Modulation of the inflammatory properties and hepatotoxicity of recombinant adenovirus vectors by the viral E4 gene products.

Liver toxicity and inflammation were assessed in C57BL/6, CBA, and BALB/c mice injected intravenously with a series of recombinant adenoviruses deleted simultaneously in E1/E3, in E1/E3/E2A, or in E1/E3/E4. All vectors were either devoid of transgenes or carried in E1 the human CFTR cDNA under the control of the CMV promoter. Injection of the E1/E3-deleted vector induced a significant liver dystrophy and inflammatory responses that were accompanied by an increased serum transaminase concentration. The vector toxicity remained elevated on additional deletion of the E2A gene and was further enhanced when hCFTR was expressed. In contrast, additional deletion of E4 led to a reduction in hepatotoxicity, suggesting an active role of E4 gene products in liver injury. However, deletion of E4 also led to a loss of transgene expression. To identify the individual E4 product(s) involved in liver toxicity and in the regulation of transgene expression, a series of isogenic E1/E3-deleted vectors, with or without the hCFTR transgene, and containing various combinations of functional E4 open reading frames (ORFs), were evaluated in vitro and in vivo. We demonstrate that liver injury was markedly reduced with vectors containing either ORF3 alone or ORF3,4 while vectors containing ORF4, ORF6,7 or ORF3,6,7 still displayed elevated hepatotoxicity and inflammatory responses. Moreover, transgene expression was restored when ORF3,4 or ORF3,6,7 was retained in the vector. These results highlight the importance of the E4 gene products in the design of improved in vivo gene transfer vectors.

Gene therapy with recombinant adenovirus vectors: evaluation of the host immune response.

E1, E3-deleted, replication-deficient recombinant adenoviruses are widely studied as vectors for their capacity to transfer therapeutic genes in vivo. They can infect a wide variety of dividing and quiescent cells from different organs and possess a large packaging capacity. One of the major limitations in the use of these vectors for gene therapy is the transient expression of the transgene in vivo and the poor transduction efficiency when re-administered. Despite the deletion of the viral E1 region, low level of early and late viral genes are expressed in vivo. Thus, viral antigens plus those derived from transgene expression in transduced cells contribute to cellular immune responses leading to the destruction of these cells. Production of anti-adenovirus antibodies, the cellular immune response as well as the early non-specific clearance of the vectors, constitute barriers to successful gene therapy. New vectors have been derived with additional deletions in the E2a or the E4 regions. Such second generation vectors were evaluated in vivo. These studies have revealed the complexity of the immune mechanisms elicited by these vectors and the importance of several parameters in these evaluations (i.e. mouse strains, nature of the transgene, route of administration...). In order to inhibit the production of neutralizing antibodies to adenovirus that prevent from further readministration of the vectors, immunosuppressive strategies were undertaken. Treatment regimens with immunosuppressive drugs (cyclophosphamide, FK506) or with monoclonal antibodies that block either the T cell receptor or costimulation pathways allow prolonged transgene expression and/or readministration of adenoviral vectors. In addition, transduction efficiencies may be increased by transiently inhibiting non-specific immune mechanisms that lead to the dramatic early clearance of the vectors. Taken together, these strategies may improve further gene therapy protocols by decreasing the host immune response to adenoviral vectors.

Jet-discharge cavity ring-down spectroscopy of ionized polycyclic aromatic hydrocarbons: progress in testing the PAH hypothesis for the diffuse interstellar band problem.

Naphthalene cations (C10H+8) were produced in a slit jet coupled with an electronic discharge, and cavity ring down was used to obtain its absorption spectrum in the region 645-680 nm. Two of the strongest C10H+8 bands previously characterized by matrix isolation spectroscopy were found, both with a fractional blue shift of about 0.5%. This is the first gas-phase electronic absorption spectrum of an ionized polycyclic aromatic hydrocarbon (PAH). This work opens the way for a direct comparison of laboratory PAH spectra with the diffuse interstellar bands (DIB), the origin of which still constitutes an open problem in astrophysics.

Laser optical feedback tomography.

We describe a new method for imaging in three-dimensional turbid media, laser optical feedback tomography. This technique is based on the resonant sensitivity of a short-cavity laser to frequency-shifted optical feedback from ballistic photons retrodiffused from the medium. The advantage of the method is that the detector is the laser source itself, which provides optical amplification with self-aligned spatial and temporal coherent detection.

Three-dimensional sensing based on a dynamically focused laser optical feedback imaging technique.

A new method analogous to three-dimensional confocally based sensing is proposed. This method uses the technique of laser optical feedback imaging, which takes advantage of the resonant sensitivity of a short-cavity laser to frequency-shifted optical feedback for highly sensitive detection, making it ideal for surface and volume measurements of noncooperative targets. Rapid depth scanning is made possible by use of an electrically controlled variable-focus lens. The system is able to detect height discontinuities, and because detection occurs along the axis of projection the system does not have problems of shadow. Preliminary results for a depth range of 15 mm and a resolution of 100 mum are presented.

Laser relaxation-oscillation frequency imaging.

