Needs for an expanded ontology-based classification of adverse drug reactions and related mechanisms.

The growing significance of bioinformatics and systems biology in drug safety research requires a system of adverse-event classification that goes beyond a simple vocabulary. This opinion piece outlines the need for development of an ontology-based framework of describing adverse drug reactions (ADRs) and describes the potential applications for such a framework.

Case definition and phenotype standardization in drug-induced liver injury.

Drug-induced liver injury (DILI) is the most frequent reason cited for the withdrawal of approved drugs from the market and accounts for up to 15% of the cases of acute liver failure. Investigators around the globe have begun to identify and study patients with DILI; several large registries and tissue banks are being established. In order to gain the maximum scientific benefit from these efforts, the definitions and terminology related to the clinical phenotypes of DILI must be harmonized. For this purpose, an international DILI Expert Working Group of clinicians and scientists reviewed current DILI terminology and diagnostic criteria so as to develop more uniform criteria that would define and characterize the spectrum of clinical syndromes that constitute DILI. Consensus was established with respect to the threshold criteria for definition of a case as being DILI, the pattern of liver injury, causality assessment, severity, and chronicity. Consensus was also reached on approaches to characterizing DILI in the setting of chronic liver diseases, including autoimmune hepatitis (AIH).

Clinically significant liver injury in patients treated with natalizumab.

BACKGROUND: Natalizumab is a recombinant monoclonal antibody approved for the treatment of patients with multiple sclerosis and patients with Crohn's disease. Because of its immunosuppressive effects, natalizumab has been associated with a number of atypical and opportunistic infections. AIM: To describe and summarize six spontaneously reported post-marketing cases of clinically significant drug induced-liver injury associated with natalizumab use. METHODS: The FDA maintains a database of adverse event reports (AERS). We searched the AERS database for reports of serious liver injury associated with natalizumab use from November 2004, when the drug was approved, through 30 June 2008. RESULTS: The search resulted in six spontaneously reported post-marketing cases of severe drug-induced liver injury. Four of six patients developed liver injury with elevations of serum transaminases and hyperbilirubinemia after only a single infusion of natalizumab. One of these patients experienced repeated increases of aminotransferases and bilirubin when natalizumab was re-administered. CONCLUSIONS: Serious hepatic injury may occur in association with natalizumab use. Health professionals should be alerted to possible serious liver injury in patients receiving natalizumab.

A src-related kinase in the brush border membranes of gastrointestinal cells is regulated by c-met.

Hepatocyte growth factor (HGF) elicits pleiotropic cellular responses by binding to c-met, a PTK transmembrane receptor. The recent identification of HGF in fluids which enter the gut lumen suggests a mechanism by which c-met molecules are accessible to ligand that is present near the apical surfaces of polarized enterocytes. A subset of c-met molecules was detected, by confocal and immunoelectron microscopic analysis, which colocalizes with a recently identified src-related gastrointestinal tyrosine kinase (gtk) in the brush border membranes of enterocytes. Furthermore, treatment of c-met/gtk-transfected cells with a chemical cross-linking agent revealed that c-met forms a physical complex with gtk, in vivo. Not surprisingly, activation of the receptor molecules with HGF rapidly stimulated gtk enzymatic activity. Similarly, the stimulation of gtk activity occurred when nontransfected primary hepatocytes were exposed to ligand. These findings suggest a model in which HGF binding to luminally accessible c-met stimulates gtk activity. This brush border-associated c-met-linked pathway may be associated with a defined set of epithelial cell responses.

FUSE-binding protein is developmentally regulated and is highly expressed in mouse and chicken embryonic brain.

GAP-43 modulates axon guidance and neuronal plasticity. In vitro, FUSE-binding protein (FBP) binds to a segment of GAP-43 mRNA which regulates the stability of the transcript. FBP has also been shown to bind to a c-myc cis element and regulate transcription. In the current work, analysis of RNA and protein expression indicated that FBP is expressed in a distinct spatial temporal pattern during embryonic development. Expression was particularly high in the brain. In the adult, expression was not detected in most tissues but was still prominent in the brain and teste. This finding is consistent with a dual role of the protein as a single-strand polynucleotide-binding protein.

GATA-6 stimulates a cell line-specific activation element in the human lactase promoter.

