Transient immunosuppression allows transgene expression following readministration of adeno-associated viral vectors

The effects of external pH (pHout) variations on the Na+ and on the Ca2+ dependent fractions of the evoked amino acid neurotransmitter release were separately investigated, using GABA as a model transmitter. In [3H]GABA loaded mouse brain synaptosomes, the external acidification (pHout 6.0) markedly decreased the Na+ dependent fraction of [3H]GABA release evoked by veratridine (10 microM) in the absence of external Ca2+, as well as the Ca2+ dependent fraction of [3H]GABA release evoked by high (20 mM) K+ in the absence of external Na+. The depolarization-induced elevation of [Na(i)] (monitored in synaptosomes loaded with the Na+ indicator dye, SBFI) and the depolarization-induced elevation of [Ca(i)] (monitored in synaptosomes loaded with the Ca2+ indicator dye fura-2) were also markedly decreased at pHout 6. On the contrary, the external alkalinization (pHout 8) facilitated all the above responses. A slight increase of the baseline release of the [3H]GABA was observed when pHout was changed from 7.4 to 8. This effect was only observed in the presence of Ca2+. pHout changes from 7.4 to 6 or to 7 did not modify the baseline release of the transmitter. All the effects of pHout variations on [3H]GABA release were independent on the presence of HCO3-. It is concluded that external H+ regulate amino acid neurotransmitter release by their actions on presynaptic Na+ channels, as well as on presynaptic Ca2+ channels.     

Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration

The ability of recombinant adeno-associated virus (AAV) vectors to integrate into the host genome and to transduce nondividing cells makes them attractive as vehicles for gene delivery. In this study, we assessed the ability of several AAV vectors to transduce airway cells in rabbits by measuring marker gene expression. AAV vectors that transferred either a beta-galactosidase (beta-gal) or a human placental alkaline phosphatase (AP) gene were delivered to one lobe of the rabbit lung by use of a balloon catheter placed under fluoroscopic guidance. We observed vector-encoded beta-gal or AP staining almost exclusively in the epithelial and smooth muscle cells in the bronchus at the region of balloon placement. The overall efficiency of transduction in the balloon-treated bronchial epithelium was low but reached 20% in some areas. The majority of the staining was in ciliated cells but was also observed in basal cells and airway smooth muscle cells. We observed an 80-fold decrease in marker-positive epithelial cells during the 60-day period after vector infusion, whereas the number of marker-positive smooth muscle cells stayed constant. Although treatment with the topoisomerase inhibitor etoposide dramatically enhanced AAV transduction in primary airway epithelial cells in culture, treatment of rabbits did not improve transduction rates in the airway. Vector readministration failed to produce additional transduction events, which correlated with the appearance of neutralizing antibodies. These results indicate that both readministration and immune modulation will be required in the use of AAV vectors for gene therapy to the airway epithelium.     

Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus

Recently, efficient and long-term in vivo gene transfer by recombinant adeno-associated virus type 2 (rAAV) vectors has been demonstrated in a variety of tissues. Further improvement in vector titer and purity will expedite this in vivo exploration and provide preclinical information required for use in human gene therapy. In an effort to obtain higher titers, we constructed a novel AAV helper plasmid which utilizes translational control of AAV Rep genes (J. Li et al., J. Virol. 71:5236-5243, 1997). To address the issue of purity, in this study we report the first rAAV production method which is completely free of adenovirus (Ad) helper virus. The new production system uses a plasmid construct which contains a mini-Ad genome capable of propagating rAAV in the presence of AAV Rep and Cap genes. This construct is missing some of the early and most of the late Ad genes and is incapable of producing infectious Ad. Transfection of 293 cells with the new mini-Ad helper and AAV packaging plasmids results in high-titer rAAV vectors with yields greater than 1,000 transducing units, or 10(5) viral particles per cell. When rAAV vectors were produced by using this production scheme and compared to traditional heat-inactivated rAAV preparations in vitro and in vivo, we observed transduction equivalent to or better than normal levels. The complete removal of infectious Ad from AAV production should facilitate a better understanding of immune response to AAV vectors in vivo, eliminate the need for developing replication-competent Ad assays, and provide a more defined reagent for clinical use.     

