rKv1.2 overexpression in the central medial thalamic area decreases caffeine-induced arousal.

The voltage-gated potassium channel Kv1.2 belongs to the shaker-related family and has recently been implicated in the control of sleep profile on the basis of clinical and experimental evidence in rodents. To further investigate whether increasing Kv1.2 activity would promote sleep occurrence in rats, we developed an adeno-associated viral vector that induces overexpression of rat Kv1.2 protein. The viral vector was first evaluated in vitro for its ability to overexpress rat Kv1.2 protein and to produce functional currents in infected U2OS cells. Next, the adeno-associated Kv1.2 vector was injected stereotaxically into the central medial thalamic area of rats and overexpression of Kv1.2 was showed by in situ hybridization, ex vivo electrophysiology and immunohistochemistry. Finally, the functional effect of Kv1.2 overexpression on sleep facilitation was investigated using telemetry system under normal conditions and following administration of the arousing agent caffeine, during the light phase. While no differences in sleep profile were observed between the control and the treated animals under normal conditions, a decrease in the pro-arousal effect of caffeine was seen only in the animals injected with the adeno-associated virus-Kv1.2 vector. Overall, our data further support a role of the Kv1.2 channel in the control of sleep profile, particularly under conditions of sleep disturbance.

Transgene expression and effective gene silencing in vagal afferent neurons in vivo using recombinant adeno-associated virus vectors.

Vagal afferent fibres innervating thoracic structures such as the respiratory tract and oesophagus are diverse, comprising several subtypes of functionally distinct C-fibres and A-fibres. Both morphological and functional studies of these nerve subtypes would be advanced by selective, effective and long-term transduction of vagal afferent neurons with viral vectors. Here we addressed the hypothesis that vagal sensory neurons can be transduced with adeno-associated virus (AAV) vectors in vivo, in a manner that would be useful for morphological assessment of nerve terminals, using enhanced green fluorescent protein (eGFP), as well as for the selective knock-down of specific genes of interest in a tissue-selective manner. We found that a direct microinjection of AAV vectors into the vagal nodose ganglia in vivo leads to selective, effective and long-lasting transduction of the vast majority of primary sensory vagal neurons without transduction of parasympathetic efferent neurons. The transduction of vagal neurons by pseudoserotype AAV2/8 vectors in vivo is sufficiently efficient such that it can be used to functionally silence TRPV1 gene expression using short hairpin RNA (shRNA). The eGFP encoded by AAV vectors is robustly transported to both the central and peripheral terminals of transduced vagal afferent neurons allowing for bright imaging of the nerve endings in living tissues and suitable for structure-function studies of vagal afferent nerve endings. Finally, the AAV2/8 vectors are efficiently taken up by the vagal nerve terminals in the visceral tissue and retrogradely transported to the cell body, allowing for tissue-specific transduction.

Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement.

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 log(10) 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-thaw cycles. 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.

Loss of epithelial integrity resulting from E-cadherin dysfunction predisposes airway epithelial cells to adenoviral infection.

Epithelial intercellular adhesion is fundamental to the formation of the airway epithelial protective barrier. In this respect, cadherins are important because these adhesion molecules regulate formation and maintenance of epithelial intercellular junctions. To study the importance of airway epithelial integrity in determining susceptibility to virus infection, we used a replication-incompetent adenovirus, RAd35, and an E-cadherin specific function-blocking antibody, SHE78-7, to disrupt intercellular contacts in human bronchial epithelial cell line 16HBE14o- and primary bronchial epithelial cells. After exposure of 16HBE14o- cell cultures to SHE78-7, disruption of the transepithelial permeability barrier was indicated by a loss of transepithelial electrical resistance and an associated increase of mannitol, inulin, and dextran paracellular flux. Subsequent exposure of SHE78-7-treated cell cultures to RAd35 showed a remarkable increase in adenoviral infection as assessed by beta-galactosidase reporter gene expression. In cultures exposed to SHE78-7, disruption of E-cadherin function resulted in infection equivalent to that in control cultures using 16-fold lower viral titers. These studies show that manipulation of E-cadherin function provides a specific means of altering epithelial integrity that in turn determines resistance of airway epithelia to adenoviral infection.

Use of transcriptional regulatory elements of the MUC1 and ERBB2 genes to drive tumour-selective expression of a prodrug activating enzyme.

In order to exploit differences in gene expression between normal and malignant cells for genetic prodrug-activation therapy, we have generated recombinant retroviruses containing the herpes simplex virus thymidine kinase coding region cloned downstream of sequences derived from the 5'-flanking regions of the MUC1 and ERBB2 genes. Transduction with retroviruses containing MUC1 promoters resulted in an increase in GCV sensitivity in MUC1 positive cells. A further increase in GCV sensitivity was achieved when MUC1-positive cells were transduced with retroviruses containing chimeric-MUC1/ERBB2 promoters. No significant sensitization to GCV was observed when MUC1-negative cells were transduced with these recombinant retroviruses. These results suggest that one may be able to develop a tumour-selective therapy by utilizing the transcriptional regulatory regions of the MUC1 and ERBB2 genes to drive the expression of suicide genes.

