Modeling the effects of a Staphylococcal Enterotoxin B (SEB) on the apoptosis pathway.

BACKGROUND: The lack of detailed understanding of the mechanism of action of many biowarfare agents poses an immediate challenge to biodefense efforts. Many potential bioweapons have been shown to affect the cellular pathways controlling apoptosis 1234. For example, pathogen-produced exotoxins such as Staphylococcal Enterotoxin B (SEB) and Anthrax Lethal Factor (LF) have been shown to disrupt the Fas-mediated apoptotic pathway 24. To evaluate how these agents affect these pathways it is first necessary to understand the dynamics of a normally functioning apoptosis network. This can then serve as a baseline against which a pathogen perturbed system can be compared. Such comparisons can expose both the proteins most susceptible to alteration by the agent as well as the most critical reaction rates to better instill control on a biological network. RESULTS: We explore this through the modeling and simulation of the Fas-mediated apoptotic pathway under normal and SEB influenced conditions. We stimulated human Jurkat cells with an anti-Fas antibody in the presence and absence of SEB and determined the relative levels of seven proteins involved in the core pathway at five time points following exposure. These levels were used to impute relative rate constants and build a quantitative model consisting of a series of ordinary differential equations (ODEs) that simulate the network under both normal and pathogen-influenced conditions. Experimental results show that cells exposed to SEB exhibit an increase in the rate of executioner caspase expression (and subsequently apoptosis) of 1 hour 43 minutes (+/- 14 minutes), as compared to cells undergoing normal cell death. CONCLUSION: Our model accurately reflects these results and reveals intervention points that can be altered to restore SEB-influenced system dynamics back to levels within the range of normal conditions.

Intensive case management for high-risk patients with first-episode psychosis: service model and outcomes.

BACKGROUND: The first episode of psychosis is a crucial period when early intervention can alter the trajectory of the young person's ongoing mental health and general functioning. After an investigation into completed suicides in the Early Psychosis Prevention and Intervention Centre (EPPIC) programme, the intensive case management subprogramme was developed in 2003 to provide assertive outreach to young people having a first episode of psychosis who are at high risk owing to risk to self or others, disengagement, or suboptimal recovery. We report intensive case management model development, characterise the target cohort, and report on outcomes compared with EPPIC treatment as usual. METHODS: Inclusion criteria, staff support, referral pathways, clinical review processes, models of engagement and care, and risk management protocols are described. We compared 120 consecutive referrals with 50 EPPIC treatment as usual patients (age 15-24 years) in a naturalistic stratified quasi-experimental real-world design. Key performance indicators of service use plus engagement and suicide attempts were compared between EPPIC treatment as usual and intensive case management, and psychosocial and clinical measures were compared between intensive case management referral and discharge. FINDINGS: Referrals were predominately unemployed males with low levels of functioning and educational attainment. They were characterised by a family history of mental illness, migration and early separation, with substantial trauma, history of violence, and forensic attention. Intensive case management improved psychopathology and psychosocial outcomes in high-risk patients and reduced risk ratings, admissions, bed days, and crisis contacts. INTERPRETATION: Characterisation of intensive case management patients validated the clinical research focus and identified a first episode of psychosis high-risk subgroup. In a real-world study, implementation of an intensive case management stream within a well-established first episode of psychosis service showed significant improvement in key service outcomes. Further analysis is needed to determine cost savings and effects on psychosocial outcomes. Targeting intensive case management services to high-risk patients with unmet needs should reduce the distress associated with pathways to care for patients, their families, and the community. FUNDING: National Health & Medical Research Council and the Colonial Foundation.

Gene transfer to subdermal tissues via a new gene gun design.

Although particle-mediated gene transfer technology (gene gun) has been applied for gene transfer to external tissues, the application of this technology to other tissues has met with limited success. Here we report the development of a new design of a gene gun that uses helium discharge to propel DNA-coated gold beads that are suspended in liquid. Higher discharge pressures allow for the delivery of DNA to deeper tissues. Using the new gene gun to deliver a luciferase expression plasmid resulted in higher levels of gene expression in the skin than observed with conventional guns, as well as in subdermal tissues, including subcutaneous tumors. Even when using as little as 125 ng of DNA, gene expression in skin and muscle reached its peak level at 24 hr postbombardment and remained for at least 1 week. The use of a LacZ expression plasmid showed that gene expression was distributed throughout the skin with no observable pathology. The new gene gun was used to deliver a model tumor rejection antigen (a modified human papilloma virus [HPV] E7 gene) to mice. All of the treated animals developed protective immunity against HPV-positive tumors. These results demonstrate that our new design can be used in standard gene gun applications and extends the reach of gene gun technology to tissues that were previously unavailable.

Behavioral and physiological effects of RDX on adult zebrafish.

