Development and characterization of novel transgenic techniques for the study of neuronal circuitry

Modern neuroscience relies heavily on sophisticated tools that allow us to visualize and manipulate cells with precise spatial and temporal control. Transgenic mouse models, for example, can be used to manipulate cellular activity in order to draw conclusions about the molecular events responsible for the development, maintenance and refinement of healthy and/or diseased neuronal circuits. Although it is fairly well established that circuits respond to activity-dependent competition between neurons, we have yet to understand either the mechanisms underlying these events or the higher-order plasticity that synchronizes entire circuits. In this thesis we aimed to develop and characterize transgenic mouse models that can be used to directly address these outstanding biological questions in different ways. We present SLICK-H, a Cre-expressing mouse line that can achieve drug-inducible, widespread, neuron-specific manipulations in vivo. This model is a clear improvement over existing models because of its particularly strong, widespread, and even distribution pattern that can be tightly controlled in the absence of drug induction. We also present SLICK-V::Ptox, a mouse line that, through expression of the tetanus toxin light chain, allows long-term inhibition of neurotransmission in a small subset (<1%) of fluorescently labeled pyramidal cells. This model, which can be used to study how a silenced cell performs in a wildtype environment, greatly facilitates the in vivo study of activity-dependent competition in the mammalian brain. As an initial application we used this model to show that tetanus toxin-expressing CA1 neurons experience a 15% - 19% decrease in apical dendritic spine density. Finally, we also describe the attempt to create additional Cre-driven mouse lines that would allow conditional alteration of neuronal activity either by hyperpolarization or inhibition of neurotransmission. Overall, the models characterized in this thesis expand upon the wealth of tools available that aim to dissect neuronal circuitry by genetically manipulating neurons in vivo.

Adenovirus F protein as a delivery vehicle for botulinum B.

BACKGROUND: Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within beta-trefoil domain (Hcbetatre), we hypothesize that immunization with the Hcbetatre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination. RESULTS: The Hcbetatre serotype B immunogen was genetically fused to Ad2F (Hcbetatre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcbetatre-Ad2F relative to Hcbetatre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcbetatre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcbetatre and Hcbetatre-Ad2F protected mice against 2.0 LD50. CONCLUSION: These results demonstrate that Hcbetatre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.

Risk factors for and estimated incidence of community-associated Clostridium difficile infection, North Carolina, USA.

We determined estimated incidence of and risk factors for community-associated Clostridium difficile infection (CA-CDI) among patients treated at 6 North Carolina hospitals. CA-CDI case-patients were defined as adults (>18 years of age) with a positive stool test result for C. difficile toxin and no hospitalization within the prior 8 weeks. CA-CDI incidence was 21 and 46 per 100,000 person-years in Veterans Affairs (VA) outpatients and Durham County populations, respectively. VA case-patients were more likely than controls to have received antimicrobial drugs (adjusted odds ratio [aOR] 17.8, 95% confidence interval [CI] 6.6-48] and to have had a recent outpatient visit (aOR 5.1, 95% CI 1.5-17.9). County case-patients were more likely than controls to have received antimicrobial drugs (aOR 9.1, 95% CI 2.9-28.9), to have gastroesophageal reflux disease (aOR 11.2, 95% CI 1.9-64.2), and to have cardiac failure (aOR 3.8, 95% CI 1.1-13.7). Risk factors for CA-CDI overlap with those for healthcare-associated infection.

Quantitative mapping of trimethyltin injury in the rat brain using magnetic resonance histology.

