Enhancement of liposome-mediated gene transfer into vascular tissue by replication deficient adenovirus.

For both in vitro and in vivo experiments, especially in vascular cells, an efficient and easy method of gene transfer into this tissue would be extremely useful. Previous methods have either yielded low levels of expression or require complicated manipulation of viral vectors. The goal of this study was to develop an easy, efficient method to introduce unmodified plasmid DNA into vascular tissue. In this report it is demonstrated that complexing unmodified plasmid DNA with replication-deficient adenovirus (Ad5 dl312) via cationic lipids enhances gene transfer up to 1000-fold in cultured bovine aortic endothelial cells (BAECs). Further, utilizing a balloon-injured rabbit femoral artery model, intense nuclear staining in both the neointimal smooth muscle cell layer and the adventitia was seen following transfection with a plasmid containing the lacZ gene and the SV40 nuclear localization signal. Control arteries demonstrated no detectable staining. Our studies suggest that complexing plasmid DNA with adenovirus via lipids greatly enhances gene transfer both in vivo and in vitro. This method could have a wide range of applications for experiments in vascular tissue.

Peripheral blood chimerism in renal allograft recipients transfused with donor bone marrow.

Experimental studies have shown that administration of antilymphocyte serum combined with donor bone marrow cells can induce tolerance to allograft tissue. We have initially reported application of these protocols in clinical studies of cadaveric renal allograft recipients who were treated with MALG and donor-specific bone marrow cells. To evaluate the effectiveness of the donor marrow cells in the production of chimerism, a detection method based on 32P-incorporated PCR was established. The 32P PCR was utilized with primers specific for the HLA class II, VNTR (D17S5 and D1S111), and/or Y-chromosome genes to detect the presence of allogeneic chimerism in the recipients. Immediately posttransplant, 26.4% of marrow recipients demonstrated the presence of allogeneic chimerism prior to the marrow transfusion as did 18% in the untransfused controls. In transfused patients, chimerism was detected most frequently during the 1-3-month interval after marrow transfusion (65%), and then diminished to 50-56% at 3-12 months posttransfusion. In the control group the frequency of allogeneic chimerism was gradually decreased and was undetectable in the majority of the patients beyond 3 months posttransplant while marrow-transfused recipients were more likely to have chimeric cells detected consistently beyond 3 months. Rejection episodes were significantly effected by the presence of chimerism in the recipients. Of the transfused patients, 91.3% who demonstrated allogeneic chimerism were rejection-free as compared with 8.7% who experienced at least one rejection episode (P = 0.01). While the presence of allogeneic chimerism in the control group was correlated with rejection-free graft survival, this difference did not reach statistical significance.

Limiting detection of an amplification signal for HLA-D region and VNTR genes by 32P-PCR.

The limiting detection signal for identification of human genetic markers, such as HLA-D and VNTR genes, was determined using DNA isolated from a series of decreasing numbers of lymphocytes carrying the target marker in the polymerase chain reaction (PCR). The PCR procedure was assembled by incorporating 32P-labeled dCTP in the reaction mixture. Primers specific for detection of MHC Class II genes such as HLA-DR1, -DR2, -DRw52 and -DRw53 were utilized when cells were mismatched by one DR type, and primers for the identification of the region of variable number of tandem repeats (VNTRs) were utilized where cells had the same DR types. The 32P-incorporated amplified DNA was analyzed by polyacrylamide gel electrophoresis followed by exposure to x-ray film. The sensitivity of the test varied for different allelic markers as evaluated by amplification of DNA from each set of a mixture of lymphocytes. The target HLA-DR markers were detectable in a cell ratio of as high as 1:100,000, whereas the VNTR markers were detectable at a 1:1000 cell ratio. The approach described here offers certain advantages: 1) increased sensitivity, 2) quantitative power, 3) reduced assay time, 4) simplified procedure and 5) less expense. This method provides valuable information for studies involving forensic specimens and marrow engraftment after allogenic bone marrow transplantation (BMT) that require discrete representation of one allele relative to another in a heterozygous sample where limited quantities of target DNA are available.