Suicide gene expression induced in tumour cells transduced with recombinant adenoviral, retroviral and plasmid vectors containing the ERBB2 promoter.

In order to exploit the tumour-specific nature of ERBB2 expression for genetic prodrug-activation therapy, we have generated recombinant adenoviral, retroviral and plasmid vectors containing an expression cassette consisting of the ERBB2 promoter and herpes simplex virus thymidine kinase coding sequence. In the case of the adenoviral vectors, the expression cassette was introduced into the E1 or E3 region of the genome. All of the vectors were capable of sensitizing ERBB2-positive cells to the action of ganciclovir. In contrast to the retroviral and plasmid vectors, however, transduction with the adenoviral vectors also resulted in sensitization of ERBB2-negative cells to ganciclovir, infection of cell lines with a beta-galactosidase expressing adenovirus showed that the sensitizing effect was not due to adenoviral infection per as in all but one of the cell lines tested. This study demonstrates that the ERBB2 promoter can be used to induce ERBB2-dependent sensitization to ganciclovir when in the context of retroviral and plasmid vectors. Observations made in this study do, however, suggest that adenoviral vectors may not be the ideal system to engineer conditional expression, and possible explanation for this phenomenon are discussed.

Adenovirus-mediated gene delivery to the corneal endothelium.

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.

The use of DNA viruses as vectors for gene therapy.

The need for efficient transfer of potentially therapeutic genes to defined cell populations has stimulated the development of vectors based on viruses. To date, most effort has been spent on the RNA-containing retroviruses. These viruses, however, possess a number of disadvantages including an inability to infect nondividing cells as well as having potential for oncogenicity and insertional mutagenesis of host cell genes due to random chromosomal integration. These disadvantages have led to the development of vectors based on DNA-containing viruses such as adenovirus, herpes simplex virus and parvovirus. These viruses possess a number of attributes favourable to their use in gene therapy. Adenoviruses, for example, were first considered as potential vectors for the genetic treatment of lung conditions due to their natural affinity for respiratory epithelium. However, other features including their ability to be prepared at high titres, to direct high levels of foreign gene expression and their extrachromosomal existence has resulted in their development for the treatment of numerous other diseases. In many studies, adenovirus vectors have been shown to efficiently infect target cell populations and to express proteins at therapeutic levels in the absence of significant toxicity. The ability of herpes simplex virus to reside in neurons in a latent state that does not appear to affect normal cellular physiology has sparked interest in this virus as a potential vector in the treatment of neurological disorders. A subgroup of parvoviruses, namely the adeno-associated viruses, have a prediliction for integration at a defined chromosomal location and may represent a safer alternative to retroviruses.

The opposite and antagonistic effects of the closely related POU family transcription factors Brn-3a and Brn-3b on the activity of a target promoter are dependent on differences in the POU domain.

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.

Transactivation of the human papilloma virus 16 octamer motif by the octamer binding protein Oct-2 requires both the N and C terminal activation domains.

The upstream regulatory region (URR) of the human papillomaviruses HPV16 and 18 contains a sequence with a seven out of eight base match to the consensus binding site for octamer binding transcription factors. This motif acts as a target for repression by the Oct-1 transcription factor and therefore inhibits promoter activity in non-cervical cells expressing only Oct-1. In contrast the HPV octamer motif activates promoter activity in cervical cells. Here we show that cervical cells express the activating form of the Oct-2 transcription factor, Oct 2.1 and that this factor can transactivate promoter activity via the HPV16 octamer. This effect is dependent upon both the N and C-terminal activation domains of Oct-2. The expression of specific octamer binding proteins such as Oct-2 in cervical cells thus allows the HPV16 motif to produce opposite effects on gene expression in cervical and non-cervical cells suggesting that it may play a role in the cervical specificity of URR driven gene expression.

Hepatitis C viral RNA in clotting factor concentrates and the development of hepatitis in recipients.

The polymerase chain reaction (PCR) was used to detect hepatitis C (HCV) viral sequences (HCV-RNA) in clotting factor concentrates that had been stored at 4 degrees C for 1 to 16 years. A total of 43 concentrates were tested, comprising 31 batches of factor VIII, 6 of factor IX, 2 of antithrombin III, 3 of FEIBA, and 1 of factor VII. HCV-RNA was detected in 13 of the 43 batches (30.2%). Concentrates that had not undergone viral inactivation during manufacture were significantly more likely to contain detectable HCV-RNA than concentrates that had been virally inactivated (56.3% v 14.5%, P = .006). HCV sequences were more commonly detected in concentrates made from paid donor plasma than in those made from volunteer donor plasma (44% v 11%, P = .041), and more commonly in virally inactivated concentrates with pre-1989 than with post-1989 expiration dates (50% v 0%, P = .004). Of the four batches of heat-treated products that were HCV-RNA positive, at least three transmitted non-A, non-B hepatitis (NANBH). An association between the presence of HCV-RNA in concentrates and the development of NANBH was demonstrated in nine previously untreated patients on prospective follow-up. HCV-RNA was detected in the concentrates administered to the six patients whose alanine aminotransferase (ALT) abnormalities met the diagnostic criteria for NANBH and who later seroconverted for HCV, but it was not detected in the concentrates administered to the three patients whose ALT abnormalities failed to satisfy the diagnostic criteria and who did not seroconvert. We suggest that the use of this PCR technique to monitor clotting factor concentrates derived from pooled blood may potentially contribute to product safety.

