Tollip, an early regulator of the acute inflammatory response in the substantia nigra.

BACKGROUND: Tollip is a ubiquitously expressed protein, originally described as a modulator of the IL-1R/TLR-NF-κB signaling pathways. Although this property has been well characterized in peripheral cells, and despite some evidence of its expression in the central nervous system, the role of Tollip in neuroinflammation remains poorly understood. The present study sought to explore the implication of Tollip in inflammation in the substantia nigra pars compacta, the structure affected in Parkinson's disease. METHODS: We first investigated Tollip distribution in the midbrain by immunohistochemistry. Then, we addressed TLR4-mediated response by intra-nigral injections of lipopolysaccharide (LPS), a TLR4 agonist, on inflammatory markers in Tollip knockout (KO) and wild-type (WT) mice. RESULTS: We report an unexpectedly high Tollip immunostaining in dopaminergic neurons of the mice brain. Second, intra-nigral injection of LPS led to increased susceptibility to neuroinflammation in Tollip KO compared to Tollip WT mice. This was demonstrated by a significant increase of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), and interferon gamma (IFN-γ) messenger RNA (mRNA) in the midbrain of Tollip KO mice upon LPS injection. Consistently, brain rAAV viral vector transduction with a nuclear factor kappa B (NF-κB)-inducible reporter gene confirmed increased NF-κB activation in Tollip KO mice. Lastly, Tollip KO mice displayed higher inducible NO synthase (iNOS) production, both at the messenger and protein level when compared to LPS-injected WT mice. Tollip deletion also aggravated LPS-induced oxidative and nitrosative damages, as indicated by an increase of 8-oxo-2'-deoxyguanosine and nitrotyrosine immunostaining, respectively. CONCLUSIONS: Altogether, these findings highlight a critical role of Tollip in the early phase of TLR4-mediated neuroinflammation. As brain inflammation is known to contribute to Parkinson's disease, Tollip may be a potential target for neuroprotection.

Overexpression of mouse IsK protein fused to green fluorescent protein induces apoptosis of human astroglioma cells.

Intracellular K(+) plays an important role in controlling ion homeostasis for maintaining cell volume and inhibiting activity of pro-apoptotic enzymes. Cytoplasmic K(+) concentration is regulated by K(+) uptake via Na(+) -K(+) -ATPase and K(+) efflux through K(+) channels in the plasma membrane. The IsK (KCNE1) protein is known to co-assemble with KCNQ1 (KvLQT1) protein to form a K(+) channel underlying the slowly activating delayed rectifier K(+) outward current which delays voltage activation. In order to further study the activity and cellular localization of IsK protein, we constructed a C-terminal fusion of IsK with EGFP (enhanced green fluorescent protein). Expression of the fusion protein appeared as clusters located in the plasma membrane and induced degeneration of both transiently or stably transfected cells.

A system for detection of genetic and epigenetic alterations in Escherichia coli induced by DNA-damaging agents.

In order to compare the genetic and epigenetic effects of genotoxic agents, we have constructed Escherichia coli K12 strains that allow the detection of mutagenesis, SOS induction (epigenetic effect) and genetic recombination in the same genetic background. The epigenetic effect was detected in a similar way to any genetic alteration, i.e. by counting altered clones (colonies), using a gene fusion system that responds to a temporary epigenetic effect by a stable, heritable switch. The gene fusion consists of the E. coli gal operon and a partially deleted prophage lambda, resulting in the gal operon coming under the control of the cI and cro genes. It allows the detection of SOS induction and forward mutagenesis in the cI gene. Even a temporary inactivation of the CI repressor in this particular system leads to a stable epigenetic switch transmitted to the cellular progeny, which can be detected as Gal+ (red) colonies. The genetic (mutational inactivation of gene cI) and epigenetic (proteolytic inactivation of the product of gene cI) mechanisms leading to gal expression can be distinguished. Genetic recombination between two heteroallelic lacZ genes, one located in the bacterial chromosome, the other on an F'lac plasmid, can be detected as Lac+ colonies. Radiation and several chemical mutagens show very different capacities in generating mutants, inductants and recombinants; therefore, a dose range of any physical or chemical agent generates a set of relative values for the generation of mutants, inductants and recombinants that are characteristic of the agent.

