Background: The antiviral effect of anti-influenza drugs such as zanamivir may be demonstrated in patients as an increased rate of decline in viral load over a time course of treatment as compared with placebo. Historically this was measured using plaque assays, or Culture Enhanced Enzyme Linked Immunosorbent Assay (CE-ELISA). Objectives: to develop and characterise real time quantitative PCR (qPCR) assays to measure influenza A and B viral load in clinical samples, that offer improvements over existing methods, in particular virus infectivity assays. Study design: The dynamic range and robustness were established for the real time qPCR assays along with stability of the assay components. Cross validation of the real time PCR assays with CE-ELISA was performed by parallel testing of both serial dilutions of three different subtypes of cultured virus and a panel of influenza positive throat swab specimens. Results: the assays were specific for influenza A and B and the dynamic ranges were at least seven logs. The assay variability was within acceptable limits but increased towards the lower limit of quantification, which was 3.33 log10 viral cDNA copies/ml of virus transport medium (ten viral RNA copies/PCR). The components of the assay were robust enough to withstand extended storage and several freeze-thawcycles. For the real time PCR assays the limit of quantification was equivalent to the virus infectivity cut off, which equates to a 93-fold increase in sensitivity. Conclusion: Well characterised real time PCR assays offer significant improvements over the existing methods for measuring the viral load of strains of influenza A and B in clinical specimens.

The Brn-3a, Brn-3b, and Brn-3c POU family transcription factors are closely related to one another and are members of the group IV subfamily of POU factors. Here we show that despite this close relationship, the factors have different effects on the activity of a target promoter. Brn-3a and Brn-3c stimulate the promoter whereas Brn-3b represses it. Moreover, Brn-3b can antagonize the stimulatory effect of Brn-3a on promoter activity and can also inhibit promoter activation by the Oct-2.1 POU factor. The difference in the transactivation activities of Brn-3a and Brn-3b is dependent upon the C-terminal region containing the POU domain of the two proteins, since exchange of this domain between the two factors converts Brn-3a into a repressor and Brn-3b into an activator.The POU (named for Pit, Oct, and Unc) family of transcription factors was originally defined on the basis of a conserved region of approximately 150 to 160 amino acids which was identified in the Pit-1, Oct-1, Oct-2, and Unc-86 regulatory proteins (for reviews, see references 12 and 33). This central POU domain constitutes the DNA binding domain of these proteins and allows them to bind to sequences related to the octamer motif ATGCAAAT in their target genes and thereby influence transcription (33,35).In several cases, the modulation of gene expression by POU factors has been shown to play a critical role in the development of specific cell types. Thus, of the original POU family members, the Pit-1 factor has been shown to be essential for the correct development of the pituitary gland, and its inactivity results in congenital dwarfism in both mice and humans (14,24). Similarly the unc-86 mutation in the nematode results in the absence of specific neuronal cell types (7).The critical roles identified for the initial members of the POU family led to efforts aimed at isolating novel members of this family. For differences from Bm-3 within the POU domain (15). Hence, the factor encoded by these clones represents a novel POU family member which we refer to as Brn-3b, to distinguish it from the original factor isolated by He et al. (11), which we refer to as . A third member of the Brn-3 family, Brn-3c, has recently been isolated by using a similar approach (23).The close homology of the different forms of Brn-3 within the POU region isolated in the original experiments (11, 15, 23) has led to their being grouped in a separate subfamily (group IV) amongst the POU proteins, together with the product of the unc-86 gene and the Drosophila factors I-POU and twin of I-POU (35). Interestingly, however, the isolation of full-length murine Brn-3a, Brn-3b, and Brn-3c cDNA clones has shown that the three factors are less closely related outside the POU domain and are encoded by three different genes (29). A similar conclusion has been reached by comparing the cDNA clones for human Brn-3a (also known as RDC-1) (3) and .The existence of three different closely related forms of Brn-3 suggests that they may have different functions. In previous experiments, the POU ...

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The resistance of cancers to conventional therapies has inspired the search for novel strategies.

Genetic manipulation of donor cornea prior to transplantation has the potential to modulate the allogeneic response, as well as the endothelial cell function. This study examined the feasibility of gene transfer to corneal endothelial cells using replication-defective recombinant adenoviral vectors. Adult rabbits corneas were infected with recombinant adenovirus RAd35, containing the Escherichia coli beta-galactosidase (lacZ) gene. Localization of gene transfer was assessed by histochemical staining for beta-galactosidase and recombinant protein production was quantified by a soluble assay. In initial experiments, the efficiency of gene transfer and kinetics of expression were studied ex vivo, using organ culture of transfected corneas. Following coculture of whole corneal fragments with RAd35, high levels of gene expression were evident on days 107, diminishing after that time. Gene transfer was found to be almost entirely restricted to corneal endothelial cells, with scattered expression in epithelial cells. Following these ex vivo studies, genetically modified corneas were transplanted as orthotopic allografts in rabbits. Similar kinetics of gene expression were seen after transplantation as in the ex vivo experiment, with maximal levels of gene expression in endothelial cells on days 1-4 after grafting. Corneal function following transplantation was not affected by the gene transfer, with the corneas attaining clarity within 1 day of grafting, and thereafter showing the expected thinning on ultrasonic pachymetry. In the absence of any immunosuppression, no inflammation was evident in graft recipient eyes, with the exception of allograft rejection in 1 animal 23 days after grafting. In this study we show that gene transfer to nonreplicating corneal endothelial cells is feasible using recombinant adenovirus vectors, and so may have potential application in the setting of corneal transplantation.