Faculty Bibliography

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19-21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work

Two proteins, organic solute transporter (OST) alpha and beta, have recently been identified that mediate bile acid export from the ileal enterocyte. It is unclear whether these two proteins associate directly and how they interact to mediate transport function and membrane localization. In this study, the protein-protein interaction, transport function and membrane localization of human OST-alpha and beta proteins were examined. The results demonstrated that the co-expression of hOST-alpha and hOSTbeta in transfected cells resulted in a 3-5 fold increase of the initial rate of taurocholate influx or efflux compared with cells expressing each protein individually and non-transfected cells. Confocal microscopy demonstrated plasma membrane co-localization of hOST-alpha and hOSTbeta proteins in cells co-transfected with hOST-alpha and hOSTbeta cDNAs. Protein-protein interaction between hOST-alpha and hOSTbeta was demonstrated by mammalian two-hybrid and co-immunoprecipitation analyses. Truncation of the amino-terminal 50 amino acid extracellular residues of hOST-alpha abolished its interaction with hOSTbeta and led to an intracellular accumulation of the two proteins and to only background levels of taurocholate transport. In contrast, carboxyl-terminal 28 amino acid truncated hOST-alpha still interacted with hOSTbeta and majority of this cytoplasmic tail truncated protein was expressed on the basolateral membrane when stably co-transfected with hOSTbeta protein in MDCK cells. In summary, human OST-alpha and beta proteins are physically associated. The intracellular carboxyl-terminal domain of human OST-alpha is not essential for this interaction with hOSTbeta. The extracellular amino-terminal fragment of human OST-alpha may contain important information for assembly of the heterodimer, and trafficking to the plasma membrane. Key words: bile acid transporter, protein interaction, membrane localization

Invasive fungal infections cause morbidity and mortality in severely ill patients, and limited drug classes restrict treatment choices. The echinocandin drugs are the first new class of antifungal compounds that target the fungal cell wall by blocking beta-1,3-d-glucan synthase. Elevated MIC values with occasional treatment failure have been reported for strains of Candida. Yet, an uncertain correlation exists between clinical failure and elevated MIC values for the echinocandin drugs. Fungi display several adaptive physiological mechanisms that result in elevated MIC values. However, resistance to echinocandin drugs among clinical isolates is associated with amino acid substitutions in two "hot-spot" regions of Fks1, the major subunit of glucan synthase. The mutations, yielding highly elevated MIC values, are genetically dominant and confer cross-resistance to all echinocandin drugs. Prominent Fks1 mutations decrease the sensitivity of glucan synthase for drug by 1000-fold or more, and strains harboring such mutations may require a concomitant increase in drug to reduce fungal organ burdens in animal infection models. The Fks1-mediated resistance mechanism is conserved in a wide variety of Candida spp. and can account for intrinsic reduced susceptibility of certain species. Fks1 mutations confer resistance in both yeasts and moulds suggesting that this mechanism is pervasive in the fungal kingdom.

Recent advances in cancer cell biology have focused on histone deacetylase inhibitors (HDACi's) because they target pathways critical to the development and progression of disease. In particular, HDACi's can induce expression of epigenetically silenced genes that promote growth arrest, differentiation and cell death. In glioma cells, one such repressed gene is the tetraspanin CD81, which regulates cytostasis in various cell lines and in astrocytes, the major cellular component of gliomas. Our studies show that HDACi's, trichostatin and sodium butyrate, promote growth arrest and differentiation with negligible cell death in glioma cells and induce expression of CD81 and cyclin-dependent kinase inhibitor 1A (p21(CIP/WAF-1)), another regulator of cytostasis in astrocytes. Interference RNA knock-down of CD81 abrogates cytostasis promoted by HDAC inhibition indicating that HDACi-induced CD81 is responsible for growth arrest. Induction of CD81 expression through HDAC inhibition is a novel strategy to promote growth arrest in glioma cells.

