Hidevaldo B. Machado

The influence of innate and pre‐existing immunity on adenovirus therapy

Recombinant adenovirus serotype 5 (Ad5) vectors have been studied extensively in preclinical gene therapy models and in a range of clinical trials. However, innate immune responses to adenovirus vectors limit effectiveness of Ad5 based therapies. Moreover, extensive pre‐existing Ad5 immunity in human populations will likely limit the clinical utility of adenovirus vectors, unless methods to circumvent neutralizing antibodies that bind virus and block target cell transduction can be developed. Furthermore, memory T cell and humoral responses to Ad5 are associated with increased toxicity, raising safety concerns for therapeutic adenovirus vectors in immunized hosts. Most preclinical studies have been performed in naïve animals; although pre‐existing immunity is among the greatest hurdles for adenovirus therapies, it is also one of the most neglected experimentally. Here we summarize findings using adenovirus vectors in naïve animals, in Ad‐immunized animals and in clinical trials, and review strategies proposed to overcome innate immune responses and pre‐existing immunity. J. Cell. Biochem. 108: 778–790, 2009.

A selection platform for carbon chain elongation using the CoA-dependent pathway to produce linear higher alcohols

Production of green chemicals and fuels using metabolically engineered organisms has been a promising alternative to petroleum-based production. Higher chain alcohols (C4-C8) are of interest because they can be used as chemical feedstock as well as fuels. Recently, the feasibility of n-hexanol synthesis using Escherichia coli has been demonstrated by extending the modified Clostridium CoA-dependent n-butanol synthesis pathway, thereby elongating carbon chain length via reactions in reversed β-oxidation, (or β-reduction). Here, we developed an anaerobic growth selection platform that allows selection or enrichment of enzymes for increased synthesis of C6 and C8 linear alcohols. Using this selection, we were able to improve the carbon flux towards the synthesis of C6 and C8 acyl-CoA intermediates. Replacement of the original enzyme Clostridium acetobutylicum Hbd with Ralstonia eutropha homologue PaaH1 increased production of n-hexanol by 10-fold. Further directed evolution by random mutagenesis of PaaH1 improved n-hexanol and n-octanol production. This anaerobic growth selection platform may be useful for selecting enzymes for production of long-chain alcohols and acids using this CoA-dependent pathway.

Spectrally resolved bioluminescence tomography with the third-order simplified spherical harmonics approximation

Bioluminescence imaging has been extensively applied to in vivo small animal imaging. Quantitative three-dimensional bioluminescent source information obtained by using bioluminescence tomography can directly and much more accurately reflect biological changes as opposed to planar bioluminescence imaging. Preliminary simulated and experimental reconstruction results demonstrate the feasibility and promise of bioluminescence tomography. However, the use of multiple approximations, particularly the diffusion approximation theory, affects the quality of in vivo small animal-based image reconstructions. In the development of new reconstruction algorithms, high-order approximation models of the radiative transfer equation and spectrally resolved data introduce new challenges to the reconstruction algorithm and speed. In this paper, an SP(3)-based (the third-order simplified spherical harmonics approximation) spectrally resolved reconstruction algorithm is proposed. The simple linear relationship between the unknown source distribution and the spectrally resolved data is established in this algorithm. A parallel version of this algorithm is realized, making BLT reconstruction feasible for the whole body of small animals especially for fine spatial domain discretization. In simulation validations, the proposed algorithm shows improved reconstruction quality compared with diffusion approximation-based methods when high absorption, superficial sources and detection modes are considered. In addition, comparisons between fine and coarse mesh-based BLT reconstructions show the effects of numerical errors in reconstruction image quality. Finally, BLT reconstructions using in vivo mouse experiments further demonstrate the potential and effectiveness of the SP(3)-based reconstruction algorithm.

Synaptotagmin IV overexpression inhibits depolarization-induced exocytosis in PC12 cells

Depolarization‐induced vesicle exocytosis is a complex mechanism involving a number of proteins. In this process, synaptotagmins work as members of the Ca2+‐sensing system that triggers the fusion of the synaptic vesicle with the plasma membrane. Synaptotagmin IV (SytIV), an immediate‐early gene induced by depolarization in PC12 pheochromocytoma cells and in the hippocampus, has been suggested to work as a negative regulator of neurotransmitter release. Unlike other synaptotagmins, SytIV has an evolutionarily conserved substitution of an aspartate to a serine in the Ca2+ coordination site of its C2A domain, preventing SytIV from binding anionic lipids in a Ca2+‐dependent fashion. We used the secretion of human growth hormone (hGH) as a reporter system with which to examine the effects of overexpressing SytIV and other depolarization‐induced immediate‐early genes (the protein kinases KID‐1, SIK, and PIM‐1 and the transcription factors rTLE3 and Nurr1) on depolarization‐induced vesicle exocytosis in PC12 cells. SytIV overexpression resulted in decreased depolarization‐induced hGH release. However, conversion of the unique serine in SytIV to an aspartate eliminated this inhibitory activity. In addition, rTLE3 overexpression produced only a modest increase in spontaneous vesicle exocytosis, whereas KID‐1, SIK, PIM‐1, and Nurr1 overexpression had no effect on depolarization‐induced exocytosis.

