Faculty Bibliography

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.

3D cell cultures are rapidly becoming the method of choice for the physiologically relevant modeling of many aspects of non-malignant and malignant cell behavior ex vivo. Nevertheless, only a limited number of distinct cell types have been evaluated in this assay to date. Here we report the first large scale comparison of the transcriptional profiles and 3D cell culture phenotypes of a substantial panel of human breast cancer cell lines. Each cell line adopts a colony morphology of one of four main classes in 3D culture. These morphologies reflect, at least in part, the underlying gene expression profile and protein expression patterns of the cell lines, and distinct morphologies were also associated with tumor cell invasiveness and with cell lines originating from metastases. We further demonstrate that consistent differences in genes encoding signal transduction proteins emerge when even tumor cells are cultured in 3D microenvironments

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.

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

Antifungal efficacies of the echinocandin drugs caspofungin, micafungin, and anidulafungin were reduced significantly in the presence of 50% human serum, which yielded nearly equivalent MICs or minimum effective concentrations against diverse Candida spp. and Aspergillus spp. Consistent with a direct drug interaction, serum decreased the sensitivity of glucan synthase to echinocandin drugs.

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.

The catecholamines play key roles in orchestrating the response to stress. While this is crucial to handle emergency situations, stress becomes maladaptive when prolonged or repeated, increasing allosteric load and susceptibility to a wide range of serious diseases. The time frame of the regulation of gene expression, especially as it relates to catecholamine (CA) biosynthetic enzymes are compared in three crucial catecholaminergic locations, the adrenal medulla, sympathetic ganglia and locus coeruleus in male animals. The adrenal medulla displays very rapid response to stress and gene profiling reveals a wide repertoire of target genes, many of them activated by single and not by repeated stress. In contrast to the adrenal medulla, the sympathetic ganglia are especially responsive to activation of the HPA axis, and ACTH may have a direct effect. The locus coeruleus, origin of most of the noradrenergic neurons innervating much of the brain, displays activation of additional signalling pathways and transcription factor with repeated compared to single exposure to stress. Most of the studies have been performed in males. However, there is considerable evidence that females respond differently to stress. Estradiol can regulate TH, DBH and GTPCH gene expression, as well as to modulate its response to other second messenger such as cAMP. Prior treatment with estradiol was found to alter the response of CA biosynthetic enzymes to stress. This emphasizes the tissue and sex specific features of the mechanistic underpinning of the adaptation or maladaptation of the catecholaminergic systems to stress and provides the basis for specific interventions. Key words: Adrenal medulla - Catecholamine biosynthesis - Estrogen - Sympathetic ganglia - Locus coeruleus - Stress - Transcription factors.

Ras activation as a consequence of antigen receptor (T-cell receptor; TCR) engagement on T lymphocytes is required for T-cell development, selection and function. Lymphocyte function-associated antigen-1 (LFA-1) mediates lymphocyte adhesion, stabilization of the immune synapse and bidirectional signalling. Using a fluorescent biosensor we found that TCR activation with or without costimulation of CD28 led to activation of Ras only on the Golgi apparatus, whereas costimulation with LFA-1 induced Ras activation on both the Golgi and the plasma membrane. Ras activation on both compartments required RasGRP1, an exchange factor regulated by calcium and diacylglycerol (DAG), but phospholipase C (PLC) activity was required only for activation on the Golgi. Engagement of LFA-1 increased DAG levels at the plasma membrane by stimulating phospholipase D (PLD). PLD2 and phosphatidic acid phosphatase (PAP) were required for Ras activation on the plasma membrane. Thus, LFA-1 acts through PLD2 to reshape the pattern of Ras activation downstream of the TCR