Evaluation of risks related to the use of adeno-associated virus-based vectors.

Recombinant AAV efficacy has been demonstrated in numerous gene therapy preclinical studies. As this vector is increasingly applied to human clinical trials, it is a priority to evaluate the risks of its use for workers involved in research and clinical trials as well as for the patients and their descendants. At high multiplicity of infection, wild-type AAV integrates into human chromosome 19 in approximately 60% of latently infected cell lines. However, it has been recently demonstrated that only approximately 1 out of 1000 infectious units can integrate. The mechanism of this site-specific integration involves AAV Rep proteins which are absent in vectors. Accordingly, recombinant AAV (rAAV) do not integrate site-specifically. Random integration of vector sequences has been demonstrated in established cell lines but only in some cases and at low frequency in primary cultures and in vivo. In contrast, episomal concatemers predominate.Therefore, the risks of insertional mutagenesis and activation of oncogenes are considered low. Biodistribution studies in non-human primates after intramuscular, intrabronchial, hepatic artery and subretinal administration showed low and transient levels of vector DNA in body fluids and distal organs. Analysis of patients body fluids revealed rAAV sequences in urine, saliva and serum at short-term. Transient shedding into the semen has been observed after delivery to the hepatic artery. However, motile germ cells seemed refractory to rAAV infection even when directly exposed to the viral particles, suggesting that the risk of insertion of new genetic material into the germ line is absent or extremely low. Risks related to viral capsid-induced inflammation also seem to be absent since immune response is restricted to generation of antibodies. In contrast, transgene products can elicit both cellular and humoral immune responses, depending on the nature of the expressed protein and of the route of vector administration. Finally, a correlation between early abortion as well as male infertility and the presence of wt AAV DNA in the genital tract has been suggested. Although no causal relationship has been established, this issue stresses the importance of using rAAV stocks devoid of contaminating replication-competent AAV. This review comprehensively examines virus integration, biodistribution, immune interactions, and other safety concerns regarding the wild-type AAV and recombinant AAV vectors.

Neuroprotective gene therapy for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterised by a progressive loss of the dopaminergic neurones in the substantia nigra pars compacta. Accumulating evidence indicates that apoptosis contributes to neuronal cell death in PD patients' brain. Excitotoxicity, oxidative stress, and mitochondrial respiratory failure are thought to be the key inducers of the apoptotic cascade. Even though the initial cause and the mechanism of degeneration are poorly understood, neuroprotection can be achieved by interfering with neuronal cell death either directly or by preventing neuronal dysfunction. Potential agents for neuroprotection are neurotrophic factors, inhibitors of apoptosis or anti-oxidative agents. However, the existence of the blood-brain barrier precludes systemic delivery of these factors. In situ gene delivery provides strategies for local and sustained administration of protective factors at physiologically relevant doses. Viral vectors mediating stable gene expression in the central nervous system exist and are still under development. Efficacy of these vectors has repeatedly been demonstrated in the animal models both ex vivo and in vivo. Ex vivo gene delivery could furthermore be combined with cell replacement therapies by transplanting genetically modified cells compensating for the lost neuronal cell population in order to provide neuroprotection to both the grafted cells and degenerating host neurones. However, several aspects of gene transfer, such as uncontrolled diffusion, axonal transport, unpredictable site of integration and immunological responses, still raise safety concerns and justify further development of viral and non-viral vectors as well as genetic elements with tightly controlled gene expression. Various relevant animal models for Parkinson's disease are available for the evaluation of gene therapy strategies. These include induction of cell death in specific neurone population through administration of toxins either directly in the brain or systemically, as well as transgenic mice expressing human disease-associated mutations.

Tropism of AAV-2 vectors for neurons of the globus pallidus.

A recombinant AAV-2 vector encoding the green fluorescent protein (gfp) under the control of the cytomegalovirus (CMV) promoter was injected into the striatum at varying antero-posterior coordinates. When the virus was delivered to the anterior part of the striatum, transduction efficiency was low and limited to the vicinity of the needle tract. In contrast, after injection into the posterior part of the striatum, in addition to a localized transduced area in the striatum, efficient and widespread transduction was observed at distance from the injection site, in the globus pallidus. In the latter case, labelled cells were also detected in the internal capsule and in the stria terminalis. The number of transduced cells in the striatum increased up to I month and then decreased whereas in the globus pallidus, transduction was maximal as early as 2 weeks post-injection. In the striatum and in the globus pallidus, the labelled cells had a neuron-like morphology. In contrast, in the internal capsule, labelled cells had a glial-like morphology.