Lactase-phlorizin hydrolase (LPH) synthesis is restricted to differentiated small intestinal enterocytes and is highly regulated during development. Analysis of expression of LPH promoter segments fused with luciferase transfected in Caco-2 cells, a line that uniquely expresses LPH mRNA, mapped an 18-base pair (bp) segment 100 bp upstream of the transcription start site that is required for transactivation. Remarkably, the LPH upstream element (LUE) has no stimulatory activity in both human intestinal and nonintestinal lines in which LPH mRNA is absent. Electrophoretic analysis of sequence-specific DNA-nuclear protein complexes demonstrated the presence of a Caco-2 cell-specific protein(s) (CCP), which is uniformly absent in LPH nonproducer cell lines. Mutational analysis of the LUE demonstrated that bases contained within a GATA consensus motif are critical for both CCP binding and transcription from the LPH promoter. Caco-2 cells express high levels of GATA-6 mRNA in a cell line-specific manner, suggesting that GATA-6 is a CCP that complexes with the LUE. When expressed by a plasmid, GATA-6 transactivated the LPH promoter. The stimulation was abrogated with mutations in the GATA consensus motif as well as mutations in a flanking downstream element. These studies are consistent with an important role of an intestinal GATA binding protein in cell type-specific transactivation of the LPH promoter.

A new system for defining endoscopic complications emphasizing the measure of importance.

BACKGROUND: Currently, there are no satisfactory systems for defining, classifying, and/or scoring endoscopic complications, although it would be important for quality assurance, comparative studies, and outcomes research. Recently the term "negative outcomes" was proposed rather than "complications," and an approach that incorporates "measures of importance" was added to compare negative outcomes. METHODS: A system was developed that defines, classifies, and grades negative outcomes with a scoring system based on measures of importance. Information was recorded on a Morbidity and Mortality (M & M) form, which was used at a monthly quality assurance (M & M) conference. Several measures of importance related to the immediate negative outcome (O) were quantified (effect of the complication on completion of the endoscopy, change in level of care, change in number of hospital days, necessity for new invasive procedures). The disability (D), defined as a residual or chronic negative outcome caused by the complication, was characterized and scored. Death (D) was also characterized, the value varying with circumstances. As a quantitative measure, an overall ODD score was used. RESULTS: One hundred twenty-three negative outcomes were retrospectively classified using the new M & M form and the ODD score was applied for 117 complications. Complications were ranked according to the ODD score. CONCLUSION: A system for defining, classifying, and grading negative outcomes of endoscopic procedures is proposed with a quantitative scoring system that emphasizes measures of importance. The ODD score looks at the immediate negative outcome and also the separate long-term issues of disability and death.

The apical membranes of maturing gut columnar epithelial cells contain the enzymatically active form of a newly identified fyn-related tyrosine kinase.

Recently, we isolated a new src family member from a rat small intestinal cDNA library which by RNase protection analysis is selectively expressed in the columnar epithelium of gut. Complete nucleotide sequencing of the gastrointestinal associated tyrosine kinase (gtk) has revealed that it is a rat homologue of frk/rak-a fyn related human tyrosine kinase. Unlike frk/rak, gtk is myristylated, in vivo. Furthermore, by immunohistochemical analysis, the kinase is concentrated in the brush border membranes of epithelial cells, throughout the maturation axis of the adult small intestine. In vitro analysis revealed that gtk kinase activity is present in intestinal cells throughout their maturation, suggesting that the enzyme might influence signal transduction pathways in both mitotic and post-mitotic states. Gtk is expressed in all regions of the gastrointestinal tract which contain columnar epithelium, but is absent in the stratified epithelium of the esophagus. Moreover, during gestation, the kinase dramatically appears at high levels in plasma membranes, at the time of transition of gut cells from an undifferentiated to a simple columnar phenotype. After solubilization of cellular membranes with Triton X-100, sucrose gradient analysis of gtk revealed that it partitions differently than c-yes, demonstrating that the brush border src kinases associate with different components of the plasma membranes. These findings suggest that gtk plays a specialized role in the growth/differentiation of gut columnar epithelial cells.

High-resolution chromoendoscopy for the diagnosis of diminutive colon polyps: implications for colon cancer screening.

BACKGROUND & AIMS: A visual, nonbiopsy technique that could reliably determine the histology of diminutive colorectal polyps could greatly reduce the cost of colon cancer screening. This study was designed to report our experience using a high-resolution colonoscope combined with indigo carmine dye to diagnosis diminutive colorectal polyps. METHODS: Colonoscopy using a Fujinon EC-400 HM/HL was performed in 36 patients with polyps <10mm in diameter. Polyps from the first 12 patients (phase 1) were sprayed with 10 mL of 0.2% indigo carmine dye, and a biopsy was performed or a specimen removed and submitted for histological analysis. The morphological data were used to predict polyp histology in the subsequent 24 patients (phase 2). RESULTS: Hyperplastic polyps had a characteristic surface "pit pattern" of orderly arranged "dots" that resembled the surrounding, nonpolypoid mucosa. Adenomatous polyps had surface "grooves" or "sulci." Sensitivity and specificity of our techniques in distinguishing adenomatous from nonadenomatous colorectal polyps were 93% and 95% respectively. CONCLUSIONS: High-resolution chromoendoscopy provides morphological detail of diminutive colorectal polyps that correlates well with polyp histology. If incorporated into colon cancer screening, these techniques may limit the need for biopsy and/or subsequent colonoscopy and ultimately decrease costs.