We describe a new imaging technique based on modification of laser relaxation frequency induced by coherent optical feedback from an external target. A direct comparison (both theoretical and experimental) is made with laser feedback interferometry techniques, in which there is a modification of the laser's steady state. We show that, for a laser with a cavity damping rate gamma(c) higher than the population damping rate, gamma(1) , the modification of the laser relaxation frequency can be several orders of magnitude more sensitive than the perturbation of the laser's output power. Application of this technique to imaging is reported.

Transient blocking of both B7.1 (CD80) and B7.2 (CD86) in addition to CD40-CD40L interaction fully abrogates the immune response following systemic injection of adenovirus vector.

Blockade of the CD40-CD40L and CD80/CD86-CD28 costimulatory pathways represents a strategy to inhibit the immune response against Ad vectors designed for gene therapy applications. Since most previous studies have used a CTLA4-Ig fusion molecule binding to both CD80 and CD86, the respective roles of these B7 molecules remained undefined. We have studied the effect of blocking monoclonal Abs (mAbs) directed against the costimulatory molecules CD40L, CD80 and CD86, alone or in different combinations, on the humoral and cellular immune responses against Ad. Groups of mice were transiently treated with each combination of blocking mAbs upon systemic injection of a first Ad vector. Combinations of anti-CD80 + anti-CD86 or anti-CD40L + anti-CD86 mAbs resulted in strong inhibition of the immune response against Ad. Using either of these mAb pairs, a second vector could be administered 1 month after the first injection but with lower efficiency than in naive animals. Thus, CD86 stands as the pivotal B7 molecule involved in the development of the immune response against Ad. However, only the blockade of both CD80 and CD86 in addition to CD40L fully inhibited the humoral and cellular responses against the Ad vector, such that readministration after 1 month was as efficient as in naive animals. At the time of readministration, treated animals had regained their ability to mount a normal immune response to the second Ad vector, showing that tolerance was not induced.

High-sensitivity intracavity laser absorption spectroscopy with vertical-external-cavity surface-emitting semiconductor lasers.

We report the demonstration of high-sensitivity intracavity laser absorption spectroscopy with multiple-quantum-well vertical-external-cavity surface-emitting semiconductor lasers (VECSEL's). A detection limit of 3 x 10(-10) cm (-1) has been achieved. The spectrotemporal dynamics of a VECSEL in the 1030-nm wavelength region has been studied. The laser was operating cw at room temperature, with a baseline signal-to-noise ratio as high as 400. The laser was optically pumped with a threshold as low as 80 mW and was broadly tunable over a spectral range of approximately 75 nm .

High sensitivity intracavity stimulated emission pumping.

Small optical gains have been measured by an intracavity laser technique. The iodine molecule is used to probe and calculate the different mechanisms occurring in the intracavity stimulated emission pumping technique. We observed and measured the gain of lines: R(15), P(13),P(17)(43-26). Absorption from nu'' = 6,7, and 8 was also obtained. We show that collision processes and random coincidences of the cavity modes govern the sensitivity of this technique.

Intracavity laser absorption measurements at ultrahigh spectral resolution.

Intracavity laser absorption spectroscopy with ultrahigh sensitivity and enhanced spectral resolution is demonstrated. It entails use of an intracavity étalon that selects equally spaced modes of the cavity. These modes are reduced in intensity when they occur at spectral locations where absorption that is due to intracavity species also occurs. We obtained absorption spectra by tuning the étalon in small steps across its free spectral range, recording intracavity spectra at each step, and summing the result. The maximum resolution is determined by the width of the étalon peaks, which was ~0.01 cm(-1). When the finesse of the étalon is increased, the resolution equal to the width of a single laser mode can be achieved. With this technique, spectra at Doppler-free resolution such as that required for studies of high vibrational-overtone transitions of molecules in supersonic jets are possible.

In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1/E2A, or E1/E4 deleted.

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.

Inhibition of costimulation allows for repeated systemic administration of adenoviral vector in rhesus monkeys.

Immunogenicity of recombinant adenoviral (Ad) vectors severely hampers the clinical development of gene therapy protocols using repeated vector administrations. Inhibition of costimulation by APCs was explored as a strategy to circumvent the immune response against Ad particles. This strategy was tested in rhesus monkeys, treated transiently with chimeric anti-human CD40 and anti-human CD86 antagonist monoclonal antibodies (MAbs) at the time of systemic administration of a recombinant Ad vector. After Ad vector administration in the absence of immunosuppressive treatment, transgene expression in the serum lasted about 3-4 weeks. All control animals developed a strong neutralizing antibody (NAb) response to the Ad particles, which totally prevented efficient administration of a second vector, as shown by the lack of transgene expression. Treatment with anti-CD40 and anti-CD86 chimeric MAbs delayed or blocked the development of a humoral response against Ad and the infiltration of CD8(+) lymphocytes into the liver. This resulted in (i) increased persistence of Ad-transduced cells after injection of a first vector encoding a nonimmunogenic transgene, and (ii) the possibility of readministering a second Ad vector with significant efficacy. In both respects, the combined blockade of CD40 and CD86 was more efficient than treatment with anti-CD40 alone. This study shows for the first time in non-human primates that blocking CD40 and CD86 costimulatory molecules represents a promising strategy to inhibit immune responses against an Ad vector injected systemically.