Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model.

Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in preclinical tests. For clinical translation, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, whereas human adenovirus type 5 (HAdV5) showed increased tropism toward glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity.

Impact of adenovirus life cycle progression on the generation of canine helper-dependent vectors.

Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a ~4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus-cell interaction.

Bioprocess development for canine adenovirus type 2 vectors.

Canine adenovirus type 2 (CAV-2) vectors overcome many of the clinical immunogenic concerns related to vectors derived from human adenoviruses (AdVs). In addition, CAV-2 vectors preferentially transduce neurons with an efficient traffic via axons to afferent regions when injected into the brain. To meet the need for preclinical and possibly clinical uses, scalable and robust production processes are required. CAV-2 vectors are currently produced in E1-transcomplementing dog kidney (DK) cells, which might raise obstacles in regulatory approval for clinical grade material production. In this study, a GMP-compliant bioprocess was developed. An MDCK-E1 cell line, developed by our group, was grown in scalable stirred tank bioreactors, using serum-free medium, and used to produce CAV-2 vectors that were afterwards purified using column chromatographic steps. Vectors produced in MDCK-E1 cells were identical to those produced in DK cells as assessed by SDS-PAGE and dynamic light scatering measurements (diameter and Zeta potential). Productivities of ∼10(9) infectious particles (IP) ml(-1) and 2 × 10(3) IP per cell were possible. A downstream process using technologies transferable to process scales was developed, yielding 63% global recovery. The total particles to IP ratio in the purified product (<20:1) was within the limits specified by the regulatory authorities for AdV vectors. These results constitute a step toward a scalable process for CAV-2 vector production compliant with clinical material specifications.

Evidence of founder chromosomes in fragile X syndrome.

The mutation responsible for fragile X syndrome and myotonic dystrophy involves the amplification of a simple trinucleotide repeat sequence, which increases in successive generations of affected pedigrees accounting for increasing penetrance of both disorders. This common molecular basis suggests that the two diseases may share other genetic features, but whereas myotonic dystrophy exhibits a significant founder chromosome effect, fragile X syndrome apparently has a very high mutation frequency. By haplotype analysis of microsatellite markers which flank the fragile X unstable element, we have uncovered evidence of founder chromosomes of the fragile X 'mutation'. Disorders caused by heritable unstable elements may therefore exhibit common genetic properties including anticipation and founder chromosomes.

[Indices of matrix metalloproteinases (MMP-2, MMP-9, TIMP-1) with juvenile arthritis in children].

UNLABELLED: One of the perspectives of modern rheumatology is the study of matrix metalloproteinases (MMP) in juvenile arthritis--enzymes that play a key role in the process of joint destruction. AIM: To analyse the content of matrix MMP-2 and MMP-9 and their tissue inhibitor (TIMP-1) in blood serum and synovial fluid in various embodiments of juvenile arthritis in children. PATIENTS AND METHODS: The study involved 82 children with juvenile arthritis, and 20 healthy children. The level of MMP-2, MMP-9 and TIMP-1 were determined in serum and synovial fluid by ELISA. RESULTS: It was found out that with any form of arthritis serum concentrations of MMP-2, MMP-9 and TIMP-1 was significantly higher than control values, but the level of MMP-2 in a subset of enthesitis-related arthritis, didn't differ from the control. Studied parameters in the synovial fluid were much higher than the serum level. With the development of uveitis TIMP-1 in blood serum was lower than in the absence of eye damage. On treatment of patients significant changes in the studied enzymes weren't established. On a good response to therapy the level of MMP-9 in serum decreased, on the lack of effectiveness - increased. CONCLUSIONS: The analysis results confirm the involvement of MMP-2, MMP-9 and TIMP-1 in the processes of inflammatory changes of the joints in juvenile idiopathic arthritis (JIA) and reactive arthritis, regardless the patients' sex or age.

[Immunophenotypic characteristic of dendritic cells in bronchial asthma in conditions of extract opisthorchis felineus in vitro].

