Molecular Detection of Multiple Emerging Pathogens in Sputa from Cystic Fibrosis Patients

Background: There is strong evidence that culture-based methods detect only a small proportion of bacteria present in the respiratory tracts of cystic fibrosis (CF) patients. Methodology/Principal Findings: Standard microbiological culture and phenotypic identification of bacteria in sputa from CF patients have been compared to molecular methods by the use of 16S rDNA amplification, cloning and sequencing. Twenty-five sputa from CF patients were cultured that yield 33 isolates (13 species) known to be pathogens during CF. For molecular cloning, 760 clones were sequenced (7.263.9 species/sputum), and 53 different bacterial species were identified including 16 species of anaerobes (30%). Discrepancies between culture and molecular data were numerous and demonstrate that accurate identification remains challenging. New or emerging bacteria not or rarely reported in CF patients were detected including Dolosigranulum pigrum, Dialister pneumosintes, and Inquilinus limosus. Conclusions/Significance: Our results demonstrate the complex microbial community in sputa from CF patients, especially anaerobic bacteria that are probably an underestimated cause of CF lung pathology. Metagenomic analysis is urgentl

Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles

Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways - but in opposite directions - suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer

Early virion-associated suppression of cellular protein synthesis by herpes simplex virus is accompanied by inactivation of mRNA.

Vero cells infected with herpes simplex virus in the presence of actinomycin or cycloheximide, or with u.v.-inactivated virus, suffered a rapid loss of functional mRNA as determined by translation in vitro. It is suggested that a virion-associated factor causes a structural change in the mRNA of the host cell

Progress and prospects: Immunobiology of gene therapy for neurodegenerative disease: prospects and risks.

Gene therapy for neurological, and in particular neurodegenerative, disease is now a reality. A number of early phase clinical trials have been completed and several are currently in progress. In view of this, it is critically important to evaluate the immunological risk associated with neurological gene therapy, which has clear implications for trial safety and efficacy. Moreover, it is imperative in particular to identify factors indicating potential high risk. In the light of recent advances in understanding immune regulation in the central nervous system (CNS) and with the continued development of new gene delivery vectors, this review critically assesses the current knowledge of immunobiology within the CNS in terms of likely immunological risk pertaining to viral vectors and gene therapy applications for neurodegenerative disease

Th1 cytokines are upregulated by adenoviral vectors in the brains of primed mice.

A major disadvantage of first generation adenoviral vectors for gene therapy in the brain is the immune response they elicit. Human adenovirus is a common respiratory virus and earlier exposure to it has important implications for gene therapy. We show that the immune response against E1-deleted adenoviral vectors in the brain is more deleterious in animals previously exposed to the virus. Analysis of cytokine mRNA revealed enhanced and prolonged upregulation of the Th1 proinflammatory cytokines, IFN-gamma, TNF-alpha and IL-12 whereas, effects on Th2 cytokines were negligible. This was associated with reduced reporter gene expression, decreased expression of the dopamine transporter protein and demyelination. This knowledge of the molecular regulation of the immune response provides insight into targets, which could be manipulated to reduce inflammation in immunologically primed animals

A gamma34.5 mutant of herpes simplex 1 causes severe inflammation in the brain.

A number of viral vectors are currently being evaluated as potential gene therapy vectors for gene delivery to the brain. As well as evaluating their ability to express a transgene for extended periods of time it is also essential to examine any cytotoxic immune response to such vectors as this may not only limit transgene expression but also cause irreparable harm. This work describes the effect of inoculating a gamma34.5 mutant of herpes simplex type 1 (1716lacZ) into the brain of different strains of rats and mice. Animals were monitored for weight loss and signs of illness, and their brains were evaluated for inflammation, beta-galactosidase expression and recoverable infectious virus. We report that there is (i) a powerful immune response consisting of an early non-specific phase and a later presumably T-cell-mediated phase; (ii) significant weight loss in some animals strains accompanied by severe signs of clinical illness and (iii) transient reporter gene expression in all animal strains examined. To be useful for gene therapy we suggest this virus requires further modification, it should be tested in several animal strains and the dose of virus used may be critical in order to limit damage

Readministration of adenoviral gene delivery to dopamine neurons.

An approach currently being explored as treatment for Parkinson's disease is gene therapy. An important question concerns the duration of transgene expression in dopamine neurons and the issues of vector persistence, neuronal damage and the feasibility of readministering vector to the same neuronal population. We show, using an adenoviral vector expressing the LacZ reporter gene, that transgene expression declined over time but with minimal loss of dopamine neurons or vector DNA. Readministration of vector resulted in low levels of transgene delivery to the neurons. Moreover, the neurons to which vector had already been delivered were unable to transport the retrograde tracer fluorogold. Our findings indicate that transgene expression declined in dopamine neurons despite the persistence of virus, and the capacity to readminister vector to these neurons was limited

Neuropathological consequences of delivering an adenoviral vector in the rat brain.

BACKGROUND: Adenoviruses have many advantages as vehicles for gene delivery to the central nervous system (CNS) and retrograde transport of vectors to axonally linked sites has been postulated as a method for targeting neurons in remote brain regions. To investigate optimisation of this we injected different doses of vector and have documented the neuropathological side effects. METHODS: Increasing doses of a first-generation adenoviral vector, expressing the lacZ gene, were inoculated in the rat striatum and beta-galactosidase expression was examined at the primary and secondary sites. Subsequently, at the highest dose of vector, transgene expression, the inflammatory response, tyrosine hydroxylase (TH) expression and the rotational behaviour of animals were studied over time. RESULTS: When a high dose of an adenoviral vector was delivered to the rat striatum, high levels of transgene expression were seen at 5 days in the injection site and in the substantia nigra. Smaller doses gave lower levels of expression with little expression detectable in the substantia nigra. At later time points, with the high dose, a marked reduction in transgene expression was detected and was accompanied by cytopathic damage, a strong inflammatory response and animal weight loss. This was associated with depletion in TH levels and abnormal motor behaviour in animals. CONCLUSIONS: Neuropathological damage in the dopaminergic system, caused by high doses of adenoviral vectors, has not previously been documented. To minimise damage and prolong transgene expression, it is important that the dose of vectors to be delivered is carefully optimised