AAV2 vectors mediate efficient and sustained transduction of rat embryonic ventral mesencephalon.

The success of transplantation of human embryonic mesencephalic tissue to treat parkinsonian patients is limited by the poor survival of the transplant. We show that an AAV2 vector mediates efficient expression of the egfp reporter gene in organotypic cultures of freshly explanted solid fragments of rat embryonic ventral mesencephalon (VM). We observed early and sustained transgene expression (4 days to > or = 6 weeks). Furthermore, rAAV-infected rat embryonic VM transplanted in the adult striatum continued to express EGFP for > or = 3 months. More than 95% of the transduced cells were neurons. Dopaminergic neurons were transduced at low frequency at earlier time points. This method of gene delivery could prove useful to achieve local, continuous secretion of neurotrophic factors at physiologically relevant doses to treat Parkinson's disease.

Efficient early and sustained transduction of human fetal mesencephalon using adeno-associated virus type 2 vectors.

The success of transplantation of human fetal mesencephalic tissue into the putamen of patients with Parkinson's disease (PD) is still limited by the poor survival of the graft. In animal models of fetal transplantation for PD, antiapoptotic agents, such as growth factors or caspase inhibitors, or agents counteracting oxidative stress enhance the survival and reinnervation potential of the graft. Genetic modification of the transplant could allow a local and continuous delivery of these factors at physiologically relevant doses. The major challenge remains the development of strategies to achieve both early and sustained gene delivery in the absence of vector-mediated toxicity. We recently reported that E14 rat fetal mesencephalon could be efficiently tranduced by adeno-associated virus type 2 (AAV2) vectors and that gene expression was maintained until at least 3 months after transplantation in the adult rat striatum. Here we report that an AAV2 vector can mediate the expression of the EGFP reporter gene under the control of a CMV promoter in organotypic cultures of freshly explanted solid fragments of human fetal mesencephalic tissue as early as 3 days to at least 6 weeks postinfection. These results suggest that AAV2 vectors could be used to genetically modify the human fetal tissue prior to transplantation to Parkinson's patients to promote graft survival and integration.

Genotoxic potency of monofunctional alkylating agents in E. coli: comparison with carcinogenic potency in rodents.

A quantitative correlation between carcinogenicity and genotoxicity was investigated by a comparison between the carcinogenic potency in rodents and the mutagenic (M), recombinogenic (R) and SOS-inducing (I) potencies in a bacterial test (E. coli multitest) for 9 monofunctional alkylating agents: N-nitroso-N-methylurethane, N-nitroso-N-ethylurea, epichlorohydrin, N-nitroso-N-methylurea, N-nitroso-N-methyl-N'-nitroguanidine, methyl methanesulfonate, diethylsulfate, dimethylsulfate, ethyl methanesulfonate. A significant positive correlation between the carcinogenic potency and the product of the mutagenic and recombinogenic potencies was found for all tested compounds. Thus, the E. coli multitest may be used as a simple test to search for correlations between carcinogenicity and genotoxicity of DNA-damaging agents.

The E. coli multitest: a set of strains to characterize diverse genotoxic effects.

A set of E. coli strains was developed by Toman et al. (1985) to study the effects of chemical and physical agents on forward mutation, homologous recombination and induction of the SOS system. New tester strains have been constructed to improve this test system in order to explore quantitative genotoxicity spectra. Through the use of these strains: (i) SOS induction can be specifically detected without interference from mutagenesis; (ii) SOS-dependent and SOS-independent mutational events can be distinguished; (iii) the sensitivity of the recombination system has been considerably increased.

recA-independent mutagenicity induced by chloroethylene oxide in E. coli.

The mechanism of mutagenicity of chloroethylene oxide (CEO), an ultimate carcinogenic metabolite of vinyl chloride, was investigated in 3 Escherichia coli strains (E. coli "multitest"). In this system, the mutagenicity of CEO was found to be mainly SOS-independent. CEO did not induce recombinational events at a detection level of about 10(-2) recombinants/survivor. Our results indicate that CEO- (or vinyl chloride-) induced bacterial mutagenesis arises mainly from miscoding DNA adducts.