This work shows comparative characteristics of immunogenic properties of the extract of Opisthorchis felineus in different severity of asthma using antigen presenting dendritic cells in vitro. New data on the effect of Opisthorchis felineus on the expression of surface markers of dendritic cells (CD209, HLA-DR, CD83, CD86) were received. Pronounced expression of CD209, CD86 and HLA-DR on the surface of dendritic cells in mild and severe asthma compared with healthy individuals was shown. In the stimulation of dendritic cells with extract Opisthorchis felineus in vitro weakening of CD86 expression in mild and in severe asthma was found. CD86 molecule may be a regulatory factor in the co-stimulation of dendritic cells which

Human innate lymphoid cell activation by adenoviruses is modified by host defense proteins and neutralizing antibodies.

Innate lymphoid cells (ILCs), the complements of diverse CD4 T helper cells, help maintain tissue homeostasis by providing a link between innate and adaptive immune responses. While pioneering studies over the last decade have advanced our understanding how ILCs influence adaptive immune responses to pathogens, far less is known about whether the adaptive immune response feeds back into an ILC response. In this study, we isolated ILCs from blood of healthy donors, fine-tuned culture conditions, and then directly challenged them with human adenoviruses (HAdVs), with HAdVs and host defense proteins (HDPs) or neutralizing antibodies (NAbs), to mimic interactions in a host with pre-existing immunity. Additionally, we developed an ex vivo approach to identify how bystander ILCs respond to the uptake of HAdVs ± neutralizing antibodies by monocyte-derived dendritic cells. We show that ILCs take up HAdVs, which induces phenotypic maturation and cytokine secretion. Moreover, NAbs and HDPs complexes modified the cytokine profile generated by ILCs, consistent with a feedback loop for host antiviral responses and potential to impact adenovirus-based vaccine efficacy.

Early production of table olives at a mid-7th millennium BP submerged site off the Carmel coast (Israel).

We present here the earliest evidence for large-scale table olive production from the mid-7th millennium BP inundated site of Hishuley Carmel on the northern Mediterranean coast of Israel. Olive pit size and fragmentation patterns, pollen as well as the architecture of installations associated with pits from this site, were compared to finds from the nearby and slightly earlier submerged Kfar Samir site. Results indicate that at Kfar Samir olive oil was extracted, while at Hishuley Carmel the data showed that large quantities of table olives, the oldest reported to date, were prepared. This process was most probably facilitated by the site's proximity to the Mediterranean Sea, which served as a source of both sea water and salt required for debittering/pickling/salting the fruit, as experimentally demonstrated in this study. Comparison of pit morphometry from modern cultivars, wild-growing trees and the archaeological sites, intimates that in pit morphology the ancient pits resemble wild olives, but we cannot totally exclude the possibility that they derive from early cultivated trees. Our findings demonstrate that in this region, olive oil production may have predated table olive preparation, with each development serving as a milestone in the early exploitation of the olive.

Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n.

The sequence of a Pst I restriction fragment was determined that demonstrate instability in fragile X syndrome pedigrees. The region of instability was localized to a trinucleotide repeat p(CCG)n. The sequence flanking this repeat were identical in normal and affected individuals. The breakpoints in two somatic cell hybrids constructed to break at the fragile site also mapped to this repeat sequence. The repeat exhibits instability both when cloned in a nonhomologous host and after amplification by the polymerase chain reaction. These results suggest variation in the trinucleotide repeat copy number as the molecular basis for the instability and possibly the fragile site. This would account for the observed properties of this region in vivo and in vitro.

Fragile X genotype characterized by an unstable region of DNA.

DNA sequences have been located at the fragile X site by in situ hybridization and by the mapping of breakpoints in two somatic cell hybrids that were constructed to break at the fragile site. These hybrids were found to have breakpoints in a common 5-kilobase Eco RI restriction fragment. When this fragment was used as a probe on the chromosomal DNA of normal and fragile X genotype individuals, alterations in the mobility of the sequences detected by the probe were found only in fragile X genotype DNA. These sequences were of an increased size in all fragile X individuals and varied within families, indicating that the region was unstable. This probe provides a means with which to analyze fragile X pedigrees and is a diagnostic reagent for the fragile X genotype.