1,3,5-Trinitroperhydro-1,3,5-triazine (RDX) is a nitroamine explosive, with common toxic effects including seizures. Here, we explore the behavioral effects of acute RDX exposure in adult zebrafish Danio rerio, a rapidly developing model in neuroscience and neurotoxicology research. Overall, a 30-min exposure to RDX low dose of 0.1 mM evoked behavioral activation in zebrafish, while a higher dose of 1 mM markedly reduced exploration, increased freezing and evoked seizure-like responses (i.e., bouts of hyperactivity, spasms, and corkscrew swimming). Likewise, whole-body cortisol levels were also significantly elevated in fish exposed to 1 mM (but not 0.1 mM) RDX. In line with clinical and animal data, our study demonstrates the dose-dependent behavioral activation and pro-convulsant effects of RDX in zebrafish-based models.

Effects of piracetam on behavior and memory in adult zebrafish.

Piracetam, a derivative of γ-aminobutyric acid, exerts memory-enhancing and mild anxiolytic effects in human and rodent studies. To examine the drug's behavioral profile further, we assessed its effects on behavioral and endocrine (cortisol) responses of adult zebrafish (Danio rerio)--a novel model species rapidly gaining popularity in neurobehavioral research. Overall, acute piracetam did not affect zebrafish novel tank and light-dark box behavior at mild doses (25-400mg/L), but produced nonspecific behavioral inhibition at 700mg/L. No effects on cortisol levels or inter-/intra-session habituation in the novel tank test were observed for acute or chronic mild non-sedative dose of 200mg/L. In contrast, fish exposed to chronic piracetam at this dose performed significantly better in the cued learning plus-maze test. This observation parallels clinical and rodent literature on the behavioral profile of piracetam, supporting the utility of zebrafish paradigms for testing nootropic agents.

Behavioral and physiological effects of acute ketamine exposure in adult zebrafish.

Ketamine is a non-competitive glutamatergic antagonist used to induce sedation and analgesia. In sub-anesthetic doses, it induces hyperlocomotion, impairs memory and evokes stereotypic circling in rodents. Zebrafish (Danio rerio) emerged as a promising new animal model to screen the effects of psychotropic compounds. Here, we investigated the effects of sub-anesthetic doses of ketamine on anxiety, locomotion, habituation and social behavior of adult zebrafish. Acute 20-min exposure to 20 and 40 mg/L (but not 2 mg/L) of ketamine reduced anxiety, impaired intra-session habituation, evoked circular swimming and disrupted zebrafish shoaling. Additionally, ketamine reduced whole-body cortisol levels and elevated brain c-fos expression in zebrafish. Our findings demonstrate the sensitivity of zebrafish to behavioral and physiological effects of sub-anesthetic doses of ketamine, further supporting the utility of this species as a model for neuropharmacological research, including testing ketamine and related drugs.

Co-design in synthetic biology: a system-level analysis of the development of an environmental sensing device.

The concept of co-design is common in engineering, where it is necessary, for example, to determine the optimal partitioning between hardware and software of the implementation of a system features. Here we propose to adapt co-design methodologies for synthetic biology. As a test case, we have designed an environmental sensing device that detects the presence of three chemicals, and returns an output only if at least two of the three chemicals are present. We show that the logical operations can be implemented in three different design domains: (1) the transcriptional domain using synthetically designed hybrid promoters, (2) the protein domain using bi-molecular fluorescence complementation, and (3) the fluorescence domain using spectral unmixing and relying on electronic processing. We discuss how these heterogeneous design strategies could be formalized to develop co-design algorithms capable of identifying optimal designs meeting user specifications.

Homebase behavior of zebrafish in novelty-based paradigms.

Zebrafish (Danio rerio) are emerging as a promising model species in neuroscience research. Many traditional rodent behavioral paradigms may be adapted for zebrafish testing. Exposing zebrafish to three different "open field" tanks for 30 min, we showed that fish display robust homebase behavior, in which one area of the tank is chosen as a preferred point of reference during the test, which the fish frequently return to and spend a longer duration in. This phenotype strikingly resembles rodent homebase behavior, confirming that both species use homebases as "reference points" for their exploration. Our study introduces a simple method for zebrafish homebase phenotyping, and further supports the utility of these fish in neurobehavioral and cognitive research.

Modeling seizure-related behavioral and endocrine phenotypes in adult zebrafish.

Larval zebrafish (Danio rerio) have recently been suggested as a high-throughput experimental model of epilepsy-related pathogenetic states. Here we use adult zebrafish to study behavioral symptoms associated with drug-evoked seizures. Experimental epilepsy-like states were evoked in zebrafish by exposure for 20min to three chemoconvulsant drugs: caffeine (250mg/L; 1.3mM), pentylenetetrazole (1.5g/L; 11.0mM) and picrotoxin (100mg/L; 0.17mM). Fish behavior was analyzed using manual and video-tracking methods (Noldus Ethovision XT7). Compared to their respective controls, all three drug-treated groups showed robust seizure-like responses (hyperactivity bouts, spasms, circular and corkscrew swimming) accompanied by elevated whole-body cortisol levels (assessed by ELISA). In contrast, control fish did not display seizure-like behaviors and had significantly lower cortisol levels. Paralleling behavioral and endocrine phenotypes observed in clinical and rodent studies, our data implicates adult zebrafish as an emerging experimental model for epilepsy research.