Optimization of packaging of adeno-associated virus gene therapy vectors using plasmid transfections

A method is described for the analysis of amino acids, monoamines and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC-ED) from individual brain areas. The chromatographic separations were achieved using microbore columns. For amino acids we used a 100x1 mm I.D. C8, 5 microm column. A binary mobile phases was used: mobile phase A consisted of 0.1 M sodium acetate buffer (pH 6.8)-methanol-dimethylacetamide (69:24:7, v/v) and mobile phase B consisted of sodium acetate buffer (pH 6.8)-methanol-dimethylacetamide (15:45:40, v/v). The flow-rate was maintained at 150 microl/min. For monoamines and metabolites we used a 150X1 mm I.D. C18 5 microm reversed-phase column. The mobile phase consisted of 25 mM monobasic sodium phosphate, 50 mM sodium citrate, 27 microM disodium EDTA, 10 mM diethylamine, 2.2 mM octane sulfonic acid and 10 mM sodium chloride with 3% methanol and 2.2% dimethylacetamide. The potential was +700 mV versus Ag/AgCl reference electrode for both the amino acids and the biogenic amines and metabolites. Ten rat brain regions, including various cortical areas, the cerebellum, hippocampus, substantia nigra, red nucleus and locus coeruleus were microdissected or micropunched from frozen 300-microm tissue slices. Tissue samples were homogenized in 50 or 100 microl of 0.05 M perchloric acid. The precise handling and processing of the tissue samples and tissue homogenates are described in detail, since care must be exercised in processing such small volumes while preventing sample degradation. An aliquot of the sample was derivatized to form the tert.-butylthiol derivatives of the amino acids and gamma-aminobutyric acid. A second aliquot of the same sample was used for monamine and metabolite analyses. The results indicate that the procedure is ideal for processing and analyzing small tissue samples.     

Efficient expression of CFTR function with adeno-associated virus vectors that carry shortened CFTR genes

Adeno-associated virus (AAV)-based vectors have been shown to be effective in transferring the cystic fibrosis gene (CFTR) into airway epithelial cells in animal models and in patients. However, the level of CFTR gene expression has been low because the vector cannot accommodate the CFTR gene together with a promoter. In this study, we described a strategy to reduce the size of the CFTR cDNA to allow the incorporation of an effective promoter with the CFTR gene into AAV vectors. We engineered and tested 20 CFTR mini-genes containing deletions that were targeted to regions that may contain nonessential sequences. Functional analyses showed that four of the shortened CFTRs (one with combined deletions) retained the function and the characteristics of a wild-type CFTR, as measured by open probability, time voltage dependence, and regulation by cAMP. By using an AAV vector with a P5 promoter, we transduced these short forms of CFTR genes into target cells and demonstrated high levels of CFTR expression. We also demonstrated that smaller AAV/CFTR vectors with a P5 promoter expressed the CFTR gene more efficiently than larger vectors or a vector in which CFTR gene was expressed from the AAV inverted terminal repeat sequence. The CFTR mini-gene with combined deletions was packaged into AAV virions more efficiently, generated higher titers of transducing virions, and more effectively transferred CFTR function into target cells. These new vectors should circumvent the limitations of AAV vector for CFTR expression. Our strategy also may be applicable to other genes, the sizes of which exceed the packaging limit of an AAV vector.     