Implications of FRA16A structure for the mechanism of chromosomal fragile site genesis.

Fragile sites are chemically induced nonstaining gaps in chromosomes. Different fragile sites vary in frequency in the population and in the chemistry of their induction. DNA sequences encompassing and including the rare, autosomal, folate-sensitive fragile site, FRA16A, were isolated by positional cloning. The molecular basis of FRA16A was found to be expansion of a normally polymorphic p(CCG)n repeat. This repeat was adjacent to a CpG island that was methylated in fragile site-expressing individuals. The FRA16A locus in individuals who do not express the fragile site is not a site of DNA methylation (imprinting), which suggests that the methylation associated with fragile sites may be a consequence and not a cause of their genesis.

A Poincaré's approach for plasmonics: the plasmon localization.

A Poincaré's approach is employed to characterize the excitation of a plasmon, which in essence corresponds to a zero of a complex S-matrix. Throughout this work we study the plasmonic behaviour of gold, as this metal not only is frequently used in experimental arrays, but also requires an accurate dispersion model to properly excite plasmons. We investigate the plasmonic behaviour of gold nanogratings by means of Born's approximation and the Finite-Elements Method. Also, a method based on the Poincaré's approach is proposed to optimize this kind of structures.

Prevention of bleomycin-induced pulmonary fibrosis after adenovirus-mediated transfer of the bacterial bleomycin resistance gene.

A serious limitation in the use of the DNA-cleaving, antitumoral-antibiotic, bleomycin during chemotherapy is pulmonary toxicity. Lung injury induced by bleomycin is characterized by an increased deposition of interstitial extracellular matrix proteins in the alveolar wall that compromises respiratory function. Several drugs have been tested in animal models to prevent the pulmonary toxicity of bleomycin, but have not led to a useful clinical treatment because of their adverse effects on other tissues. We have shown that transgenic mice expressing Streptoalloteichus hindustanus (Sh) ble bleomycin resistance protein in pulmonary epithelial cells in the lungs are protected against bleomycin-induced toxicity in lungs. In the present study, we used intranasal administration by adenovirus-mediated gene transfer of the bleomycin resistance Sh ble gene to mouse lung for prevention of bleomycin-induced pulmonary fibrosis. We constructed recombinant adenoviruses Ad.CMVble and Ad.RSVble harboring the bleomycin resistance Sh ble gene under the control of the cytomegalovirus early promoter and the Rous sarcoma virus early promoter, respectively. Transgene expression was detected in epithelia of conducting airways and alveolar septa by immunostaining with a rabbit polyclonal antibody directed against the bleomycin resistance protein and persisted for the duration of drug treatment; i.e., up to 17 d. No toxic effect was seen in adenovirus-treated mice. Pretreatment of mice with Ad.CMVble or Ad.RSVble completely prevented collagen deposition 42-133 d after bleomycin treatment, as measured by lung OH-proline content. Histologic studies indicated that there was little or no lung injury in the adenovirus/bleomycin-treated mice compared with the bleomycin-treated mice. These observations may lead to new approaches for the prevention of bleomycin-induced pulmonary fibrosis.

The conundrum between immunological memory to adenovirus and their use as vectors in clinical gene therapy.

In the context of clinical gene transfer using viral vectors, the risk of memory antivector immunity is often poorly appreciated. The immunological past of the patient, the site of injection, and the vector dose will play intertwined and decisive roles in the safety and efficacy of treatment. To circumvent the drawbacks due to the ubiquitous human adenovirus (HAd) memory immunity, we believe that vectors derived from canine adenovirus type 2 (CAV-2) will be more clinically useful than those derived from HAds based, in part, on the potential lack of immunological memory. CAV-2 is not a human pathogen in spite of the approx 100,000 yr of cohabitation of humans with dogs. During the last 8 yr, we found that CAV-2 vectors preferentially transduced neurons in the central nervous system (CNS) of several species, and had a surprisingly efficient level of axoplasmic transport. CAV-2 vectors also lead to greater than 1 yr transgene expression in the immunocompetent rat CNS-without immunosuppression. However, more immediate harm can be caused to a patient via an acute and/or chronic vector-induced cellular infiltration in the CNS than by the normal progression of most neurodegenerative disorders. In this context, we continue to assess the clinical potential of CAV-2. This mini-review addresses our analysis of the interaction of CAV-2 vectors with human memory immunity and monocyte-derived dendritic cells.

