Photoreceptor degeneration, structural remodeling and glial activation: a morphological study on a genetic mouse model for pericyte deficiency.

Interaction between pericytes and endothelial cells via platelet-derived growth factor B (PDGF-B) signaling is critical for the development of the retinal microvasculature. The PDGF-B retention motif controls the spatial distribution range of the growth factor in the vicinity of its producing endothelial cells allowing its recognition by PDGF receptor beta-(PDGFR-ß)-carrying pericytes; this promotes recruitment of pericytes to the vascular basement membrane. Impairment of the PDGF-B signaling mechanism causes development of vascular abnormalities, and in the retina this consequently leads to defects in the neurological circuitry. The vascular pathology in the pdgf-b(ret/ret) (PDGF-B retention motif knockout) mouse retina has been previously reported; our study investigates the progressive neuronal defects and changes in the retinal morphology of this pericyte-deficient mouse model. Immunohistochemical analysis revealed retinal injuries to occur as early as postnatal day (P) 10 with substantial damage progressing from P15 and onward. Vascular abnormalities were apparent from P10, however, prominent neuronal defects were mostly observed from P15, beginning with the compromised integrity of the laminated retinal structure characterized by the presence of rosettes and focally distorted regions. Photoreceptor degeneration was observed by loss of both rod and cone cells, including the disassembly and altered structure of their synaptic terminals. Significant shortening of cone outer segments was observed from P10 and later stages; however, decrease in cone density was only observed at P28. Disorganization and dendrite remodeling of rod bipolar cells also added to the diminished neural and synaptic integrity. Moreover, in response to retinal injuries, Müller and microglial cells were observed to be in the reactive phenotype from P15 and onward. Such a sequence of events indicates that the pdgf-b(ret/ret) mouse model displays a short time frame between P10 and P15, during which the retina shifts to a retinopathic phase by the development of prominently altered morphological features.

Receptor-mediated endocytosis of iron-oxide particles provides efficient labeling of dendritic cells for in vivo MR imaging.

Dendritic cells (DCs) function as antigen presenting cells in vivo and play a fundamental role in numerous diseases. New methods are described for high-efficiency intracellular labeling of DCs with superparamagnetic iron-oxide (SPIO) utilizing a receptor-mediated endocytosis (RME) mechanism. Bone marrow-derived DCs or a fetal skin-derived DC line were incubated with SPIO conjugated to anti-CD11c monoclonal antibody (mAb) under conditions favoring RME. These cells exhibited approximately a 50-fold increase in uptake relative to DCs incubated with SPIO without the mAb. Flow cytometry studies assaying cell surface markers showed a down-modulation of CD11c, but no other changes in phenotype. Immunological function of the DCs was unmodified by the labeling, as determined by cytokine secretion assays. The RME mechanism was confirmed using electron microscopy, endocytosis inhibition assays, and incubation experiments with SPIO conjugated to mAbs against accessory molecules that are not expressed on DCs. Labeled DCs were injected into murine quadriceps and monitored in vivo for several days using MR microimaging at 11.7 T. DCs were observed to remain within the muscle for >24 hr. The use of RME is an efficient way to label immune cells for in vivo MRI and can be applied to a wide variety of cell types.

Gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin 12.

The use of gene therapy to enhance antitumor immunity has emerged as a promising procedure to fight cancer. In this study we have tested the ability of an adenovirus carrying interleukin 12 (IL-12) gene (AdCMVIL-12) to eliminate tumoral lesions in 3 animal models of orthotopic hepatocellular carcinoma (HCC). Intratumoral injection of AdCMVIL-12 in animals with a single big tumor nodule implanted in the liver resulted in significant inhibition of tumor growth in a dose-dependent manner. Fifty percent of animals that received a dose of 5 x 10(9) plaque-forming units, showed complete regression of the tumor 2 weeks after treatment. In animals with 2 independent tumor nodules in the left liver lobe, injection in only one of them of 5 x 10(9) pfu AdCMVIL-12 induced, 15 days after therapy, complete regression of 50% of treated tumors and also of 50% of untreated lesions, with 60% long-term survival. Rats that were tumor free after therapy with AdCMVIL-12 showed protection against tumor rechallenge. A group of rats received the carcinogen diethylnitrosamine and developed multiple hepatic dysplasic nodules of 1 to 5 mm in diameter. These animals were treated by intrahepatic artery injection of either AdCMVIL-12 (5 x 10(9) pfu) or control vector. In this model AdCMVIL-12 induced complete tumor regression in 20% of treated rats and inhibited tumor growth in 60% of cases with an increase in rat survival. Activation of natural killer (NK) cells and inhibition of angiogenesis were found to be antitumor mechanisms set in motion by AdCMVIL-12. Our data indicate that experimental HCC can be efficiently treated by intratumoral or intravascular injection of adenovirus expressing IL-12.