Preferred-pattern formation during the initial transient in cellular solidification

Critical information on the pending problem of preferred-pattern formation and wavelength selection in cellular directional solidification is currently expected from the study of initial transients. Therefore, experiments were carried out in lead—thallium alloys to analyse the dynamical process by which a periodic array of cells is formed by using two classical but different experimental procedures, one violent and one soft. The statistical analysis of pattern characteristics (average wavelength, level of disorder) shows that both procedures converge towards a common asymptotic state, which means that a preferred pattern does exist. Although different, the initial transients are qualitatively similar. It is found that in both cases, the solid—liquid interface is first dendritic and then restabilizes into a cellular array. Oscillations, in periodicity and in the sense of disorder, are observed, that are associated to the repetition of phases of cell divisions, probably by tip-splitting, separated by rearrangement periods.     

Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors

Recombinant adeno-associated virus (AAV) vectors have been used to transduce murine skeletal muscle as a platform for secretion of therapeutic proteins. The utility of this approach for treating alpha-1-antitrypsin (AAT) deficiency was tested in murine myocytes in vitro and in vivo. AAV vectors expressing the human AAT gene from either the cytomegalovirus (CMV) promoter (AAV-C-AT) or the human elongation factor 1-alpha promoter (AAV-E-AT) were examined. In vitro in C2C12 murine myoblasts, the expression levels in transient transfections were similar between the two vectors. One month after transduction, however, the human elongation factor 1 promoter mediated 10-fold higher stable human AAT expression than the CMV promoter. In vivo transduction was performed by injecting doses of up to 1.4 x 10(13) particles into skeletal muscles of several mouse strains (C57BL/6, BALB/c, and SCID). In vivo, the CMV vector mediated higher levels of expression, with sustained serum levels over 800 micrograms/ml in SCID and over 400 micrograms/ml in C57BL/6 mice. These serum concentrations are 100,000-fold higher than those previously observed with AAV vectors in muscle and are at levels which would be therapeutic if achieved in humans. High level expression was delayed for several weeks but was sustained for over 15 wk. Immune responses were dependent upon the mouse strain and the vector dosage. These data suggest that recombinant AAV vector transduction of skeletal muscle could provide a means for replacing AAT or other essential serum proteins but that immune responses may be elicited under certain conditions.     

Robust, but transient expression of adeno-associated virus-transduced genes during human T lymphopoiesis

Recombinant adeno-associated viruses (rAAV) have been proposed to be gene transfer vehicles for hematopoietic stem cells with advantages over other virus-based systems due to their high titers and relative lack of dependence on cell cycle for target cell integration. We evaluated rAAV vector containing a LacZ reporter gene under the control of a cytomegalovirus (CMV) promoter in the context of primary human CD34+CD2- progenitor cells induced to undergo T-cell differentiation using an in vitro T-lymphopoiesis system. Target cells from either adult bone marrow or umbilical cord blood were efficiently transduced, and 71% to 79% CD2+ cells expressed a LacZ marker gene mRNA and produced LacZ-encoded protein after exposure to rAAV-CMV-LacZ. The impact of transgene expression on the differentiation of T cells was assessed by sequential quantitation of immunophenotypic subsets of virus-exposed cells and no alteration was noted compared with control. The durability of transgene expression was assessed and found to decay by day 35 with kinetics dependent on the multiplicity of infection. In addition, vector DNA was absent from CD4 or CD8 subselected CD3+ cells by DNA-polymerase chain reaction. These data suggest that rAAV vectors may result in robust transgene expression in primitive cells undergoing T-cell lineage commitment without toxicity or alteration in the pattern of T-cell differentiation. However, expression is transient and integration of the transgene unlikely. Recombinant AAV vectors are potentially valuable gene transfer tools for the genetic manipulation of events during T-cell ontogony but their potential in gene therapy strategies for diseases such as acquired immunodeficiency syndrome is limited.     

