Specific immune induction following DNA-based immunization through in vivo transfection and activation of macrophages/antigen-presenting cells

The initiation of an adaptive immune response requires Ag presentation in combination with the appropriate activation signals. Classically, Ag presentation and immune activation occur in the lymph node and spleen, where a favorable organ architecture and rich cellular help can enhance the process. Recently, several investigators have reported the use of DNA expression cassettes to elicit cellular and humoral immunity against diverse pathogens. Although the immune mechanisms involved are still poorly understood, plasmid inoculation represents a model system for studying immune function in response to invading pathogens. In this report, we demonstrate the presence of activated macrophages or dendritic cells in the blood lymphocyte pool and peripheral tissues of animals inoculated with DNA expression cassettes. These cells are directly transfected in vivo, present Ag, and display the surface proteins CD80 and CD86. Our studies indicate that these cells function as APC and can activate naive T lymphocytes. They may represent an important first step APC in genetic immunization and natural infection.     

Mouse deformed epidermal autoregulatory factor 1 recruits a LIM domain factor, LMO-4, and CLIM coregulators

Nuclear LIM domains interact with a family of coregulators referred to as Clim/Ldb/Nli. Although one family member, Clim-2/Ldb-1/Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain factor is known to be expressed in epidermis. Therefore, we used the conserved LIM-interaction domain of Clim coregulators to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and isolated a factor that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in overlapping manner with Clim-2 in epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because LMO/Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins, we used LMO-4 as a bait to screen for such DNA-binding proteins in epidermis and isolated the mouse homologue of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element. The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse DEAF-1, distinct from the helix-loop-helix and GATA domains previously shown to interact with LMOs, thus defining an additional LIM-interacting domain.     

Arrangement of subunits in 20 S particles consisting of NSF, SNAPs, and SNARE complexes

The present study attempts to identify the antigen-presenting cells in the retina, utilizing bone marrow-transplanted chimeric rats. Two types of chimeras were used: one produced by transplanting bone marrow cells from F1 hybrids of Lewis and Brown Norway (BN) into sublethally irradiated Brown Norway rats (LBN/F1-->BN), followed by adoptive transfer of S-antigen-specific T cells obtained from Lewis rats; the second produced by transplanting bone marrow cells from BN rats into sublethally irradiated F1 hybrids (BN-->LBN/F1), followed by adoptive transfer of S-antigen-specific T cells obtained from F1 hybrids. As controls, Lewis, F1 hybrids and BN rats also received adoptive transfer of syngeneic uveitogenic T cell lines. All animals were killed on the seventh day after adoptive transfer and their eyes and pineal glands were analysed immunohistochemically, utilizing antibody directed against Lewis specific MHC class II molecules(OX-3). The analyses revealed the development of uveoretinitis and pinealitis in both types of chimeras and in the Lewis and F1 hybrid rats. BN rats did not develop uveoretinitis. OX-3-positive cells were found in the retina and the pineal glands of both types of chimeras, and in the Lewis and F1 hybrid rats but not in the BN rats. These cells in the retina expressed dendritic morphology and perivascular distribution. Retinal pigment epithelia, Müller cells and the vascular endothelia of both chimeras, the two strains, and the F1 hybrid rats did not demonstrate OX-3-positive staining. These results suggest that the bone marrow-derived cells in the retina and pineal gland may present S-antigen to T cells, initiating the cascade of uveoretinitis and pinealitis.     

In vivo high resolution MRI of the calcaneus: differences in trabecular structure in osteoporosis patients

The purpose of this study was to use high resolution (HR) magnetic resonance (MR) images of the calcaneus to investigate the trabecular structure of patients with and without osteoporotic hip fractures and to compare these techniques with bone mineral density (BMD) in differentiating fracture and nonfracture patients. Axial and sagittal HR MR images of the calcaneus were obtained in 50 female (23 postmenopausal patients with osteoporotic hip fractures and 27 postmenopausal controls). A three-dimensional gradient-echo sequence was used with a slice thickness of 500 micron and in plane resolution of 195 x 195 micron. Texture analysis was performed using morphological features, analogous to standard histomorphometry and fractal dimension. Additionally, BMd measurements of the hip (dual-energy X-ray absorptiometry) were obtained in all patients. Significant differences between both patient groups were obtained using morphological parameters and fractal dimension as well as hip BMD (p < 0.05). Odds ratios for the texture parameters apparent (app.) bone volume/total volume and app. trabecular separation were higher than for hip BMD. Receiver operator characteristic values of texture measures and hip BMD were comparable. In conclusion, trabecular structure measures derived from HR MR images of the calcaneus can differentiate between postmenopausal women with and without osteoporotic hip fractures.     