Comparative survival of Cryptosporidium, coxsackievirus A9 and Escherichia coli in stream, brackish and sea waters.

Discharge of inadequately treated wastewater into streams may result in the dissemination of pathogens and the contamination of surface water sources. Determining the die-off rate of pathogenic microorganisms in stream and sea waters may serve as the basis for evaluating the health risks posed by the presence of pathogens in seawater. This study was conducted to determine the effect of microbial load, temperature, salinity and turbidity on the die-off of oocysts of Cryptosporidium as compared to that of coxsackie A9 virus (Cox A9) and E. coli. The test microorganisms were seeded into stream, outfall or sea waters and incubated at either 30 degrees C (summer) or 15 degrees C (winter). At 30 degrees C, the fastest die-off was observed for Cox A9 where < 5-log was reduced regardless of the water quality. At 1 degrees C Cox A9 persistence was similar to that of Cryptosporidium where no change was detected in the concentration of either throughout the study period. E. coli die-off reached 5 orders of magnitude within 10d then its concentration remained unchanged. The die-off of E. coli was faster than observed for Cox A9 at 15 degrees C regardless of the water quality. No decrease was observed in the viability of Cryptosporidium under all tested conditions throughout the study period indicating the unsuitability of E. coli to serve as an indicator for the presence of parasites and viruses in stream and marine waters. The prolonged persistence of pathogenic microorganisms in marine waters suggested that discharge of contamination into streams may present a serious environmental health risk.

[The medical oncology clinic]. / La Clinique d'Oncologie Médicale.

The clinic of medical oncology is mainly devoted to the development of new anticancer treatments based on molecular biology and immunology. The clinic was the first in Belgium to start a protocol of gene therapy. Scientific contributions deal with the role of various oncogens in cell transformation, the interaction between cancer and the immune system and, new tools for the molecular diagnosis of cancers. Focus was particularly put on the development of new vectors for gene therapy and antitumor cell vaccines for cell therapy.

Controlled delivery of glial cell line-derived neurotrophic factor by a single tetracycline-inducible AAV vector.

An autoregulated tetracycline-inducible recombinant adeno-associated viral vector (rAAV-pTet(bidi)ON) utilizing the rtTAM2 reverse tetracycline transactivator (rAAV-rtTAM2) was used to conditionally express the human GDNF cDNA. Doxycycline, a tetracycline analog, induced a time- and dose-dependent release of GDNF in vitro in human glioma cells infected with rAAV-rtTAM2 serotype 2 virus. Introducing the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) downstream to the rtTAM2 coding sequence, resulted in a more rapid induction and a higher basal expression level. In vivo, 8 weeks after a single injection of the rAAV-rtTAM2-GDNF vector encapsidated into AAV serotype 1 capsids in the rat striatum, the GDNF protein level was 60 pg/mg tissue in doxycycline-treated animals whereas in untreated animals, it was undistinguishable from the endogenous level ( approximately 4 pg/mg tissue). However, a residual GDNF expression in the uninduced animals was evidenced by a sensitive immunohistochemical staining. As compared to rAAV1-rtTAM2-GDNF, the rAAV1-rtTAM2-WPRE-GDNF vector expressed a similar concentration of GDNF in the induced state (with doxycycline) but a basal level (without doxycycline) approximately 2.5-fold higher than the endogenous striatal level. As a proof for biological activity, for both vectors, downregulation of tyrosine hydroxylase was evidenced in dopaminergic terminals of doxycycline-treated but not untreated animals. In conclusion, the rAAV1-rtTAM2 vector which expressed biologically relevant doses of GDNF in the striatum in response to doxycycline with a basal level undistinguishable from the endogenous striatal level, as measured by quantitative ELISA assay, constitutes an interesting tool for local conditional transgenesis.

Quantification of the genotoxic effects of alkylating agents in bacterial assays.