NMDA receptor-mediated refinement of a transient retinotectal projection during development requires nitric oxide

We recently developed a simple new method which is designed to separate and concentrate bacteria from a sample by centrifugation in a gel system. Bacterial enzyme activity is then detected inside the gel without further manipulation using a colorimetric or fluorogenic substrate. The method provides a rapid, direct means of detecting bacteria in clinical samples, dispensing with the 24-h period normally required to isolate colonies on agar. Various applications of the method are described below, e.g. screening of negative urine samples, identification of Escherichia coli in urine samples, identification of Staphylococcus aureus in blood culture broths and detection of oxacillin-resistant S. aureus in blood culture broths. The advantages of the gel system and other applications are discussed.     

In vitro cultivation of mouse embryos during the initial stages of organogenesis

Optimal conditions for in vitro cultivation of mouse embryos were sought. The embryos at 8--9.5 days of development were explanted into the culture in rotating tubes. A number of nutritional media were studied: the human umbilical blood serum; the rat blood serum, as well as its mixture (1 : 1) with Eagle's medium. It was demonstrated that the optimal medium for cultivation of 8-day-old embryos was the mixture of the rat blood serum with Eagle's medium, and for 8.5--9.5-day-old embryos--the rat blood serum, as well. Comparing in vitro and in vivo embryonic development, it was evident that in the cultivated embryos the parameters characterizing their growth decreased significantly. Possible causes of this phenomenon are discussed.     

The specificity of immunosuppression by alloantibody in the mouse

The ability of alloantibody to suppress the humoral immune response to transplantation antigens in the mouse has been studied in relation to its specificity. It was found that antibody was able to suppress the cytotoxic antibody response to determinants other than those against which it was directed when there was some common antigenicity between the different H-2 types used for immunising. It was also found that suppression of the response to both parental haplotypes of an F1-hybrid cell could be achieved by antibody to only one parental haplotype, the antibody being made in the other parental strain to avoid any possibility of cross-reactivity. Such nonspecific immunosuppression was not achieved when cells of the two parental strains were administered mixed together.     

Efficient coexpression and secretion of anti-atherogenic human apolipoprotein AI and lecithin-cholesterol acyltransferase by cultured muscle cells using adeno-associated virus plasmid vectors

Plasma apolipoprotein AI (apoAI) and lecithin-cholesterol acyltransferase (LCAT) play important roles in reverse cholesterol transport, promoting the removal of excess cholesterol from peripheral cells and reducing formation of atherosclerotic lesions. Gene augmentation of either apoAI or LCAT, or both, are thus attractive targets for prevention or treatment of atherosclerosis. With the eventual aim of safe and efficient gene delivery to skeletal muscle, our chosen secretory platform for systemic delivery of anti-atherogenic proteins, we have constructed conventional and AAV-based plasmid vectors containing human apoAI or LCAT cDNAs; their efficacy was tested by lipoplex transfection of mouse C2C12 muscle cells or human 293 cells. The secretion of apoAI or LCAT by transduced cultures was two- to five-fold higher using AAV-based plasmid vectors than conventional plasmid vectors. Additionally, cells transfected with a bicistronic AAV-based vector containing an internal ribosome entry site (IRES) efficiently expressed both apoAI and LCAT simultaneously. Furthermore, AAV-based vector sequences were retained by host cells, whereas those of conventional plasmid vectors were lost. These studies indicate that ectopic overexpression of apoAI and LCAT in muscle tissue using AAV-based plasmid vectors might provide a feasible anti-atherogenic strategy in vivo.     

The regulatory rep protein of adeno-associated virus binds to sequences within the c-H-ras promoter