Developmental regulation of cryptdin, a corticostatin/defensin precursor mRNA in mouse small intestinal crypt epithelium.

Cryptdin mRNA codes for the apparent precursor to a corticostatin/defensin-related peptide that accumulates to high levels in mouse intestinal crypt epithelium during postnatal development. The primary structure, intestinal cell distribution, and developmental appearance of cryptdin mRNA have been determined. Cryptdin mRNA is 450-480 nucleotides long. Translation of the partial cryptdin cDNA sequence reveals a 70-amino acid open reading frame that includes 32 carboxy-terminal residues that align with those in the consensus sequence, C.CR...C....ER..G.C....CCR, which is a common feature of leukocyte defensins and lung corticostatins (Selsted, M. E., D. M. Brown, R. J. DeLange, S. S. L. Harwig, and R. I. Lehrer. 1985. J. Biol. Chem. 260:4579-4584; Zhu, Q., J. Hu, S. Mulay, F. Esch, S. Shimasaki, and S. Solomon. 1988. Proc. Natl. Acad. Sci. USA. 85:592-596). In situ hybridization of cryptdin cDNA to paraformaldehyde-fixed, frozen sections of adult jejunum and ileum showed intense and specific labeling of epithelial cells in the base of all crypts. Analysis of sections from suckling mice showed that cryptdin mRNA is detectable in 10-20% of crypts in 10-d-old mice, in approximately 80% of crypts in 16-d-old mice, and in all crypts of mice 20 d and older. During the fourth week, the sequence accumulates in crypts to the maximal adult level. Cryptdin mRNA content in adult small intestine is independent both of T cell involvement and luminal bacteria. The role of cryptdin in small bowel physiology remains to be determined: cryptdin may inhibit bacterial translocation, modulate intestinal hormone synthesis, influence hormonal sensitivity of the intestinal epithelium, or exhibit a multiplicity of related activities.

Neonatal atria and ventricles secrete atrial natriuretic factor via tissue-specific secretory pathways.

The cellular mechanisms regulating secretion of the peptide hormone atrial natriuretic factor (ANF) differ in neonatal atrial and ventricular cardiocytes. We demonstrate that although both cell types synthesize and secrete ANF, only atrial cells store peptide in abundant secretory granules. Neonatal ventricular cells secrete ANF rapidly after synthesis and lack secretory granules. We propose that ventricular ANF is released by a constitutive secretory pathway whereas atrial ANF is stored and released by a regulated pathway. Furthermore, ventricular ANF mRNA and hormone concentrations decrease during the first week of life. Developmental variation in the use of ANF secretory pathways may reflect changing requirements for maintenance of intravascular volume and pressure. Tissue-specific modulation of hormone secretory pathways appears to be a novel response to developmentally induced changes in the requirements for a peptide hormone.

Surface morphology of isolated cardiac myocytes from hypertrophied hearts of aging spontaneously hypertensive rats.

Morphologic features of the cell surface were studied in isolated cardiac myocytes obtained from 12 and 48 week old Spontaneously Hypertensive Rats (SHR) and normotensive Wistar-Kyoto (WKY) control rats. Hearts obtained from ether-anesthetized animals were perfused with Ca++-free Hanks' solution containing EGTA and 0.1% collagenase. The hearts were minced and the suspended cells fixed in 2% phosphate buffered glutaraldehyde. Cells mounted on Nuclepore membrane filters were dehydrated in alcohol and critical point dried for SEM. Quantitative evaluation of myocyte length, width and volume was done with a sonic digitizer on H and E stained cells by light microscopy. At both ages studied there was a significant increase in heart weight to body weight ratio, systolic blood pressure and cell size in SHR compared to WKY controls. In the older animals there appeared to be increased numbers of cell junction areas and deep grooves in the cell surface which appeared more pronounced in SHR than in WKY. The cell surface of the myocytes from 48 week old animals had deep capillary grooves surrounded by protruding longitudinal bundles of myofibrils. These changes would result in increased surface area of the larger cells and diminish the effect of increased cell size on diffusion distance from capillary to tissue. These changes in cell morphology were interpreted as providing a protective effect against development of functional impairment in the hypertrophied hearts.