The growing exposure to chemicals in our environment and the increasing concern over their impact on health have elevated the need for new methods for surveying the detrimental effects of these compounds. Today's gold standard for assessing the effects of toxicants on the brain is based on hematoxylin and eosin (H&E)-stained histology, sometimes accompanied by special stains or immunohistochemistry for neural processes and myelin. This approach is time-consuming and is usually limited to a fraction of the total brain volume. We demonstrate that magnetic resonance histology (MRH) can be used for quantitatively assessing the effects of central nervous system toxicants in rat models. We show that subtle and sparse changes to brain structure can be detected using magnetic resonance histology, and correspond to some of the locations in which lesions are found by traditional pathological examination. We report for the first time diffusion tensor image-based detection of changes in white matter regions, including fimbria and corpus callosum, in the brains of rats exposed to 8 mg/kg and 12 mg/kg trimethyltin. Besides detecting brain-wide changes, magnetic resonance histology provides a quantitative assessment of dose-dependent effects. These effects can be found in different magnetic resonance contrast mechanisms, providing multivariate biomarkers for the same spatial location. In this study, deformation-based morphometry detected areas where previous studies have detected cell loss, while voxel-wise analyses of diffusion tensor parameters revealed microstructural changes due to such things as cellular swelling, apoptosis, and inflammation. Magnetic resonance histology brings a valuable addition to pathology with the ability to generate brain-wide quantitative parametric maps for markers of toxic insults in the rodent brain.

Heat-labile enterotoxin: beyond G(m1) binding.

Enterotoxigenic Escherichia coli (ETEC) is a significant source of morbidity and mortality worldwide. One major virulence factor released by ETEC is the heat-labile enterotoxin LT, which is structurally and functionally similar to cholera toxin. LT consists of five B subunits carrying a single catalytically active A subunit. LTB binds the monosialoganglioside G(M1), the toxin's host receptor, but interactions with A-type blood sugars and E. coli lipopolysaccharide have also been identified within the past decade. Here, we review the regulation, assembly, and binding properties of the LT B-subunit pentamer and discuss the possible roles of its numerous molecular interactions.

Bordetella pertussis from functional genomics to intranasal vaccination.

Whooping cough still represents a major health problem, despite the use of effective vaccines for several decades. Being classically a typical childhood disease, whooping cough in young adults is now more common than it used to be, suggesting that protection after vaccination wanes during adolescence. As an alternative to the current vaccines, we wish to develop live attenuated vaccines to be delivered by the nasal route, such as to mimic the natural route of infection and to induce long lasting immunity. Bordetella pertussis, the etiological agent of whooping cough, produces a number of virulence factors, including toxins. Its recently determined genome sequence makes it now possible to apply functional genomics, such as transcriptomics and systematic knock-out mutagenesis. The expression of most known B. pertussis virulence genes is controlled by the two-component system BvgA/S. DNA microarray analyses have led to the identification of novel genes in the BvgA/S regulon, some of which are activated by BvgA/S and others are repressed by BvgA/S. In addition, some genes appear to be differentially modulated by nicotinic acid and MgSO4, both known to modulate the expression of BvgA/S-regulated genes. Among others, the functional genomics approach has uncovered two strongly BvgA/S-activated genes, named hotA and hotB (for 'homolog of toxin'), the products of which show high sequence similarities to pertussis toxin subunits. The identification of the full array of virulence factors, as well as an integrated understanding of the bacterial physiology should allow us to design attenuated B. pertussis strains useful for intranasal vaccination. A first generation of attenuated strains has already shown full protection in mice after a single intranasal administration. Such strains may also serve as vaccine carriers for heterologous antigens, in order to vaccinate against several different pathogens simultaneously.

Environmental and Human Pathogenic Microorganisms

As the study of interactions between pathogenic microorganisms and their environment is part of microbial ecology, this chapter reviews the different types of human pathogens found in the environment, the different types of fecal indicators used in water quality monitoring, the biotic and abiotic factors affecting the survival and the infectivity of pathogenic microorganisms during their transportation in the environment, and the methods presently available to detect rare microorganisms in environmental samples. This chapter exclusively focuses on human pathogens.

