SPECT/CT imaging of baculovirus biodistribution in rat.

Non-invasive imaging provides essential information regarding the biodistribution of gene therapy vectors and it can also be used for the development of targeted vectors. In this study, we have utilized micro Single-photon emission computed tomography to visualize biodistribution of a (99m)Tc-polylys-ser-DTPA-biotin-labelled avidin-displaying baculovirus, Baavi, after intrafemoral (i.f.), intraperitoneal (i.p.), intramuscular (i.m.) and intracerebroventricular (i.c.v.) administration. The imaging results suggest that the virus can spread via the lymphatic network after different administration routes, also showing accumulation in the nasal area after systemic administration. Extensive expression in the kidneys and spleen was seen after i.p. administration, which was confirmed by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Additionally, transduction of kidneys was seen with i.m. and i.f. administrations. We conclude that baculovirus may be beneficial for the treatment of kidney diseases after i.p. administration route.

Cerebroretinal microangiopathy with calcifications and cysts.

BACKGROUND: Extensive cerebral calcifications and leukoencephalopathy have been reported in two rare disorders Coats plus and leukoencephalopathy with calcifications and cysts. In the latter, a progressive formation of parenchymal brain cysts is a special feature, whereas Coats plus is characterized by intrauterine growth retardation, bilateral retinal telangiectasias and exudations (Coats disease), sparse hair, and dysplastic nails without cyst formation. METHODS: We identified 13 patients, including two pairs of siblings, with extensive cerebral calcifications and leukoencephalopathy. We reviewed clinical, ophthalmologic, radiologic and neuropathologic data of seven deceased patients and studied five patients prospectively. RESULTS: Eleven patients were small for gestational age; the other symptoms emerged from infancy to adolescence. All patients had neurologic symptoms including seizures, spasticity, dystonia, ataxia, and cognitive decline. Progressive intracerebral calcifications involved deep gray nuclei, brainstem, cerebral and cerebellar white matter, and dentate nuclei and were accompanied by diffuse white matter signal changes and, in five patients, cerebral cysts. Eleven patients had retinal telangiectasias or angiomas. Additional features were skeletal and hematologic abnormalities, intestinal bleeding, and poor growth. Neuropathologic examination showed extensive calcinosis and abnormal small vessels with thickened, hyalinized wall and reduced lumen. CONCLUSIONS: Our data suggest that Coats plus syndrome and leukoencephalopathy with calcifications and cysts belong to the same spectrum. The primary abnormality seems to be an obliterative cerebral angiopathy involving small vessels, leading to dystrophic calcifications via slow necrosis and finally to formation of cysts and secondary white matter abnormalities.

Magnetic resonance imaging of viral particle biodistribution in vivo.

We describe here a technique for the visualization of viral vector delivery by magnetic resonance imaging (MRI) in vivo. By conjugating avidin-coated baculoviral vectors (Baavi) with biotinylated ultra-small superparamagnetic iron oxide particles (USPIO), we are able to produce vector-related MRI contrast in the choroid plexus cells of rat brain in vivo over a period of 14 days. Ten microlitres of 2.5 x 10(10) PFU/ml nuclear-targeted LacZ-encoding Baavi with bUSPIO coating was injected into rat brain ventricles and visualized by MRI at 4.7 T. As baculoviruses exhibit restricted cell-type specificity in the rat brain, altered MRI contrast was detected in the choroid plexus of the injected ventricles. No specific signal loss was detected when wild-type baculoviruses or intact biotinylated USPIO particles were injected into the lateral ventricles. Cryosectioned brains were stained for nuclear-targeted beta-galactosidase gene expression, which was found to colocalize with MRI contrast. This study provides the first proof of principle for robust and non-invasive viral vector MRI by using avidin-displaying viruses in vivo. Considering the widespread use of MRI in current medical imaging, the approach is likely to provide numerous future applications in imaging of therapeutic gene transfer.

T2 relaxation reveals spatial collagen architecture in articular cartilage: a comparative quantitative MRI and polarized light microscopic study.

It has been suggested that orientational changes in the collagen network of articular cartilage account for the depthwise T2 anisotropy of MRI through the magic angle effect. To investigate the relationship between laminar T2 appearance and collagen organization (anisotropy), bovine osteochondral plugs (N = 9) were T2 mapped at 9.4T with cartilage surface normal to the static magnetic field. Collagen fibril arrangement of the same samples was studied with polarized light microscopy, a quantitative technique for probing collagen organization by analyzing its ability to rotate plane polarized light, i.e., birefringence (BF). Depthwise variation of safranin O-stained proteoglycans was monitored with digital densitometry. The spatially varying cartilage T2 followed the architectural arrangement of the collagen fibril network: a linear positive correlation between T2 and the reciprocal of BF was established in each sample, with r = 0.91 +/- 0.02 (mean +/- SEM, N = 9). The current results reveal the close connection between the laminar T2 structure and the collagen architecture in histologic zones.

1H NMR visible lipids in the life and death of cells.

A functionally and metabolically interesting class of cell lipid can be observed by 1H nuclear magnetic resonance (NMR) spectroscopy in situ. These prominent resonances are not only associated with malignancy and cell death, but also act as heralds of benign processes, such as cell activation and proliferation. Originally, these NMR observations were explained with a membrane lipid microdomain model. However, recent studies have identified intracellular droplets, so called lipid bodies, as important contributors to these resonances. This finding bears novel implications for our understanding and assessment of lipid biochemistry in the life and death of cells.

Quantitative MR microscopy of enzymatically degraded articular cartilage.

