha chain mRNA is down-regulated by transforming growth factor beta 1.

Interleukin-5 is a T cell-derived cytokine with actions restricted to the eosinophil/basophil lineage and a subset of murine B cells. High affinity receptors have been identified and shown to comprise an IL-5-specific alpha chain (IL-5R alpha) in association with a beta chain which is shared with the receptors for IL-3 and GM-CSF. Nothing is currently known of the factors which regulate the transcription and subsequent expression of the IL-5 receptor alpha chain; this study was undertaken, therefore, in order to identify agents which modulate IL-5R alpha mRNA levels, with the goal of understanding the regulation of this gene in vivo. The human IL-5-dependent erythroleukemia TF-1 was used as a source of mRNA which was analysed by northern blotting using a cDNA probe for IL-5R alpha. A range of cytokines and pharmacological agents were used in 20 hour cultures of TF-1 followed by northern analysis. Of these, only TGF-beta 1 and PMA showed any effect, which was a selective downregulation, although the PMA displayed some cytotoxicity over the long culture period. The remainder (interleukins 1 to 11, G-CSF, GM-CSF, LIF, SCF, erythropoetin, IFN-gamma, RANTES, MIP-1 alpha, FGF, EGF, PDGF, dexamethasone, forskolin, retinoic acid and cyclosporin A) failed to alter expression.(ABSTRACT TRUNCATED AT 250 WORDS)

[Influence of visual cues on upright postural control: differentiated effects of eyelids closure]. / Influence des informations visuelles sur le contrôle de la posture debout: effet différencié de la fermeture des paupières.

In most protocols aimed at testing balance abilities, patients are generally required to close their eyes in order to gain insight about proprioceptive cues and the way the central nervous system (CNS) uses this information. However, one should not exclude possible interaction with the physiological mechanisms involved in eyelid closure, thus leading to a biased neurological evaluation. To assess this possible involvement, 15 healthy adults were required to keep their eyes open in the dark (YOn), to close normally (YF) and forcibly their eyelids (YFF), respectively in random order. The analysis was focused on elementary motions computed from the complex center of pressure (CP) trajectories, i.e. the horizontal motions of the center of gravity (CG(h)) and the difference between the CP and the vertical projection of the center of gravity (CP-CG(v)). The former is recognized as the main variable in postural control whilst several interesting features can be extracted from the latter: their link with the horizontal accelerations communicated to the CG, the level of muscular activity and the implied ankle stiffness expressed by their frequential distribution. The results indicate that the amplitudes of the CP-CG(v) spectra are statistically reduced in YOn when compared to the YF condition, especially in the antero-posterior direction. On the other hand, no shift in the frequential bandwidth was observed on these spectra, signifying a constancy in ankle stiffness over all conditions. Interestingly, the CNS does not really seem to gain from these reduced horizontal accelerations at the CG level since the CG(h) amplitudes are only slightly reduced. However, it is important to emphasize that the CP trajectories alone are not able to demonstrate any statistical trend. It can thus be hypothesized that, despite the fact that visual information is still unavailable, the proprioceptive cues nonetheless continue to play a minor role in the detection-correction process aimed at controlling body sway. Past studies on the physiology of eyes blinking have suggested the probability that the cerebellar cortex or brain stem structures (such as the reticular formation) are involved in these commands. Because of its dual facilitating-inhibiting function, the latter is indeed a fair candidate for modulating the descending command operating through the postural muscles. These data are of interest for the practician in order to assess with precision the role played by proprioceptive and visual cues in possible balance disfunctioning.

Separation and characterization of a protein antigen from cells of Streptococcus mutans.

A protein antigen, I/II, was purified from cells and culture supernatants of Streptococcus mutans (serotype c) by solubilization in urea followed by ion exchange chromatography and gel filtration. Immunological activity was retained after further purification by preparative sodium dodecyl sulphate--polyacrylamide gel electrophoresis (SDS--PAGE). The sedimentation coefficient was estimated to be approximately 8.7S by sucrose gradient centrifugation. The use of staining procedures, as well as the linear migration of this protein through different concentrations of acrylamide during SDS--PAGE, indicated that the antigen is probably not a glycoprotein. A lower molecular weight protein containing the free antigen I determinant was shown to have extensive homology with intact antigen I/II which implied that the former was a degradation product of the intact 185 000 dalton antigen I/II. Antigen II, although previously defined by its resistance to proteases, could be further digested with trypsin after denaturation by SDS--PAGE. Antigen I/II could not be correlated with a group of glucosyltransferases isolated from whole cells and culture supernatants. The cell surface location of antigen I/II was established by the lactoperoxidase-catalysed iodination of intact cells followed by protein analysis using SDS--PAGE. Previously, the potential importance of antigen I/II has been established by its immunogenicity and capacity to induce a protective immune response against dental caries.