Effects of nitric oxide on neuroendocrine function and behavior

Nitric oxide (NO) is an unusual chemical messenger. NO mediates blood vessel relaxation when produced by endothelial cells. When produced by macrophages, NO contributes to the cytotoxic function of these immune cells. NO also functions as a neurotransmitter and neuromodulator in the central and peripheral nervous systems. The effects on blood vessel tone and neuronal function form the basis for an important role of NO on neuroendocrine function and behavior. NO mediates hypothalamic portal blood flow and, thus, affects oxytocin and vasopression secretion; furthermore, NO mediates neuroendocrine function in the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes. NO influences several motivated behaviors including sexual, aggressive, and ingestive behaviors. Learning and memory are also influenced by NO. Taken together, NO is emerging as an important chemical mediator of neuroendocrine function and behavior.     

The inverted repeat regions of the simian varicella virus and varicella-zoster virus genomes have a similar genetic organization

Simian varicella virus (SVV) causes a varicella-like disease in nonhuman primates. The DNA sequence and genetic organization of the inverted repeat region (RS) of the SVV genome was determined. The SVV RS is 7559 bp in size with 56% guanine+cytosine (G+C) content and includes 3 open reading frames (ORFs). The SVV RS1 ORF encodes a 1279 amino acid (aa) protein with 58 and 39% identity to the varicella-zoster virus (VZV) gene 62 and herpes simplex virus type 1 (HSV-1) ICP4 homologs, respectively. The predicted 261 aa SVV RS2 polypeptide possesses 52% identity with the VZV gene 63 homolog and 23% identity with the HSV-1 ICP22. The SVV RS3 encodes a 187 aa polypeptide with 56% and 28% identity to the VZV gene 64 and the HSV-1 US10 homologs, respectively, and includes an atypical zinc finger motif. A G+C-rich 16 base-pair (bp) sequence which is repeated 7 times and a putative SVV origin of replication were identified between the RS1 and RS2 ORFs. Comparison with the VZV RS indicates the SVV and VZV RS regions are similar in size and genetic organization.     

Reduction of renal uptake of monoclonal antibody fragments by amino acid infusion

The renal uptake of radiolabeled antibody fragments and peptides presents a problem in radioimmunodetection and therapy, compromising lesion sensitivity, especially with intracellularly-retained isotopes. Previously, we showed that cationic amino acids and their derivatives are capable of significantly reducing kidney uptake in animals. We report our initial clinical results of successful renal uptake reduction in five patients who underwent cancer radioimmunodetection with 99mTc-anti-CEA Fab' fragments. METHODS: The patients were infused with two liters of a commercially-available nutritive amino acid solution (containing approximately 2.25 g/liter lysine-glutamate and 2.50 g/liter arginine), whereas 75 control patients received the same volume of saline (quantification of organ and tumor kinetics from conjugate whole-body views by ROI technique). RESULTS: The renal uptake in the amino acid group was significantly lower (p<0.05) than in the control group (11.1 +/- 2.0% injected dose versus 17.7 +/- 7.0% injected dose at 24 hr postinjection), whereas the uptake of all other organs remained unaffected. Gel filtration chromatography of the urine taken from amino-acid-treated patients showed that a significantly higher amount of excreted activity was bound to intact Fab' (53% of excreted activity) in contrast to only less than 10% in the control group. CONCLUSION: The renal uptake of monoclonal antibody fragments in patients can be reduced significantly by amino acid infusion, even at considerably lower doses than those that were safe and effective in animals. As was found in animals, the mechanism seems to rely on an inhibition of the re-absorption of tubularly-filtered proteins by the proximal tubule cells. These results encourage further clinical trials to lower the renal uptake experienced in radioimmunodetection, as well as in therapeutic trials with antibody fragments and peptides.     

Adsorption of chitin derivatives onto liposomes: optimization of adsorption conditions

A 2(4) factorial design is used to optimize the adsorption conditions of the hydrophilic anionic polyelectrolytes. Carboxymethylchitin (CMC) and Carboxymethyl/Glycolchitin (CO) onto liposomes at physiological ionic strength (I) and pH using phosphate buffered saline (PBS, I = 154 mM, pH = 7.4). Positive ([+]) or high surface affinity liposomes (DSPC:CHOL:DMTAP, 5:4:1), and Neutral ([N]) or low surface affinity liposomes (DSPC:CHOL, 1:1) were used as adsorption surfaces. Results of the calculations of the main effects indicate that polymer molecular weight (mwt), Surface Affinity (S), Number of Adsorption Shots (Sh), Temperature (T), and the combinations mwt x S, mwt x Sh are the most important process parameters. Results of a study conducted at T = 37 degrees C show that no loss occurs from the positive surface at the highest particle concentration, Np = 4.043 x 10(11). Finally, the extent of polymer-induced particle aggregation is decreased when the diameter of the uncoated liposomes is doubled from 0.22 to 0.45 micron. These results are as expected, given the stiffness and the dimensions of the macromolecules.