Acute hepatitis C infection in patients undergoing therapy for haematological malignancies: a clinical and virological study.

Patients receiving multiple transfusions are at risk of acquiring hepatitis C (HCV) infection from a donor population which is unscreened for hepatitis C antibodies (anti-HCV). Prior to the introduction of blood donor screening for anti-HCV in the U.K., a group of patients undergoing therapy for haematological malignancies, with repeatedly abnormal liver function tests, were investigated for acute HCV infection. Thirty-two patients had repeatedly raised serum transaminases, and eight of these (25%) had evidence of an acute HCV infection. The diagnosis was made by the detection of HCV-RNA in the patients' serum using a complementary DNA/polymerase chain reaction (cDNA/PCR) procedure. All eight patients had received myeloablative chemotherapy and three had undergone bone marrow transplantation. HCV infection contributed significantly to the morbidity of this group of patients in the short term whilst they were undergoing treatment for their underlying haematological condition. The long-term effects have yet to be evaluated. In an attempt to decrease hepatic damage due to HCV, three patients were placed on interferon therapy. None showed a sustained reduction in serum transaminases or HCV viraemia. It is hoped that the introduction of anti-HCV screening of blood donors, will reduce the frequency of transfusion-acquired HCV infections. Early observations suggest that this is the case, as we have seen no new cases of HCV infection in our unit since the introduction of donor screening in September 1991.

The octamer binding site in the HPV16 regulatory region produces opposite effects on gene expression in cervical and non-cervical cells.

The upstream regulatory region (URR) of the tumorigenic human papillomaviruses HPV 16 and 18 contains an octamer binding site which is located adjacent to a binding site for the ubiquitous transcription factor NFI. The octamer site binds both the constitutively expressed transcription factor Oct-1 and a novel cervical octamer binding protein. In contrast the URR of the non-tumorigenic viruses HPV6 and HPV11 lacks the octamer binding site although the adjacent NFI site is conserved. Inactivation of the octamer binding site results in a higher level of gene expression in cells which contain only Oct-1 and a lower level in cells containing the cervical octamer binding protein indicating that that whilst Oct-1 binding reduces promoter activity, the cervical protein increases it. In agreement with this, over-expression of Oct-1 reduces the level of gene activity directed by this region of the HPV 16/18 URR and inhibits its activation by NFI whilst having no effect on the corresponding region of the HPV 6/11 URR. The significance of these effects is discussed in terms of the cervical-specific activity of the HPV16/18 URR and its role in HPV-mediated transformation.

Alternative splicing determines the carboxy terminus of the Epstein-Barr virus nuclear antigen 5 species expressed in the Burkitt's lymphoma cell line Daudi.

The Daudi strain of Epstein-Barr virus (EBV) possesses a genomic deletion, relative to the B95-8 EBV prototype, that removes the entire Epstein-Barr virus nuclear antigen 2 (EBNA2) open reading frame (ORF) and the sequences encoding the carboxy terminus of EBNA5. Immunoblot analysis carried out in this study indicates that two species of EBNA5 (31K and 37K) are expressed in Daudi cells. Nucleotide sequence analysis of Daudi cDNA clones has confirmed that, as a consequence of the genomic deletion, exons usually appearing further downstream in EBNA messages (exons U or HF) are spliced directly onto the truncated EBNA5 ORF. Furthermore, the use of alternative splicing suggests that the two EBNA5 species expressed in Daudi cells possess different carboxy termini.

Detection of hepatitis C viraemia in Caucasian patients with hepatocellular carcinoma.

Potential risk factors for the development of hepatocellular carcinoma were analysed in 40 Caucasian patients with this malignancy. A higher proportion (14 of 40; 35%) had evidence of hepatitis C virus (HCV) infection than had evidence of either hepatitis B virus (HBV) carriage (17.5%) or alcohol abuse (30%). In all 14 patients whose sera were reactive by HCV ELISA (Ortho second generation test), the presence of antibodies to HCV were confirmed by recombinant immunoblot assay (Ortho RIBA-2). Furthermore, two independent laboratories detected HCV-RNA in 10 of the 14 (71%) anti-HCV positive sera. Two additional sera were shown to contain HCV-RNA when reanalysed by a modified PCR using oligonucleotide primers designed to amplify a shorter fragment of the 5' noncoding region of the genome. Seven of the anti-HCV positive patients also had evidence of prior HBV infection and 2 admitted to alcohol abuse. HCV infection was the only identifiable risk factor in 6 patients. These data confirm the association between HCV infection and hepatocellular carcinoma and suggest that persistent viral replication accompanies tumour development in the majority of patients whose serum contains anti-HCV.