Characterization of behavioral and endocrine effects of LSD on zebrafish.

Lysergic acid diethylamide (LSD) is a potent hallucinogenic drug that strongly affects animal and human behavior. Although adult zebrafish (Danio rerio) are emerging as a promising neurobehavioral model, the effects of LSD on zebrafish have not been investigated previously. Several behavioral paradigms (the novel tank, observation cylinder, light-dark box, open field, T-maze, social preference and shoaling tests), as well as modern video-tracking tools and whole-body cortisol assay were used to characterize the effects of acute LSD in zebrafish. While lower doses (5-100 microg/L) did not affect zebrafish behavior, 250 microg/L LSD increased top dwelling and reduced freezing in the novel tank and observation cylinder tests, also affecting spatiotemporal patterns of activity (as assessed by 3D reconstruction of zebrafish traces and ethograms). LSD evoked mild thigmotaxis in the open field test, increased light behavior in the light-dark test, reduced the number of arm entries and freezing in the T-maze and social preference test, without affecting social preference. In contrast, LSD affected zebrafish shoaling (increasing the inter-fish distance in a group), and elevated whole-body cortisol levels. Overall, our findings show sensitivity of zebrafish to LSD action, and support the use of zebrafish models to study hallucinogenic drugs of abuse.

Measuring behavioral and endocrine responses to novelty stress in adult zebrafish.

Several behavioral assays are currently used for high-throughput neurophenotyping and screening of genetic mutations and psychotropic drugs in zebrafish (Danio rerio). In this protocol, we describe a battery of two assays to characterize anxiety-related behavioral and endocrine phenotypes in adult zebrafish. Here, we detail how to use the 'novel tank' test to assess behavioral indices of anxiety (including reduced exploration, increased freezing behavior and erratic movement), which are quantifiable using manual registration and computer-aided video-tracking analyses. In addition, we describe how to analyze whole-body zebrafish cortisol concentrations that correspond to their behavior in the novel tank test. This protocol is an easy, inexpensive and effective alternative to other methods of measuring stress responses in zebrafish, thus enabling the rapid acquisition and analysis of large amounts of data. As will be shown here, fish anxiety-like behavior can be either attenuated or exaggerated depending on stress or drug exposure, with cortisol levels generally expected to parallel anxiety behaviors. This protocol can be completed over the course of 2 d, with a variable testing duration depending on the number of fish used.

Lipid-protamine-DNA-mediated antigen delivery to antigen-presenting cells results in enhanced anti-tumor immune responses.

Vaccination with antigenic peptides encoding tumor antigens has the potential to be an effective treatment for cancer. To induce tumor-specific cellular immune responses, a peptide antigen must be presented by antigen-presenting cells (APCs) to T-cells in the lymphatic tissues. Effective in vivo delivery of peptide antigens to APCs has been problematic. Here we use a model antigen from the HPV16 E7 protein to formulate LPD/E7 particles that upon iv administration are internalized by CD11c(+) and CD11b(+) cells in the marginal zone of the spleen. Either iv or sc vaccination with LPD/E7 particles induces E7-specific CTL responses stronger than those obtained using previously described liposome/peptide strategies and prevents the establishment of E7-expressing tumors. Furthermore, the administration of LPD/E7 particles to tumor-bearing mice caused complete tumor regression in 100% of the treated animals. Based on these studies, the entrapment of peptide antigens inside LPD particles may be an effective and generally applicable strategy for the enhancement of peptide vaccine potency.

Lipid-mediated delivery of oligonucleotide to pulmonary endothelium.

Pulmonary endothelium plays an important role in the maintenance of normal pulmonary physiology and its dysfunction is involved in a number of pulmonary diseases. Correction of endothelial dysfunction via antisense oligodeoxyonucleotides (ODN) is dependent on the development of a delivery vehicle that can efficiently deliver the ODN to pulmonary endothelium with minimal toxicity. To this end, we have developed a lipidic vector (LPD) that is composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes, protamine, and ODN. This formulation is highly efficient in delivering ODN to the lung via the vascular route. The efficiency of delivery is a function of lipid composition and of the charge ratio between lipid and ODN. Immunofluorescence staining of BrdU-labeled ODN suggested efficient accumulation of ODN in the alveolar capillary region. Transmission electron microscopy of immunogold localization of BrdU-labeled ODN confirmed that pulmonary endothelial cells were indeed targeted by the vector. Furthermore, this formulation is associated with minimal proinflammatory cytokine response and other hematologic toxicities when the ODN lack a potent unmethylated CpG motif. Pretreatment of mice with LPD containing an ODN against intercellular adhesion molecule-1 (ICAM-1) significantly decreased ICAM-1 expression in the lung following LPS challenge. These results provide a basis for lipid-mediated delivery of ODN for the treatment of pulmonary diseases.