Intracellular fate of bioresponsive poly(amidoamine)s in vitro and in vivo

Linear poly(amidoamine)s (PAAs) have been designed to exhibit minimal non-specific toxicity, display pH-dependent membrane lysis and deliver genes and toxins in vitro. The aim of this study was to measure PAA cellular uptake using ISA1-OG (and as a reference ISA23-OG) in B16F10 cells in vitro and, by subcellular fractionation, quantitate intracellular trafficking of (125)I-labelled ISA1-tyr in liver cells after intravenous (i.v.) administration to rats. The effect of time after administration (0.5-3h) and ISA1 dose (0.04-100mg/kg) on trafficking, and vesicle permeabilisation (N-acetyl-b-D-glucosaminidase (NAG) release from an isolated vesicular fraction) were also studied. ISA1-OG displayed approximately 60-fold greater B16F10 cell uptake than ISA23-OG. Passage of ISA1 along the liver cell endocytic pathway caused a transient decrease in vesicle buoyant density (also visible by TEM). Increasing ISA1 dose from 10mg/kg to 100mg/kg increased both radioactivity and NAG levels in the cytosolic fraction (5-10 fold) at 1h. Moreover, internalised ISA1 provoked NAG release from an isolated vesicular fraction in a dose-dependent manner. These results provide direct evidence, for the first time, of PAA permeabilisation of endocytic vesicular membranes in vivo, and they have important implications for potential efficacy/toxicity of such polymeric vectors.

Oceans and Human Health (OHH): a European Perspective from the Marine Board of the European Science Foundation (Marine Board-ESF)

The oceans and coastal seas provide mankind with many benefits including food for around a third of the global population, the air that we breathe and our climate system which enables habitation of much of the planet. However, the converse is that generation of natural events (such as hurricanes, severe storms and tsunamis) can have devastating impacts on coastal populations, while pollution of the seas by pathogens and toxic waste can cause illness and death in humans and animals. Harmful effects from biogenic toxins produced by algal blooms (HABs) and from the pathogens associated with microbial pollution are also a health hazard in seafood and from direct contact with water. The overall global burden of human disease caused by sewage pollution of coastal waters has been estimated at 4 million lost person-years annually. Finally, the impacts of all of these issues will be exacerbated by climate change. A holistic systems approach is needed. It must consider whole ecosystems, and their sustainability, such as integrated coastal zone management, is necessary to address the highly interconnected scientific challenges of increased human population pressure, pollution and over-exploitation of food (and other) resources as drivers of adverse ecological, social and economic impacts. There is also an urgent and critical requirement for effective and integrated public health solutions to be developed through the formulation of politically and environmentally meaningful policies. The research community required to address "Oceans & Human Health" in Europe is currently very fragmented, and recognition by policy makers of some of the problems, outlined in the list of challenges above, is limited. Nevertheless, relevant key policy issues for governments worldwide include the reduction of the burden of disease (including the early detection of emerging pathogens and other threats) and improving the quality of the global environment. Failure to effectively address these issues will impact adversely on efforts to alleviate poverty, sustain the availability of environmental goods and services and improve health and social and economic stability; and thus, will impinge on many policy decisions, both nationally and internationally. Knowledge exchange (KE) will be a key element of any ensuing research. KE will facilitate the integration of biological, medical, epidemiological, social and economic disciplines, as well as the emergence of synergies between seemingly unconnected areas of science and socio-economic issues, and will help to leverage knowledge transfer across the European Union (EU) and beyond. An integrated interdisciplinary systems approach is an effective way to bring together the appropriate groups of scientists, social scientists, economists, industry and other stakeholders with the policy formulators in order to address the complexities of interfacial problems in the area of environment and human health. The Marine Board of the European Science Foundation Working Group on "Oceans and Human Health" has been charged with developing a position paper on this topic with a view to identifying the scientific, social and economic challenges and making recommendations to the EU on policy-relevant research and development activities in this arena. This paper includes the background to health-related issues linked to the coastal environment and highlights the main arguments for an ecosystem-based whole systems approach.