Experimental Bioluminescence Tomography with Fully Parallel Radiative-transfer-based Reconstruction Framework

Bioluminescence imaging is a very sensitive imaging modality, used in preclinical molecular imaging. However, in its planar projection form, it is non-quantitative and has poor spatial resolution. In contrast, bioluminescence tomography (BLT) promises to provide three dimensional quantitative source information. Currently, nearly all BLT reconstruction algorithms in use employ the diffusion approximation theory to determine light propagation in tissues. In this process, several approximations and assumptions that are made severely affect the reconstruction quality of BLT. It is therefore necessary to develop novel reconstruction methods using high-order approximation models to the radiative transfer equation (RTE) as well as more complex geometries for the whole-body of small animals. However, these methodologies introduce significant challenges not only in terms of reconstruction speed but also for the overall reconstruction strategy. In this paper, a novel fully-parallel reconstruction framework is proposed which uses a simplified spherical harmonics approximation (SPN). Using this framework, a simple linear relationship between the unknown source distribution and the surface measured photon density can be established. The distributed storage and parallel operations of the finite element-based matrix make SPN-based spectrally resolved reconstruction feasible at the small animal whole body level. Performance optimization of the major steps of the framework remarkably improves reconstruction speed. Experimental reconstructions with mouse-shaped phantoms and real mice show the effectiveness and potential of this framework. This work constitutes an important advance towards developing more precise BLT reconstruction algorithms that utilize high-order approximations, particularly second-order self-adjoint forms to the RTE for in vivo small animal experiments.

In Vivo Mouse Bioluminescence Tomography with Radionuclide-Based Imaging Validation

IntroductionBioluminescence imaging, especially planar bioluminescence imaging, has been extensively applied in in vivo preclinical biological research. Bioluminescence tomography (BLT) has the potential to provide more accurate imaging information due to its 3D reconstruction compared with its planar counterpart.MethodsIn this work, we introduce a positron emission tomography (PET) radionuclide imaging-based strategy to validate the BLT results. X-ray computed tomography, PET, spectrally resolved bioluminescence imaging, and surgical excision were performed on a tumor xenograft mouse model expressing a bioluminescent reporter gene.ResultsWith different spectrally resolved measured data, the BLT reconstructions were acquired based on the third-order simplified spherical harmonics (SP3) approximation and the diffusion approximation (DA). The corresponding tomographic images were obtained for validation of bioluminescence source reconstruction.ConclusionOur results show the strength of PET imaging compared with other validation methods for BLT and improved source localization accuracy based on the SP3 approximation compared with the diffusion approximation.

The MAPK pathway is required for depolarization-induced "promiscuous" immediate-early gene expression but not for depolarization-restricted immediate-early gene expression in neurons.

Depolarization, growth factors, neurotrophins, and other stimuli induce expression of immediate early genes (IEGs) in neurons. We identified a subset of IEGs, IPD‐IEGs, which are induced preferentially by depolarization, but not by neurotrophins or growth factors, in PC12 cells. The “promiscuous” IEGs Egr1 and c‐fos, induced by growth factors and neurotrophins, in addition to depolarization, require activation of the MAP kinase signaling pathway for induction in response to KCl depolarization in PC12 cells; MEK1/2 inhibitors block KCl‐induced Egr1 and c‐fos expression. In contrast, MEK1/2 inhibition has no effect on KCl‐induced expression of the known IPD‐IEGs in PC12 cells. Additional “candidate” IDP‐IEGs were identified by a microarray comparison of genes induced by KCl in the presence vs. the absence of an MEK1/2 inhibitor in PC12 cells. Northern blot analyses demonstrated that representative newly identified candidate IPD‐IEGs, as with the known IPD‐IEGs, are also induced by a MAP kinase‐ independent pathway in response to depolarization, both in PC12 cells and in rat primary cortical neurons. Nerve growth factor and epidermal growth factor are unable to induce the expression of the Crem/Icer, Nur77, Nor1, Rgs2, Dusp1 (Mkp1), and Dscr1 genes in PC12 cells, validating their identification as IPD‐IEGs. Inhibiting calcium/calmodulin‐dependent kinase II (CaMKII), calcineurin, or protein kinase A (PKA) activity prevents KCl‐induced IPD‐IEG mRNA accumulation, suggesting that the IPD‐IEG genes are induced by depolarization in neurons via a combination of calcineurin/PKA‐ and CaMKII‐dependent pathways.