Copper complexes with heterocyclic sulfonamides: synthesis, spectroscopic characterization, microbiological and SOD-like activities: crystal structure of [Cu(sulfisoxazole)2(H2O)4] . 2H2O.

The synthesis, characterization and comparative biological study of a series of antibacterial copper complexes with heterocyclic sulfonamides were reported. Two kinds of complexes were obtained with the stoichiometries [Cu(L)2] . H2O and [Cu(L)2(H2O)4] . nH2O. They were characterized by infrared and electronic spectroscopies and the crystal structure of [Cu(sulfisoxazole)2(H2O)4] . 2H2O was determined by single crystal X-ray diffraction. It crystallized in the C2/c with Z = 8 monoclinic space group C2/c with Z = 8. The Cu(II) is in a slightly tetragonal distorted octahedron formed by four oxygen atoms from water molecules and two nitrogen atoms from two isoxazole rings. The antimicrobial activity was evaluated for all the synthesized complexes and ligands using the agar dilution test. The results showed that the complexes with five-membered heterocyclic rings were more active than the free sulfonamides while the pyrimidine, pyridine and pyridazine complexes had similar or less activity than the free ligands. In order to find an explanation for this behavior lipophilicity and superoxide dismutase-like activity were tested, showing that the [Cu(sulfamethoxazol)2(H2O)4] . 3H2O presented the highest antimicrobial potency and a superoxide dismutase-like activity comparable with pharmacological active compounds.

Preferential transduction of neurons by canine adenovirus vectors and their efficient retrograde transport in vivo.

In the central nervous system (CNS), there are innate obstacles to the modification of neurons: their relative low abundance versus glia and oligodendrocytes, the inaccessibility of certain target populations, and the volume one can inject safely. Our aim in this study was to characterize the in vivo efficacy of a novel viral vector derived from a canine adenovirus (CAV-2). Here we show that CAV-2 preferentially transduced i) rat olfactory sensory neurons; ii) rodent CNS neurons in vitro and in vivo; and, more clinically relevant, iii) neurons in organotypic slices of human cortical brain. CAV-2 also showed a high disposition for retrograde axonal transport in vivo. We examined the molecular basis of neuronal targeting by CAV-2 and suggest that due to CAR (coxsackie adenovirus receptor) expression on neuronal cells-and not oligodendrocytes, glia, myofibers, and nasal epithelial cells-CAV-2 vectors transduced neurons preferentially in these diverse tissues.

Strategies for cancer gene therapy using adenoviral vectors.

Modification of tumor cells using gene transfer either to enhance host immunity or to act directly on tumor cells is being intensively studied in animal models. Remarkable results have yielded to approved clinical protocols in the treatment of cancer patients using this approach. Several methods of gene delivery have been developed. This article is particularly devoted to the interest of the use of adenoviral vectors in the different strategies of cancer gene therapy.

Hydroxyapatite induces autolytic degradation and inactivation of matrix metalloproteinase-1 and -3.