Toxic marine microalgae and shellfish poisoning in the British isles: history, review of epidemiology, and future implications

The relationship between toxic marine microalgae species and climate change has become a high profile and well discussed topic in recent years, with research focusing on the possible future impacts of changing hydrological conditions on Harmful Algal Bloom (HAB) species around the world. However, there is very little literature concerning the epidemiology of these species on marine organisms and human health. Here, we examine the current state of toxic microalgae species around the UK, in two ways: first we describe the key toxic syndromes and gather together the disparate reported data on their epidemiology from UK records and monitoring procedures. Secondly, using NHS hospital admissions and GP records from Wales, we attempt to quantify the incidence of shellfish poisoning from an independent source. We show that within the UK, outbreaks of shellfish poisoning are rare but occurring on a yearly basis in different regions and affecting a diverse range of molluscan shellfish and other marine organisms. We also show that the abundance of a species does not necessarily correlate to the rate of toxic events. Based on routine hospital records, the numbers of shellfish poisonings in the UK are very low, but the identification of the toxin involved, or even a confirmation of a poisoning event is extremely difficult to diagnose. An effective shellfish monitoring system, which shuts down aquaculture sites when toxins exceed regularity limits, has clearly prevented serious impact to human health, and remains the only viable means of monitoring the potential threat to human health. However, the closure of these sites has an adverse economic impact, and the monitoring system does not include all toxic plankton. The possible geographic spreading of toxic microalgae species is therefore a concern, as warmer waters in the Atlantic could suit several species with southern biogeographical affinities enabling them to occupy the coastal regions of the UK, but which are not yet monitored or considered to be detrimental.

Vulnerability of coastal ecosystems to changes in harmful algal bloom distribution in response to climate change: projections based on model analysis

Harmful algal blooms (HABs), those proliferations of algae that can cause fish kills, contaminate seafood with toxins, form unsightly scums, or detrimentally alter ecosystem function have been increasing in frequency, magnitude, and duration worldwide. Here, using a global modeling approach, we show, for three regions of the globe, the potential effects of nutrient loading and climate change for two HAB genera, pelagic Prorocentrum and Karenia, each with differing physiological characteristics for growth. The projections (end of century, 2090-2100) are based on climate change resulting from the A1B scenario of the Intergovernmental Panel on Climate Change Institut Pierre Simon Laplace Climate Model (IPCC, IPSL-CM4), applied in a coupled oceanographic-biogeochemical model, combined with a suite of assumed physiological 'rules' for genera-specific bloom development. Based on these models, an expansion in area and/or number of months annually conducive to development of these HABs along the NW European Shelf-Baltic Sea system and NE Asia was projected for both HAB genera, but no expansion (Prorocentrum spp.), or actual contraction in area and months conducive for blooms (Karenia spp.), was projected in the SE Asian domain. The implications of these projections, especially for Northern Europe, are shifts in vulnerability of coastal systems to HAB events, increased regional HAB impacts to aquaculture, increased risks to human health and ecosystems, and economic consequences of these events due to losses to fisheries and ecosystem services.

Detection of Emerging and Re-Emerging Pathogens in Surface Waters Close to an Urban Area

Current knowledge about the spread of pathogens in aquatic environments is scarce probably because bacteria, viruses, algae and their toxins tend to occur at low concentrations in water, making them very difficult to measure directly. The purpose of this study was the development and validation of tools to detect pathogens in freshwater systems close to an urban area. In order to evaluate anthropogenic impacts on water microbiological quality, a phylogenetic microarray was developed in the context of the EU project µAQUA to detect simultaneously numerous pathogens and applied to samples from two different locations close to an urban area located upstream and downstream of Rome in the Tiber River. Furthermore, human enteric viruses were also detected. Fifty liters of water were collected and concentrated using a hollow-fiber ultrafiltration approach. The resultant concentrate was further size-fractionated through a series of decreasing pore size filters. RNA was extracted from pooled filters and hybridized to the newly designed microarray to detect pathogenic bacteria, protozoa and toxic cyanobacteria. Diatoms as indicators of the water quality status, were also included in the microarray to evaluate water quality. The microarray results gave positive signals for bacteria, diatoms, cyanobacteria and protozoa. Cross validation of the microarray was performed using standard microbiological methods for the bacteria. The presence of oral-fecal transmitted human enteric-viruses were detected using q-PCR. Significant concentrations of Salmonella, Clostridium, Campylobacter and Staphylococcus as well as Hepatitis E Virus (HEV), noroviruses GI (NoGGI) and GII (NoGII) and human adenovirus 41 (ADV 41) were found in the Mezzocammino site, whereas lower concentrations of other bacteria and only the ADV41 virus was recovered at the Castel Giubileo site. This study revealed that the pollution level in the Tiber River was considerably higher downstream rather than upstream of Rome and the downstream location was contaminated by emerging and re-emerging pathogens.

