Stitching the fabric back together: child fostering attitudes in the transition to a chronic HIV epidemic

There are an estimated 56 million orphans and vulnerable children across sub-Saharan Africa. Communities typically care for orphan children through informal caring arrangements – either within or outside of kinship networks. Within Kenya, an estimated 250,000 children live on the streets. There is less research related to fostering attitudes of this special population than orphans and vulnerable children generally. Important research over the past decade has illuminated multiple ways in which children are made more vulnerable because of HIV, including parental death and street-migration from HIV-affected households. As HIV transitions from a terminal illness to a chronic, manageable one, research is also required to establish how parents living with HIV can be an asset to children. In this study, we assess whether mothers living with HIV were very willing to foster biologically-related children, and street-involved children, how these fostering attitudes differed from mothers not living with HIV, and whether differences in fostering attitudes by reported HIV status were mediated by social support, family functioning and general self-rated health. Approximately 40% of mothers living with HIV were very willing to provide long-term foster care to biologically-related or street-involved children. This was less than the percentage of mothers not living with HIV, who were very willing to foster biologically-related children (61%) or street-involved children (58%). Significant portions of these differences were explained by social support, family functioning and general self-rated health. Multi-sectoral approaches are suggested by these findings in order to improve the child-fostering capacity of mothers living with HIV. Improving social support, family functioning and general self-rated health among HIV-infected mothers may not only provide protective benefits for the mothers and their children, but also expand the community’s capacity to care for orphan and vulnerable children.     

Rat sponge implant model: a new system for evaluating angiogenic gene transfer

Therapeutic angiogenesis, either by protein injection or gene therapy, holds considerable promise for the treatment of coronary and peripheral artery diseases. Given the large number of angiogenic genes available, a simple, well defined, standard system to compare the relative angiogenic efficacy of such genes would be valuable. We have employed a replication-deficient adenovirus vector (complete E1a-, partial E1b- and partial E3-) to deliver the beta-galactosidase (beta-gal, AdLacZ) reporter gene or the human VEGF121 gene (AdGV VEGF121.10) to a rat sponge implant model of angiogenesis. beta-gal staining results reveal a transfection efficiency as high as 60% 24 h after 2x1010 particle units AdLacZ injection. Our results also indicate that a single injection of 2x1010 particle units of AdGVVEGF121.10 in the sponge results in >10, 000 pg VEGF protein expression per milligram of sponge tissue 24 h later. VEGF121 protein concentrations decreased 10-fold within 3 days and 100-fold within 7 days after injection. Significant VEGF121 protein levels were still detectable 14 days after initial virus injection. The high level of gene transfection efficiency was accompanied by enhanced angiogenesis in the sponge, a tissue devoid of any vessels before implantation. Compared to control (AdNull: adenovirus vector without the VEGF gene), AdGVVEGF121.10 induced a 2- to 3-fold up-regulation of angiogenesis at 7 and 14 days post vector injection as determined by both increased capillary number and increased tissue ingrowth. The angiogenic effects of AdGVVEGF121. 10 were dose-related in this model system. These findings demonstrate a dose-related angiogenic response to adenovirus-mediated gene therapy in this model.     

Comparison of sensitivity for human cytomegalovirus of the polymerase chain reaction, traditional tube culture and shell vial assay by sequential dilutions of infected cell lines.

Although traditional tube culture (TTC) is still considered by many as the 'gold standard' for the laboratory diagnosis of human cytomegalovirus (HCMV), the shell vial assay (SVA) offers greater speed of detection. This technique utilizes immunofluorescence (IF) to detect early or immediate early nuclear antigens (IEA). The detection capabilities of these two tests were compared with the polymerase chain reaction (PCR), a technique that amplifies enzymatically selected DNA target sequences. Serial dilutions of crude culture harvests from 2 HCMV strains, Towne and a clinical urine isolate, were made up to 1:1 000,000. Ten-microliters aliquots of the original sample and each dilution were tested by PCR, TTC and SVA. For PCR, the nested-primer approach was used. Outer primers delimited a 721-bp sequence contained within the 2nd to 4th exons of the immediate-early protein. Inner nest primers delimited a 167-bp sequence in the third exon, detected by a 32P-labelled probe. The results show that: (1) control samples which contained all PCR reagents but no DNA were uniformly negative; (2) radiolabelled-probe detection (RPD) of PCR products is, on average, 100 x more sensitive than detection by ethidium bromide; (3) PCR is, on average, 100 x more sensitive than evaluation of cytopathic effect (CPE) in the TTC; (4) the predictive value of a negative SVA result is low compared to PCR.     

Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis - acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.      

Delayed angiogenesis in aging rats and therapeutic effect of adenoviral gene transfer of VEGF

We have studied an age-related impairment in angiogenesis and evaluated the effect of overexpressing VEGF in this situation. Polyvinyl alcohol sponges were implanted subcutaneously into aged (24-month), adult (12-month), and young (2-month) rats. Blood vessel ingrowth and proliferative activity in the sponges were assessed by histology with immunostaining for von Willebrand's factor and proliferating cell nuclear antigen (PCNA), respectively. The percentage of total sponge area filled with ingrowing fibrovascular tissue was minimal in aged rats, intermediate in adult rats and highest in young rats. A similar pattern was observed for the total blood vessel numbers in the sponges from old to young animals. The percentage of total sponge endothelial cells (ECs) showing proliferative activity (PCNA positive) was lowest in the aged animals, intermediate in the adult rats and highest in the young rats. To further explore the mechanism of impaired angiogenesis in aged animals, we investigated and found a reduced level of endogenous VEGF protein expression in 12-month-old rats compared to that in 2-month-old rats. VEGF121 gene transfer significantly enhanced blood vessel and fibrovascular tissue ingrowth in adult/aged rats. Adenoviral-VEGF gene transfer also significantly stimulated EC proliferation in aged and adult rats. However, identical treatment failed to further stimulate the already more robust angiogenesis in young animals. The different angiogenic response in adult vs. young rats was not due to differences in gene transfer efficiency, since similar levels of human VEGF121 protein was detected in adult and young rats. Our results indicate that the decreased angiogenic response with aging is associated with reduced EC proliferation and reduced endogenous VEGF production. Adenoviral-VEGF121 gene transfer is effective in augmenting angiogenesis, particularly in older animals.