Expression of depolarization-induced immediate early gene proteins in PC12 cells.

Immediate early genes induced by depolarization are thought to be important in mediating neuronal functional plasticity. We previously identified a group of immediate early genes that are preferentially induced by depolarization and forskolin but not by nerve growth factor or epidermal growth factor in PC12 pheochromocytoma cells. These depolarization‐induced genes include synaptotagmin 4; the protein kinases KID‐1, PIM‐1, and SIK; an orphan transcription factor, Nurr‐1; and a transcription corepressor, rTLE‐3. All these genes are also induced in the hippocampus in response to kainic‐acid induced depolarization. To characterize further the unique functions of these genes in plasticity, we used recombinant proteins to generate and purify antibodies against KID‐1 and SIK proteins. Immunoblotting experiments were performed to examine the induced expression of the KID‐1 and SIK proteins in PC12 cells. PIM‐1 and Nurr‐1 protein expression was also examined following stimulation, using commercially available antibodies. There is an increase in synthesis, in PC12 cells, of these four IEG proteins after KCl plus forskolin treatment. Nurr‐1 protein peaks between 2 and 4 hr and decreases by 6 hr after the treatment. PIM‐1 and KID‐1 proteins rise by 1 hr, peak between 2 and 4 hr, and return to their basal levels at 6 hr. SIK protein increases significantly at 2 hr after treatment, peaks between 4 and 6 hr, and returns to the basal level at 8 hr. Immunofluorescence studies demonstrate distinct distribution patterns of each of these depolarization‐induced IEG proteins in PC12 cells.

MAPKAP kinase‐2 is a primary response gene induced by depolarization in PC12 cells and in brain

Using a combination of targeted differential display for induced protein kinases and differential library screening, we identified mitogen‐activated protein kinase activated protein kinase 2 (MAPKAPK2), as a primary response gene whose transcription is stimulated by membrane depolarization and by forskolin in rat PC12 pheochromocytoma cells. MAPKAPK3 was neither induced nor repressed by similar treatments. The increase in MAPKAPK2 mRNA is preceded by an increase in a MAPKAPK2 intron‐containing RNA precursor, indicating that the increase in message is due at least in part to increased transcription. The open reading frame of full‐length rat MAPKAPK2 cDNA is 99% identical to mouse MAPKAPK2 and 92% identical to human MAPKAPK2. The human MAPKAPK2 predicted protein contains 14 additional amino acids in the proline‐rich N‐terminal domain, when compared to murine and rat MAPKAPK2 predicted proteins. The MAPKAPK2 form found in PC12 cells corresponds to variant 2 in the human; this ortholog carries a nuclear translocation signal near its C‐terminus. MAPKAPK2 message is also induced in the dentate gyrus, CA1, and CA3 of the rat hippocampus between 2–4 hr after the onset of kainic acid‐induced seizures.

Repetitive, noninvasive imaging of cyclooxygenase-2 gene expression in living mice.

The cyclooxygenase-2 (COX-2) gene plays a role in a wide variety of normal physiologic pathways and is a major target of pharmacologic intervention in a large number of pathophysiologic contexts, including pain, fever, inflammation, and cancer. Expression of the COX-2 gene is induced in a wide range of cells, in response to an ever-increasing number of stimuli. The regulation of the COX-2 gene has been the subject of extensive study, using traditional transfection techniques with reporter gene constructs. Regulation of the COX-2 gene in living animals, however, requires sacrifice of the animal and in situ hybridization and/or immunohistochemical studies. We have utilized in vivo optical imaging technology with a cooled charged coupled device camera to image the expression of the firefly luciferase gene in tumor xenografts that are stably transfected with a chimeric gene containing the first kilobase of the murine COX-2 promoter. Induction of luciferase gene expression following systemic lipopolysaccharide/endotoxin administration can be robustly demonstrated; both a dose-response relationship and a time course for luciferase expression from the COX-2 promoter can be noninvasively analyzed in the tumor xenografts. These data suggest expression from the COX-2 promoter will be easily analyzed in transgenic mice, in knock-in mice, and in somatic cell and gene transfer experiments.