Brain-retrocerebral complexes of female crickets, Gryllus bimaculatus and Acheta domesticus, treated with antibody to allatostatin-1 from a cockroach, Diploptera punctata, show extensive immunoreactivity. The results suggest that allostatins or allatostatin-like molecules are produced in neurosecretory cells of the brain and are delivered to the corpora allata through nervous connections and/or via haemolymph. Radiochemical measurements of juvenile hormone III biosynthesis by isolated corpora cardiaca-corpora allata complexes from adult G. bimaculatus have been used to demonstrate an in vitro sensitivity of these glands to allatostatin-1 from D. punctata. Allatostatin-1 is a relatively potent inhibitor of juvenile hormone III biosynthesis in corpora allata of both young adult females and males. In glands taken from 3-day virgin females, 50% inhibition of hormone biosynthesis is reached at ca. 3 nmol.l-1 allatostatin-1. The inhibitory action of allatostatin-1 is rapid, dose-dependent and reversible. Addition of 200 mumol.l-1 farnesol to the incubation medium prevents inhibition of juvenile hormone III biosynthesis by allatostatin-1. Juvenile hormone III biosynthesis by isolated corpora allata of 3-day female house crickets, A. domesticus, is also susceptible to inhibition by 1 mumol.l-1 allatostatin-1.     

Promotion of N-nitrosodimethylamine-initiated mouse lung tumors following single or multiple low dose exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin

The environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is highly toxic to several rodent species and may have adverse health effects in exposed human populations. Further, TCDD has been shown to be a potent liver tumor promoter in the rat after repeated administration. These studies were conducted to determine the tumor promoting capability of TCDD in the Swiss mouse following single or multiple exposures. Following tumor initiation with N-nitrosodimethylamine (NDMA; 25 mg/kg), animals were given either a single dose (1.6, 16 or 48 micrograms/kg) or repeated injections (0.05 microgram/kg/week for 20 weeks) of TCDD and sacrificed at 52 weeks of age. Neither NDMA nor TCDD caused an increase in incidence of liver tumors. NDMA induced lung tumors in 100% of animals, with 12 +/- 0.1 tumors/mouse. The multiplicity of lung tumors was significantly increased by low dose TCDD treatment, with 20 +/- 2.6 tumors/mouse following a single 1.6 micrograms/kg dose (P = 0.016) and 18 +/- 1.7 (P = 0.031) following repeated 0.05 microgram/kg doses (x 20). Higher doses of TCDD did not increase multiplicity of lung tumors and, in fact, may have been toxic to the lungs of NDMA-treated mice, as evidenced by the infiltration of pigmented macrophages. These data demonstrate the potent tumor promoting capability of TCDD in mouse lung.     

The transient to steady-state transition during the spray-rolling process

From the geometrical standpoint, this article presents a qualitative theoretical analysis and prediction of the transient to steady-state transition during the spray-rolling process, a novel manufacturing technique for aluminum strips. The analytical results indicate that, when the deposited materials at the specific points on one roll surface overlap their counterparts on the other roll surface, spray rolling transits from the transient state to the steady state. The specific points are the limiting deposition positions of the atomized droplets on the roll surface initially.     

Structure of adeno-associated virus vector DNA following transduction of the skeletal muscle

The skeletal muscle provides a very permissive physiological environment for adeno-associated virus (AAV) type 2-mediated gene transfer. We have studied the early steps leading to the establishment of permanent transgene expression, after injection of recombinant AAV (rAAV) particles in the quadriceps muscle of mice. The animals received an rAAV encoding a secreted protein, murine erythropoietin (mEpo), under the control of the human cytomegalovirus major immediate-early promoter and were sacrificed between 1 and 60 days after injection. The measurement of plasma Epo levels and of hematocrits indicated a progressive increase of transgene expression over the first 2 weeks, followed by a stabilization at maximal plateau values. The rAAV sequences were analyzed by Southern blotting following neutral or alkaline gel electrophoresis of total DNA from injected muscles. While a high number of rAAV sequences were detected during the first 5 days following the injection, only a few percent of these sequences was retained in the animals analyzed after 2 weeks, in which transgene expression was maximal. Double-stranded DNA molecules resulting from de novo second-strand synthesis were detected as early as day 1, indicating that this crucial step of AAV-mediated gene transfer is readily accomplished in the muscle. The templates driving stable gene expression at later time points are low in copy number and structured as high-molecular-weight concatemers or interlocked circles. The presence of the circular form of the rAAV genomes at early time points suggests that the molecular transformations involved in the formation of stable concatemers may involve a rolling-circle type of DNA replication.     