The genotoxic effects of 11 monofunctional alkylating carcinogens in the Escherichia coli Multitest, SOS Chromotest and Salmonella assay were quantified using the 'yield versus lethal hit plot' method. In this method, the genotoxic yield-potency is defined as the integral under the yield versus lethal hit curve, whereas the genotoxic yield-efficiency is defined as the maximum yield obtained. With the series of substances tested, these two genotoxicity indices were found to be equivalent in the E. coli Multitest. The three bacterial short-term tests were compared and the following observations made: (i) the mutagenic efficiencies in the Salmonella assay and in the Multitest are correlated; (ii) the SOS-inducing efficiency in the SOS Chromotest correlates with both the SOS-inducing efficiency and the recombinogenic efficiency in the Multitest; (iii) mutagenesis and SOS induction measured by the same protocol in the Multitest are clearly distinct phenomena; (iv) when quantified with the 'yield versus lethal hit plot' method, the data from the SOS Chromotest do not correlate any more with the data from the Salmonella assay. Therefore the SOS Chromotest should be considered as a complementary rather than an alternative test to the Salmonella assay. Consequently, the Multitest appears as a relevant candidate for the basis of a battery of tests since it permits the measurement of at least two independent genotoxic effects in a single bacterial system. The method of quantification of genotoxic potencies using the 'yield versus lethal hit plot' method demonstrates the quantitative relationships existing between the data obtained using different systems measuring the same genotoxic effect and differentiates different genotoxic effects from each other.

Anthelmintic polyfunctional nitrogen-containing terpenoids from marine sponges.

The study of the anthelmintic terpenoid components from the Fiji sponge Axinyssa fenestratus (senior synonym of Leucophloeus fenestratus) and two Thailand sponges, Acanthella cavernosa and Topsentia sp., has yielded several new nitrogen-containing sesqui- and diterpenes of known carbon skeletons. Amorphane sesquiterpenes from A. fenestratus included (1R, 6S, 7S, 10S)-10-isothiocyanato-4-amorphene [(+)-1] and new metabolites (1R*, 4S*, 6R*, 7S*)-4-isothiocyanato-9-amorphene [2], 10-isothiocyanato-4,6-amorphadiene [3], and (4S*, 10S*)-10-isothiocyanato-5-amorphen-4-ol [4]. The amorphene (+)-1 of this study may be antipodal to (-)-1 previously isolated from a Hawaiian sponge. A similar relationship may exist at C-1, C-6, C-7 between (+)-2 of this study and (-)-11 isolated from a Palauian sponge. Another known sesquiterpene, axisonitrile 3 [5] was obtained from Topsentia sp. The diterpenes obtained from A. cavernosa included known kalihinols X [6] and Y [8] and new kalihinols J [7] and I [9]. Those terpenoids with potent antiparasite activity include 1, 2, 3-5, and 7-9.

Adeno-associated virus (AAV) as a vector for gene transfer into glial cells of the human central nervous system.

To examine the potential of AAV as a vector for gene transfer in glial cells, an established astrocytoma cell line and short-term cultures derived from human oligodendroglioma have been coinfected with AAV and helper adenovirus. The level of AAV replication in glioma cells was high indicating that they express receptors for AAV.

Use of an autonomous parvovirus vector for selective transfer of a foreign gene into transformed human cells of different tissue origins and its expression therein.

In this work, we report the transduction of a chloramphenicol acetyltransferase (CAT) reporter gene into a variety of normal and transformed human cells of various tissue origins. The vector used was MVM/P38cat, a recombinant of the prototype strain of the autonomous parvovirus minute virus of mice (MVMp). The CAT gene was inserted into the capsid-encoding region of the infectious molecular clone of MVMp genome, under the control of the MVM P38 promoter. When used to transfect permissive cells, the MVM/P38cat DNA was efficiently replicated and expressed the foreign CAT gene at high levels. By cotransfecting with a helper plasmid expressing the capsid proteins, it was possible to produce mixed virus stocks containing MVM/P38cat infectious particles and variable amounts of recombinant MVM. MVM/P38cat viral particles were successfully used to transfer the CAT gene and to express it in a variety of human cells. Both viral DNA replication and P38-driven CAT expression were achieved in fibroblasts, epithelial cells, T lymphocytes, and macrophages in a transformation-dependent way, but with an efficiency depending on the cell type. In transformed B lymphocytes, however, the vector was not replicated, nor did it express the CAT gene.