In the course of studies to identify a protease capable of producing a long-lived 50 kDa fragment of bone acidic glycoprotein-75 (BAG-75), it was observed that incubation of matrix metalloproteinase (MMP)-3 (stromelysin 1) with preparations of BAG-75 led to inactivation of proteolytic function, e.g., an inability to fragment 125I-labeled BAG-75 added subsequently. MMP-1 (interstitial collagenase) was also inactivated by exposure to BAG-75 preparations. Investigation of the mechanism revealed that BAG-75 preparations contained millimolar levels of inorganic phosphate which formed hydroxyapatite crystals under digestion conditions. Hydroxyapatite crystals alone and in BAG-75-hydroxyapatite complexes induced the autolytic degradation of both active and precursor forms of MMP-1 and MMP-3. Autolytic degradation in the presence of hydroxyapatite was demonstrated by a loss in catalytic function assayed with peptide and/or protein substrates, and, by fragmentation into polypeptides of <10 kDa. The fate of MMP-3 incubated with hydroxyapatite depends upon the time of incubation, the free calcium concentration, and the concentration of crystals. Specifically, hydroxyapatite-induced autolysis requires a near physiological free calcium concentration of 0.5-1.0 mM. Autolysis was maximal in the presence of 150 microg/ml hydroxyapatite where MMP-3 was only partially bound to crystals. However, autolysis also occurred at higher crystal concentrations where all input MMP-3 was bound (>1000 microg/ml), suggesting that autolysis may be mediated by bound enzyme. The effect of hydroxyapatite appears to be specific for MMP-1 and MMP-3 since the catalytic activity of chymotrypsin, trypsin, papain, and thermolysin remained unchanged after exposure to hydroxyapatite. These results document for the first time a novel catalytic role for hydroxyapatite crystals in vitro and provide an initial biochemical characterization of the intermolecular, autolytic, calcium ion-dependent, matrix metalloproteinase-specific degradative mechanism.

Trafficking and propagation of canine adenovirus vectors lacking a known integrin-interacting motif.

Canine adenovirus type 2 (CAV-2) vectors may be attractive tools for gene transfer thanks to the lack of pre-existing immunity in humans, and because of the preferential transduction of neurons when the vector is injected into the brain and some innervated tissues. The coxsackievirus-adenovirus receptor (CAR) appears to play a major role during infection of most human serotypes, whereas the alpha(v)beta(3/5) integrins have been reported to play a significant auxiliary role. We showed that CAV-2 also attaches to and uses CAR to enter cells, but CAV-2 transduction could be notably different from that of the prototype human adenovirus serotype 5 (Ad5). Initially, the CAV-2 capsid appears to be 10-fold less negatively charged than Ad5. Second, the CAV-2 penton, hexon, and fiber proteins do not contain a known integrin-interacting motif. Because of its potential interest in the clinic, we analyzed the different steps of cellular trafficking and the propagation kinetics of CAV-2 vectors. We found that Ad5 and CAV-2 vectors have comparable kinetics of binding (10 min), internalization (10 min), endosomal escape (17 min), attachment to the nuclear membrane (35 min), and formation (18 hr) and release (34 hr) of functional virions. Surprisingly, the RGD(-) CAV-2 capsid also induced the reorganization of actin filaments in HeLa cells. Actin reorganization is thought to be dependent on alpha(v)beta(3/5) integrin stimulation.

A recombinant E1-deleted canine adenoviral vector capable of transduction and expression of a transgene in human-derived cells and in vivo.

Human adenovirus (HAV) serotypes 2 and 5 are commonly used as vector backbones for adenovirus-mediated gene transfer. However, HAVs were chosen as a backbone for the vectors for historical reasons and have a number of significant disadvantages when used as a shuttle for gene transfer in humans. As an initial trial to circumvent some of the shortcomings of HAV vectors, we have produced an E1-deleted canine adenovirus type 2 (CAV-2) vector for gene transfer. Initially, we demonstrated that CAV-2 undergoes an abortive viral cycle in a wide range of human-derived cell lines. Second, we assayed human sera containing HAV-5 neutralizing antibodies for their ability to inhibit CAV-2-induced plaques on permissive cells. In the cohort tested, our data demonstrate that the humoral response directed against HAV-5 does not inhibit CAV-2 plaque formation in the majority of cases. Canine cell lines expressing the E1 region of CAV-2 were generated and characterized. A recombinant CAV vector (CAVRSVbetagal) deleted in the E1 region and harboring lacZ was constructed. We show that CAVRSVbetagal is able to transduce and direct expression of the transgene in vitro in a variety of mammalian cells, most notably primary human-derived cells. In addition, gene transfer is demonstrated in vivo using chick embryos.