Site-specific molecular design and its relevance to pharmacogenomics and chemical biology.

The emergence of the new discipline of pharmacogenomics reflects the growing convergence of chemical and genomic space. The massive information-driven growth in both computational chemistry and structural biology is leading to unprecedented opportunities in both chemical and biological design. In this paper we relate current opinion in structural biology to recent developments in computational drug design. Sequence information now permits protein structure prediction and, together with experimental protein structure determination, a complete database of ligand-binding sites and protein-protein interactions can be assembled. When aligned with site exploration and virtual screening, this information provides a foundation for structure-based pharmacogenomics. In association with chemical genomics, structure-based design will allow major new insights into a compound's biological and pharmaceutical properties.

Inositol lipid metabolism in human T lymphocytes activated via the T3 complex.

Cloned human T lymphocytes and a mitogenic monoclonal antibody (UCHT1) that binds to the T3 antigen complex were used to study the role of inositol lipid hydrolysis in T-cell activation. Binding of the T3 molecular complex with anti-T3 antibody induced the generation of inositol trisphosphate after a lag of 1 min. While this is commensurate with the rise in cytosolic Ca2+ in these cells, examination of the inositol lipid revealed that phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-bisphosphate occurred before the generation of inositol trisphosphate. Thus, activation of an inositol lipid kinase appears to be one of the primary signals in cellular activation in human T lymphocytes.

Biochemical events initiated by exposure of human T lymphocyte clones to immunogenic and tolerogenic concentrations of antigen.

Interleukin 2-dependent helper T cells, cloned from human peripheral blood lymphocytes activated with strain A influenza virus hemagglutinin, proliferate in response to a 24-residue synthetic peptide (p20) of hemagglutinin, but become unresponsive to a subsequent immunogenic challenge when pretreated with a high concentration of p20. This phenomenon is associated with a loss of the T3 antigen complex, presumably in association with the T cell receptor. We have examined this phenomenon in more detail and show that in addition to changes in the expression of T3 molecules on the cell surface, high doses of p20 cause changes in the expression of certain biosynthetically and surface-labeled proteins, although total DNA and protein synthesis was unaltered. Thus, by examining these biochemical phenomena we can begin to define some of the processes which occur during antigen activation of human T lymphocytes at the clonal level.

Tolerance in T-cell clones.

The interaction of antigen with immunologically competent cells may lead either to the induction of an immune response or to a state of antigen specific unresponsiveness complete orpartial, which is often called immunological 'tolerance'. This is believed to be a major mechanism of discrimination between self and non-self(1-3). In this review Marc Feldmann and his colleagues discuss new insights into T-cell tolerance in vivo and in vitro, and discuss their significance for our understanding of mechanisms of immune activation and regulation.

Secondary immunoglobulin responses of BALB/c mice previously stimulated with goat anti-mouse IgD.

Intravenous injection of goat antibodies to mouse IgD (GAMD) into BALB/c mice has been shown to induce vigorous T-cell dependent immunoglobulin responses, particularly of the IgG1 and IgE isotypes. We have confirmed these findings and show that IgA responses are also triggered in this model. Since the study of IgE regulation in allergic individuals is concerned with secondary and subsequent T- and B-cell responses, we boosted GAMD-primed mice with goat antibodies to IgE or IgA in an attempt to specifically retrigger IgE- and IgA-bearing memory B cells. However, we found that secondary IgG1, IgE and IgA production could be elicited equally well by either antibody preparation or by normal goat IgG (GIg). As with the primary response, GIg primed and boosted mice produced very low or undetectable IgG1, IgE and IgA responses. These data suggest that GAMD is very efficient at priming T cells specific for GIg epitopes and that once primed they can be readily re-triggered by GIg. Spleen cells taken 7 days after boosting GAMD-primed mice were found to spontaneously produce much higher levels of interleukin-6 (IL-6) in culture than cells from unboosted or GIg primed and boosted mice. In contrast to primary responses, where IgE levels return to background (less than 40 ng/ml) very quickly, circulating IgE levels in boosted mice initially declined before reaching a plateau level (approximately 1 microgram/ml) which was maintained for at least 148 days. IgG1 and IgA levels continued to fall over this same time period. Mice which had been primed (but not boosted) 10 months earlier were all found to have detectable IgE in their blood, despite the fact that following priming IgE becomes undetectable within 2-3 weeks. Since only a part of the IgE response was directed towards the antigen (GIg), these observations suggest the possibility that B cells initially primed to make IgE can be non-specifically retriggered in vivo.