Word context during initial exposure influences degree of priming in word fragment completion.

Priming in word fragment completion is revealed by the increased probability of correctly completing a fragment like "_ll_p_e' when the word "ellipse' was seen recently. Three experiments investigated the effects on priming of manipulating the context in which the words were seen. Three principal results emerged. Experiments 1 and 2 demonstrated that there was much more priming for words studied in a to-be-learned list than read in meaningful passages. In these same two experiments, low-frequency words were subject to more priming than were higher frequency words, regardless of context. Experiment 3 revealed more priming for words when they did not fit sensibly into connected discourse than when they did. The results suggest that context plays a critical role in priming: As a word moves from being contextually bound in meaningful discourse to being isolated in a list, its probability of priming increases. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Prevention of rapid reperfusion-induced lung injury with prostaglandin E1 during the initial period of reperfusion

We have found that the instantaneous restoration of blood flow causes acute dysfunction and massive edema in rat lungs after 4 hours of room temperature ischemia. This is associated with an early increase in pulmonary artery pressure (Ppa) and can be prevented by a stepwise increase in flow rate during the first 10 minutes of reperfusion. The objectives of this study were to determine whether rapid reperfusion causes lung injury after hypothermic preservation, and whether this injury can be attenuated by a short-course of prostaglandin E1 (PGE1). Rat lungs were flushed preserved with low-potassium dextran solution for 12 hours at 4 degrees C and randomly divided into three groups: (1) control (no PGE1); (2) PGE1 only in the flush solution; and (3) PGE1 in both flush solution and blood perfusate during the first 10 minutes of reperfusion. Postpreservation pulmonary function was assessed in an isolated rat lung reperfusion model developed previously. We found that rapid initiation of reperfusion led to significant pulmonary dysfunction, which was attenuated by a short-course of PGE1 in the blood perfusate. The addition of PGE1 to the flush solution alone did not have such an effect. Administration of PGE1 to the blood perfusate during the first 10 minutes resulted in significant lower Ppa and airway pressure and better gas exchange. There was a positive correlation between the peak Ppa during the first 10 minutes of reperfusion and the final shunt fraction. The physical forces generated by the rapid initiation of blood reperfusion appear to induce severe injury. The first 10 minutes of reperfusion seem to be a transition phase in which mechanical factors play an important role relating to ultimate post reperfusion lung function. A short course of PGE1 may be a useful maneuver to prevent rapid reperfusion-induced lung injury.     

Behavioral recovery in 6-hydroxydopamine-lesioned rats by cotransduction of striatum with tyrosine hydroxylase and aromatic L-amino acid decarboxylase genes using two separate adeno-associated virus vectors

Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. A promising approach to the gene therapy of PD is intrastriatal expression of enzymes in the biosynthetic pathway for dopamine. Tyrosine hydroxylase (TH) catalyzes the synthesis of L-dopa, which must be converted to dopamine by aromatic L-amino acid decarboxylase (AADC). Since the endogenous AADC activity in the striatum is considered to be low, coexpression of both TH and AADC in the same striatal cells would increase the dopamine production and thereby augment the therapeutic effects. In the present study, the TH gene and also the AADC gene were simultaneously transduced into rat striatal cells, using two separate adeno-associated virus (AAV) vectors, AAV-TH and AAV-AADC. Immunostaining showed that TH and AADC were coexpressed efficiently in the same striatal cells in vitro and in vivo. Moreover, cotransduction with these two AAV vectors resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine (6-OHDA)-lesioned rats, compared with rats receiving AAV-TH alone (p < 0.01). These findings suggest an alternative strategy for gene therapy of PD and indicate that the simultaneous transduction with two AAV vectors can extend their utility for potential gene therapy applications.