Isolation of scorpion (Androctonus amoreuxi) alpha-neurotoxins and parallel cloning of their respective cDNAs from a single sample of venom

The venoms of buthid scorpions are known to contain basic, single-chain protein toxins (alpha toxins) consisting of 60–70 amino acid residues that are tightly folded by four disulfide bridges. Here we describe isolation and sequencing of three novel putative alpha toxins (AamH1-3) from the venom of the North African scorpion, Androctonus amoreuxi, and subsequent cloning of their precursor cDNAs from the same sample of venom. This experimental approach can expedite functional genomic analyses of the protein toxins from this group of venomous animals and does not require specimen sacrifice for cloning of protein toxin precursor cDNAs.

Unmasking venom gland transcriptomes in reptile venoms

While structural studies of reptile venom toxins can be achieved using lyophilized venom samples, until now the cloning of precursor cDNAs required sacrifice of the specimen for dissection of the venom glands. Here we describe a simple and rapid technique that unmasks venom protein mRNAs present in lyophilized venom samples. To illustrate the technique we have RT-PCR-amplified a range of venom protein transcripts from cDNA libraries derived from the venoms of a hemotoxic snake, the Chinese copperhead (Deinagkistrodon acutus), a neurotoxic snake, the black mamba (Dendroaspis polylepis), and a venomous lizard, the Gila monster (Heloderma suspectum). These include a metalloproteinase and phospholipase A2 from D. acutus, a potassium channel blocker, dendrotoxin K, from D. polylepis, and exendin-4 from H. suspectum. These findings imply that the apparent absence and/or lability of mRNA in complex biological matrices is not always real and paves the way for accelerated acquisition of molecular genetic data on venom toxins for scientific and potential therapeutic purposes without sacrifice of endangered herpetofauna.

Paralytic shellfish poisoning detection by surface plasmon resonance-based biosensors in shellfish matrixes.

The detection of paralytic shellfish poisoning (PSP) toxins in contaminated shellfish is essential for human health preservation. Ethical and technical reasons have prompted the search for new detection procedures as an alternative to the mouse bioassay. On the basis of the detection of molecular interactions by surface plasmon resonance (SPR) biosensors, an inhibition assay was developed using an anti-GTX2/3 antibody (GT13-A) and a saxitoxin-CM5 chip. This assay allowed for quantification of saxitoxin (STX), decarbamoyl saxitoxin (dcSTX), gonyautoxin 2,3 (GTX2/3), decarbamoyl gonyautoxin 2,3 (dcGTX2/3), gonyautoxin 5 (GTX5), and C 1,2 (C1/2) at concentrations from 2 to 50 ng/mL. The interference of five shellfish matrixes with the inhibition assay was analyzed. Mussels, clams, cockles, scallops, and oysters were extracted with five published methods. Ethanol extracts and acetic acid/heat extracts (AOAC Lawrence method) performed adequately in terms of surface regeneration and baseline interference, did not inhibit antibody binding to the chip surface significantly, and presented STX calibration curves similar to buffer controls in all matrixes tested. Hydrochloric acid/heat extracts (AOAC mouse bioassay method) presented surface regeneration problems, and although ethanol-acetic acid/dichloromethane extracts performed well, they were considered too laborious for routine sample testing. Overall the best results were obtained with the ethanol extraction method with calibration curves prepared in blank matrix extracts. STX recovery rate with the ethanol extraction method was 60.52 ± 3.72%, with variations among species. The performance of this biosensor assay in natural samples, compared to two AOAC methods for PSP toxin quantification (mouse bioassay and HPLC), suggests that this technology can be useful as a PSP screening assay. In summary, the GT13-A-STX chip inhibition assay is capable of PSP toxin detection in ethanol shellfish extracts, with sufficient sensitivity to quantify the toxin in the range of the European regulatory limit of 80 g/100 g of shellfish meat.