Structural changes in bovine patellar articular cartilage, induced by component selective enzymatic treatments, were investigated by measuring tissue T(2) relaxation at 9.4 T. This MRI parameter was compared with Young's modulus, a measure of elastic stiffness and loadbearing ability of cartilage tissue. Collagenase was used to digest the collagen network and chondroitinase ABC to remove proteoglycans. Polarized light microscopy and digital densitometry were used to assess enzyme penetration after 44 hr of enzymatic digestion. T(2) relaxation in superficial cartilage increased significantly only in samples treated with collagenase. A statistically significant decrease in Young's modulus was observed in both enzymatically treated sample groups. These results confirm that T(2) of articular cartilage is sensitive to the integrity of collagen in the extracellular matrix. Nonetheless, it does not appear to be an unambiguous indicator of cartilage stiffness, which is significantly impaired in osteoarthrosis.

Reduction in water and metabolite apparent diffusion coefficients during energy failure involves cation-dependent mechanisms. A proton nuclear magnetic resonance study of rat cortical brain slices.

Proton (1H) nuclear magnetic resonance (NMR) diffusion spectroscopy was used to assess apparent diffusion coefficients (ADCs) in rat brain slices. Aglycemic hypoxia caused reductions in the ADC of N-acetylaspartate (NAA) (0.15 to 0.09 x 10(-3) mm2/s) and "slow" diffusion coefficient (D2) of tissue water (0.51 to 0.37 x 10(-3) mm2/s), together with a 32+/-11% increase in tissue water volume, attributable to tissue swelling. The ADC and D2 reductions were diminished, however, by removing external Ca2+, and under 10 mmol/L Mg2+, normoxic diffusion coefficients persisted until 40 minutes of hypoxia. The data suggest that the shift of water into the intracellular space alone cannot satisfactorily explain the reduced cerebral diffusion upon energy failure and that external Mg2+ and Ca2+ play crucial modulatory roles.

Quantitative 1H nuclear magnetic resonance diffusion spectroscopy of BT4C rat glioma during thymidine kinase-mediated gene therapy in vivo: identification of apoptotic response.

We have investigated the effects of thymidine kinase-mediated gene therapy in a malignant rat BT4C glioma by using 1H nuclear magnetic resonance spectroscopy in vivo. Ganciclovir has been successfully used in thymidine kinase gene therapy as treatment for various experimental malignancies. The cell damaging effect seems to be mediated by apoptosis, optimally leading to eradication of tumor tissue. In this study, we show that ganciclovir treatment of tumors transfected with the herpes simplex thymidine kinase gene causes profound changes in water, metabolites, and macromolecules observable by diffusion spectroscopy. During treatment, a 50% reduction from 0.14 +/- 0.01 x 10(-9) m2/s in the apparent diffusion coefficient of choline-containing compounds can be observed, concomitant with a 219% increase in the apparent diffusion coefficient of the rapidly diffusing water component. These changes are associated with an increase in the relative fraction of this water component from 87 to 94%. The apparent diffusion coefficients of the slowly diffusing water component and macromolecules remain unaltered. The results imply a reduction in cell size and number, a significant increase in intracellular viscosity, and a possible reduction in the hydrodynamic radii of macromolecular components, which are ascribed as biophysical signatures for apoptotic cell death.

Increased macromolecular resonances in the rat cerebral cortex during severe energy failure as detected by 1H nuclear magnetic resonance spectroscopy.

Changes in cerebral macromolecular 1H nuclear magnetic resonance (NMR) spectrum were studied in cortical brain slices in vitro. Aglycaemic hypoxia irreversibly increased various short T2 spectral components at 1.8-0.8 ppm in concordance with energy loss and independent of T1 and T2 relaxation effects. Removal of external calcium (Ca2+e) slightly attenuated the effect. The results suggest NMR-visible reorganisation of intracellular proteins due to hypoxic insult, and show that it may be possible to monitor early cytoplasmic changes due to brain energy depletion by NMR spectroscopy.

1H nuclear magnetic resonance spectroscopy study of cerebral glutamate in an ex vivo brain preparation of guinea pig.

Cerebral glutamate was monitored in a superfused cerebral cortical preparation by 1H NMR spectroscopy using a semiselective spin-echo sequence N-acetyl aspartate (NAA) as an internal concentration reference. During controlled metabolic conditions, the cerebral 1H NMR-detected glutamate-to-NAA ratio was approximately 20-30% lower than expected from the ratio of neutralized perchloric acid extracts of the preparations. Inhibition of respiration in the presence of glucose did not change the 1H NMR glutamate-to-NAA ratio in brain slice preparation. In contrast, either complete depletion of ATP during cyanide poisoning together with 0 mM glucose, anoxia in the absence of glucose, or treatment with nigericin or with a protonophore, carbonyl cyanide-m-fluorophenylhydrazone, increased 1H NMR-detected glutamate/NAA in the cerebral preparations without a change in the relative and absolute concentration ratios determined from the tissue acid extracts. Spin-spin relaxation times of glutamate and NAA peaks in anoxic slices were 749 +/- 89 and 729 +/- 94 ms, respectively, and thus, the portion of glutamate that could not be detected by 1H NMR was quantified in absolute terms. It was calculated that an increase in the glutamate-to-NAA ratio from 0.55 +/- 0.02 to 0.67 +/- 0.02 during aglycemic anoxia corresponded to some 6 mmol/kg of tissue dry weight of glutamate from the total concentration of 28 mmol/kg dry weight. It is suggested that this 22% of total glutamate pool is present in a noncytoplasmic compartment during controlled metabolic state.