A transactivator of c-myc is coordinately regulated with the proto-oncogene during cellular growth.

A recently cloned nuclear protein, which binds a far upstream element (FUSE) of the human c-myc proto-oncogene, stimulates promoter driven expression in undifferentiated cells. In concert with a loss of c-myc expression, both FUSE binding protein (FBP) mRNA and protein levels disappeared in HL60 cells after PMA-induced differentiation, due to a drop in the rate of transcription that was measured by nuclear runoff. During the differentiation of these cells, the brief half-lives of FBP mRNA (3 h) and protein (1.5 h) did not change, allowing for the rapid down-regulation of nuclear protein levels, as detected by immunohistochemical staining. Like c-myc, FBP is expressed in proliferating cells from a variety of lineages, but not in quiescent cells. When T cells and fibroblasts were stimulated to transit from G0 into the cell cycle, there was a dramatic rise of both FBP mRNA and DNA sequence specific nuclear FBP binding activity, which correlated with the appearance of c-myc mRNA. In contrast to the transient expression of many other immediate early growth response genes, both FBP and c-myc expression were sustained for more than 24 h. In fibroblasts, the coordinate expression of FBP and c-myc throughout all phases of the cell cycle is consistent with FBP's role as a growth-dependent regulator of c-myc expression.

Targeted melting and binding of a DNA regulatory element by a transactivator of c-myc.

A far upstream element (FUSE) of c-myc stimulates promoter activity when bound by a newly identified trans-acting protein, which is expressed in cycling cells. Since FUSE binding protein (FBP) binds only the noncoding strand (NCS) of its regulatory element in a sequence-specific manner, and not double-stranded (ds) DNA, formation of the protein DNA complex in vivo first requires unwinding of the DNA helix. In this report, we show evidence that FBP forces strand separation of short stretches of linear dsDNA. Because FUSE is contained within a region of helical instability that is partially unwound in negatively supercoiled DNA, it is a target for more extensive duplex strand separation by FBP, which first exposes and then selectively binds its NCS cognate sequence. In contrast, other single-stranded DNA binding proteins (SSBs) do not demonstrate this FUSE targeting activity. The novel linkage of regional dsDNA melting with cis-element binding by a transcriptional activator has broad implications in the regulation of eukaryotic gene expression.

A newly identified tyrosine kinase is preferentially expressed in the gastrointestinal tract.

We have identified a novel protein tyrosine kinase, cloned from rat intestinal mRNA, which is selectively expressed at high levels in intestinal cells, but is expressed at low or insignificant levels in a variety of other epithelial and non-epithelial organs. Gastrointestinal associated kinase (GASK) mRNA is present at high levels in stomach, small intestine and colon. GASK may play an important role in the growth, differentiation and/or development of the gastrointestinal tract.

Hepatocyte growth factor stimulates invasion across reconstituted basement membranes by a new human small intestinal cell line.

Hepatocyte growth factor/scatter factor (HGF/SF) is a protein growth factor whose pleiotropic effects on epithelial cells include the stimulation of motility, mitosis and tubulogenesis. These responses are mediated by the cell surface tyrosine kinase receptor c-met. Because both the cytokine and receptor are found in the gastrointestinal tract, we have studied the effects of HGF/SF on transformed gut epithelial cells which express c-met. Here we describe the response of a new transformed human jejunal epithelioid cell line (HIE-7) to HGF/SF. Morphologically HIE-7 cells are immature. Their epithelial lineage was confirmed by reactivity with the epithelial specific antibodies AE1/AE3, Cam 5.2, Ber-EP4 and anti-EMA and is consistent with their expression of c-met mRNA and protein. In addition, electron microscopic analysis revealed the presence of primitive junctions and rudimentary microvilli, but features of polarization were absent. When grown on reconstituted basement membranes, HIE-7 cells formed closely associated multicellular cord-like structures adjacent to acellular spaces. However, the cells did not mature structurally, form lumen-like structures or express disaccharidase mRNA, even in the presence of recombinant HGF (rHGF). On the other hand, rHGF induced HIE-7 cells to scatter and stimulated their rapid migration in a modified wound assay. To determine whether the mitogenic effect caused by rHGF is associated with HIE-7 cell invasiveness across reconstituted basement membranes, a Boyden chamber chemoinvasion assay was performed. rHGF stimulated a 10-fold increase in the number of HIE-7 cells that crossed the basement membrane barrier, while only stimulating a small increase in chemotaxis across a collagen IV matrix, suggesting that the cytokine activates matrix penetration by these cells. rHGF also stimulated the invasion of basement membranes by an undifferentiated rat intestinal cell line (IEC-6) and by two human colon cancer cell lines which are poorly differentiated (DLD-1 and SW 948). In contrast, two moderately well differentiated colon cancer cell lines (Caco-2 and HT-29) did not manifest an invasive response when exposed to rHGF. These results suggest that HGF/SF may play a significant role in the invasive behavior of anaplastic and poorly differentiated gut epithelial tumors.

A sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new DNA-binding motif.

The far upstream element (FUSE) of the human c-myc proto-oncogene stimulates expression in undifferentiated cells. A FUSE-binding protein (FBP) is present in undifferentiated but not differentiated cells. Peptide sequences from the purified protein allowed cloning of cDNAs encoding FBP. Expression of FBP mRNA declined upon differentiation, suggesting transcriptional regulation of FBP. Features in the FBP cDNA suggest that FBP is also regulated by RNA processing, translation, and post-translational mechanisms. Both cellular and recombinant FBP form sequence-specific complexes with a single strand of FUSE. Transfection of FBP into human leukemia cells stimulated c-myc-promoter-driven expression from a reporter plasmid in a FUSE-dependent manner. Deletion and insertion mutagenesis of FBP defined a novel single-strand DNA-binding domain. Analysis of the primary and predicted secondary structure of the amino acid sequence reveals four copies of a reiterated unit comprised of a 30-residue direct repeat and an amphipathic alpha-helix separated by an 18- to 21-residue spacer. The third and fourth copies of this repeat-helix unit constitute the minimum single-stranded DNA-binding domain. To determine whether the FUSE site, in vivo, possesses single-strand conformation, and therefore could be bound by FBP, cells were treated with potassium permanganate (KMnO4) to modify unpaired bases. Modification of genomic DNA in vivo revealed hyperreactivity associated with single-stranded DNA in the FUSE sequence and protection on the strand that binds FBP in vitro. The role of single-stranded DNA and single-strand binding proteins in c-myc regulation is discussed.

Specific binding of heterogeneous ribonucleoprotein particle protein K to the human c-myc promoter, in vitro.

A homopurine/homopyrimidine-like sequence is found 100-150 base pairs upstream of the human c-myc promoter P1. This element, termed the CT-element, has been shown to augment expression from P1, and it serves as a positive transcriptional element when coupled to a heterologous promoter in vivo and in vitro. Synthetic oligonucleotides comprising this element were used to form DNA-protein complexes in electrophoretic mobility shift assays. By using conventional and affinity methods, 61- and 34-kDa proteins were shown to be associated with these complexes. Amino acid sequence analysis and immunological methods have identified these proteins as heterogeneous ribonucleoprotein particle (hnRNP) proteins K and A1. Surprisingly, hnRNP protein K binds to the pyrimidine-rich strand of the CT-element in a sequence-specific manner as well as to the double-stranded molecule. Cotransfection of vectors encoding hnRNP protein K in the sense or anti-sense orientations with reporter plasmids driven by wild-type or mutant CT-elements demonstrates that hnRNP protein K augments gene expression in a cis-element-dependent manner. Taken together, these results suggest that hnRNP protein K may play a role in the transcriptional regulation of the human c-myc gene.

A far upstream element stimulates c-myc expression in undifferentiated leukemia cells.

A sensitive exonuclease assay revealed multiple sites for interaction, in vitro, of sequence specific factors with c-myc upstream elements. At one site, more than 1500 base pairs upstream of the c-myc promoter P1, binding activity was lost as dimethyl sulfoxide (Me2SO) induced shut-off of c-myc expression in HL-60 and U-937 leukemia cells. The disappearance of other specific binding activities was not noted. In addition, the binding activity was noted to be cell-line specific. The sequence binding the Me2SO-regulated factor was precisely located allowing confirmation of the temporal pattern of regulation by electrophoretic mobility shift analysis. Because the binding activity was most abundant before the decrease of c-myc expression during differentiation, it was inferred that the far upstream element (FUSE) served a positive role, potentiating c-myc expression. A 4-base pair deletion which eliminated binding to FUSE also reduced expression of a transfected, chimeric c-myc-CAT gene in untreated, but not in Me2SO-treated U-937 cells. FUSE and its binding protein may contribute to cell line- and differentiation-specific modes of c-myc regulation.