The invasion of erythrocytes by Plasmodium falciparum occurs through multiple pathways that can be studied in vitro by examining the invasion of erythrocytes treated with enzymes such as neuraminidase, trypsin, and chymotrypsin. We have studied the invasion pathways used by 31 Kenyan P. falciparum isolates from children with uncomplicated or severe malaria. Six distinct invasion profiles were detected, out of eight possible profiles. The majority of isolates (23 of 31) showed neuraminidase-resistant, trypsin-sensitive invasion, characteristic of the pathway mediated by an unknown parasite ligand and erythrocyte receptor "X." The neuraminidase-sensitive, trypsin-sensitive phenotype consistent with invasion mediated by the binding of parasite ligand erythrocyte binding antigen 175 to glycophorin A, the most common invasion profile in a previous study of Gambian field isolates, was seen in only 3 of 31 Kenyan isolates. No particular invasion profile was associated with severe P. falciparum malaria, and there was no significant difference in the levels of inhibition by the various enzyme treatments between isolates from children with severe malaria and those from children with uncomplicated malaria (P, >0.1 for all enzymes; Mann-Whitney U test). These results do not support the hypothesis that differences in invasion phenotypes play an important role in malaria virulence and indicate that considerable gaps remain in our knowledge of the molecular basis of invasion pathways in natural P. falciparum infections.

The respiratory pathogen Chlamydia (Chlamydophila) pneumoniae is associated with chronic diseases, including atherosclerosis and giant-cell arteritis, which are accompanied by the occurrence of these obligate intracellular bacteria in blood vessels. There, C. pneumoniae seems to be present in a persistent state. Persistence is characterized by modified bacterial metabolism and morphology, as well as a reversible arrest of chlamydial development. In cell culture, this persistent state can be induced by gamma interferon (IFN-gamma). To elucidate this long-term interaction between chlamydiae and their host cells, microarray screening on epithelial HeLa cells was performed. Transcription of persistently (and productively) infected cells was compared with that of mock-infected cells. Sixty-six host cell genes were regulated at 24 h and/or 96 h of IFN-gamma-induced persistence. Subsequently, a set of 17 human host cell genes related to apoptosis, cell cycle, or metabolism was identified as permanently up- or down-regulated by real-time PCR. Some of these chlamydia-dependent host cell responses were diminished or even absent in the presence of rifampin. However, other expression patterns were not altered by the inhibition of bacterial RNA polymerase, suggesting two different modes of host cell activation. Thus, in the IFN-gamma model, the persisting bacteria cause long-lasting changes in the expression of genes coding for functionally important proteins. They might be potential drug targets for the treatment of persistent C. pneumoniae infections.

The genetic pathways that partition the developing nervous system into functional systems are largely unknown. The engrailed (En) homeobox transcription factors are candidate regulators of this process in the dorsal midbrain (tectum) and anterior hindbrain (cerebellum). En1 mutants lack most of the tectum and cerebellum and die at birth, whereas En2 mutants are viable with a smaller cerebellum and foliation defects. Our previous studies indicated that the difference in phenotypes is due to the earlier expression of En1 as compared with En2, rather than differences in protein function, since knock-in mice expressing En2 in place of En1 have a normal brain. Here, we uncovered a wider spectrum of functions for the En genes by generating a series of En mutant mice. First, using a conditional allele we demonstrate that En1 is required for cerebellum development only before embryonic day 9, but plays a sustained role in forming the tectum. Second, by removing the endogenous En2 gene in the background of En1 knock-in alleles, we show that Drosophila en is not sufficient to sustain midbrain and cerebellum development in the absence of En2, whereas En2 is more potent than En1 in cerebellum development. Third, based on a differential sensitivity to the dose of En1/2, our studies reveal a genetic subdivision of the tectum into its two functional systems and the medial cerebellum into four regions that have distinct circuitry and molecular coding. Our study suggests that an ;engrailed code' is integral to partitioning the tectum and cerebellum into functional domains

Here we review how prior experience with stress alters the response to a subsequent homotypic or heterotypic stressor, focusing on the catecholaminergic systems in the adrenal medulla and the locus coeruleus (LC). The changes in response to homotypic stress differ depending on the stressor applied. With immobilization stress (IMO), transcriptional responses in the adrenal medulla to a single exposure are pronounced and several of the transcription factors and signaling kinases induced or activated are reviewed and compared to the longer term alterations with repeated stress, consistent with persistent activation of gene expression of catecholamine (CA) biosynthetic enzymes. In the LC, transcriptional and post-transcriptional activation of gene expression are shown to be important. Repeated IMO stress triggers further activation of a number of signalling pathways. Neither adrenal medulla nor LC display habituation to long term repeated stress. In contrast, gene expression for CA biosynthetic enzymes habituates to prolonged cold stress in the adrenal medulla and LC, but displays an exaggerated response with exposure to a novel or heterotypic stressor such as IMO. Some of the transcriptional pathways displaying sensitization are described.