Mouse heart valve structure and function: echocardiographic and morphometric analyses from the fetus through the aged adult

The purpose of this study is to provide standard echocardiographic and morphometric data for normal mouse valve structure and function from late fetal to aged adult stages. Cross-sectional, two-dimensional and Doppler transthoracic echocardiography was performed in C57BL6 mice anesthetized with 1% to 2% isoflurane at embryonic day 18.5 (late fetal), 10 days (neonate), 1 mo (juvenile), 2 mo (young adult), 9 mo (old adult), and 16 mo (aged adult). Normal annulus dimensions indexed to age or weight, and selected flow velocities, were established by echocardiography. After echocardiographic imaging, hearts were harvested and histological and morphometric analyses were performed. Morphometric analysis demonstrated a progressive valve thinning and elongation during the fetal and juvenile stages that plateaued during adult stages (ANOVA, P < 0.01); however, there was increased thickening of the hinge of the aortic valve with advanced age, reminiscent of human aortic valve sclerosis. There was no age-related calcification. The results of this study provide comprehensive echocardiographic and morphometric data for normal mouse valve structure and function from late fetal to aged adult stages and should prove useful as a reference standard for future studies using mouse models of progressive valve disease.     

Resection for colorectal cancer in the very old: are the risks too high?

Altogether 277 consecutive patients aged 70 or more who were admitted for resection of colorectal cancer between 1975 and 1985 were studied. The postoperative complications and mortality in the 175 patients aged 70-79 were compared with those in the 102 patients aged 80 or more. The overall mortality was 11%. Mortality was significantly higher after resection in the older age group even when deaths from widespread malignancy were excluded from the analysis. After curative resection mortality was 2% (2/120) in the younger group and 7% (4/60) in the older group, but after palliative resection of tumours with local or distant spread mortality was significantly higher, at 21% (9/44) and 38% (12/32), respectively. An equal but high proportion of patients in both age groups suffered major complications, but complications caused significantly more deaths in the older group. The length of stay in hospital was not significantly different between the age groups.Patients should not be denied resection of a colorectal cancer because of age alone, especially if a curative operation is possible. The increased risk of death from major complications, particularly after palliative resection, should, however, be taken into account when an operation on patients over 80 is being considered.     

Production of avian oncoviral subgroups after multiple infection.

The number of different oncoviral env genes that can be expressed by a single chicken embryo fibroblast was investigated. Fibroblasts were infected with one to three subgroups of Rous-associated virus, which is a nontransforming avian oncovirus, then superinfected with a transforming virus, Rous sarcoma virus, of a different subgroup. The subgroups of viruses released by the resulting clones were analyzed. When two viral subgroups were used for preinfection, all the resulting clones produced transforming virus particles having the subgroup of the superinfecting virus, and most clones produced transforming virus particles of all the infecting viral subgroups. However, when cells were preinfected with three viral subgroups, many of the resulting clones did not produce transforming virus particles having the subgroup of the superinfecting virus, and only 1 of 23 clones produced transforming particles of all the infecting viral subgroups. DNA annealing experiments showed that cells infected with three or four viral subgroups had an additional 8 to 20 copies of proviral DNA per cell. Finally, most clones resulting from cells simultaneously infected with three or four viral subgroups were able to produce virus of all infecting subgroups. It appears that the number of exogenous oncoviral env genes that can be expressed by a single cell is limited, and in the range of 4 to 8-20 per cell.     

Long-Term Reproducible Expression in Human Fetal Liver Hematopoietic Stem Cells with a UCOE-Based Lentiviral Vector

Hematopoietic Stem Cell (HSC) targeted gene transfer is an attractive treatment option for a number of hematopoietic disorders caused by single gene defects. However, extensive methylation of promoter sequences results in silencing of therapeutic gene expression. The choice of an appropriate promoter is therefore crucial for reproducible, stable and long-term transgene expression in clinical gene therapy. Recent studies suggest efficient and stable expression of transgenes from the ubiquitous chromatin opening element (UCOE) derived from the human HNRPA2B1-CBX3 locus can be achieved in murine HSC. Here, we compared the use of HNRPA2B1-CBX3 UCOE (A2UCOE)-mediated transgene regulation to two other frequently used promoters namely EF1α and PGK in human fetal liver-derived HSC (hflHSC). Efficient transduction of hflHSC with a lentiviral vector containing an HNRPA2B1-CBX3 UCOE-eGFP (A2UCOE-eGFP) cassette was achieved at higher levels than that obtained with umbilical cord blood derived HSC (3.1x; p<0.001). While hflHSC were readily transduced with all three test vectors (A2UCOE-eGFP, PGK-eGFP and EF1α-eGFP), only the A2-UCOE construct demonstrated sustained transgene expression in vitro over 24 days (p<0.001). In contrast, within 10 days in culture a rapid decline in transgene expression in both PGK-eGFP and EF1α-eGFP transduced hflHSC was seen. Subsequently, injection of transduced cells into immunodeficient mice (NOD/SCID/Il2rg ^-/-) demonstrated sustained eGFP expression for the A2UCOE-eGFP group up to 10 months post transplantation whereas PGK-eGFP and EF1α-eGFP transduced hflHSC showed a 5.1 and 22.2 fold reduction respectively over the same time period. We conclude that the A2UCOE allows a more efficient and stable expression in hflHSC to be achieved than either the PGK or EF1α promoters and at lower vector copy number per cell.     

Isolation of viral specific RNA from SV40 infected cells by viral DNA chemically linked to a cellulose matrix.

SV40 DNA fragments chemically attached to neutral cellulose powder with a water-soluble carbodiimide have been used to isolate late lytic viral specific RNA from virus infected cells. Exhaustive hybridization to SV40 DNA reveals that virtually all of the isolated RNA molecules contain SV40 specific sequences. Comparison with SV40 cRNA prepared with purified Escherichia coli RNA polymerase and a SV40 DNA I template suggests that the purity of the isolated SV40 specific RNA is very close to 100%. The background level for the nonspecific binding of RNA to a purified cellulose matrix is very low. Retention of nonspecific RNA by SV40 DNA-cellulose is only 1.5% of the viral specific RNA isolated under saturating conditions for the column. Sedimentation in neutral sucrose suggests that the major 16S viral specific RNA has been isolated largely intact.