The adenine-rich tract in the 5ʹ-end of the hepatitis C virus ORF encodes a peptide regulating the binding of the C protein to RNA

Hepatitis C virus (HCV ) core (C) protein is thought to bind to viral RNA before it undergoes oligomerization leading to RNA encapsidation. Details of these events are so far unknown. The 5ʹ-terminal C protein coding sequence that includes an adenine (A)-rich tract is a part of an internal ribosome entry site(IRES). This nucleotide sequence but not the corresponding protein sequence is needed for proper initiation of translation of viral RNA by an IRES-dependent mechanism. In this study, we examined the importance of this sequence for the ability of the C protein to bind to viral RNA. Serially truncated C proteins with deletions from 10 up to 45 N-terminal amino acids were expressed in Escherichia coli, purified and tested for binding to viral RNA by a gel shift assay. The results showed that truncation of the C protein from its N-terminus by more than 10 amino acids abolished almost completely its expression in E. coli. The latter could be restored by adding a tag to the N-terminus of the protein. The tagged proteins truncated by 15 or more amino acids showed an anomalous migration in SDS-PAGE. Truncation by more than 20 amino acids resulted in a complete loss of ability of tagged C protein to bind to viral RNA. These results provide clues to the early events in the C protein - RNA interactions leading to C protein oligomerization, RNA encapsidation and virion assembly.

Early users of fertility treatment with hormones and IVF : women who live in major cities and have private health insurance

Objective: To identify early users (women aged <34 years) of fertility treatment with hormones and in vitro fertilisation (IVF). Methods: A cross-sectional survey of infertile women from fertility clinics (n=59) and from the community (Australian Longitudinal Study on Women's Health participants) who had (n=121) or had not (n=110) used hormones/IVF as treatment for infertility. Associations between socio-demographic, reproductive and lifestyle factors, medical conditions and recurrent symptoms and using treatment (or not) were analysed using multivariable logistic regression. Results: Among infertile women who had used treatment (community vs clinic), women from clinics had lower odds of living outside major cities, using hormones only, i.e., not IVF, or recurrent headaches/migraines, severe tiredness, or stiff/painful joints; and higher odds of recent diagnoses of urinary tract infection or anxiety disorder. Compared to infertile women who had not used treatment, women from clinics had lower odds of living outside major cities, recurrent allergies or severe tiredness; and higher odds of having private health insurance for hospital or ancillary services, recent diagnosis of polycystic ovary syndrome or recurrent constipation. Conclusions: Compared to infertile women in the community, living in major cities and having private health insurance are associated with early use of treatment for infertility at specialist clinics by women aged <34 years. Implications: These results provided evidence of inequity of services for infertile women.

Using expedited 10g protein counter (EP-10) for meal planning

Precise protein quantification and recommendation is essential in clinical dietetics, particularly in the management of individuals with chronic kidney disease, malnutrition, burns, wounds, pressure ulcers, and those in active sports. The Expedited 10g Protein Counter (EP-10) was developed to simplify the quantification of dietary protein for assessment and recommendation of protein intake.1 Instead of using separate protein exchanges for different food groups to quantify the dietary protein intake of an individual, every exchange in the EP-10 accounts for an exchange each of 3g non-protein-rich food and 7g protein-rich food (Table 1). The EP-10 was recently validated and published in the Journal of Renal Nutrition recently.1 This study demonstrated that using the EP-10 for dietary protein intake quantification had clinically acceptable validity and reliability when compared with the conventional 7g protein exchange while requiring less time.2 In clinical practice, the use of efficient, accurate and practical methods to facilitate assessment and treatment plans is important. The EP-10 can be easily implemented in the nutrition assessment and recommendation for a patient in the clinical setting. This patient education tool was adapted from materials printed in the Journal of Renal Nutrition.1 The tool may be used as presented or adapted to assist patients to achieve their recommended daily protein intake.

Nutritional intervention incorporating expedited 10g protein counter (EP-10) to improve the albumin and transferrin of chronic hemodialysis patients

Objective: The expedited 10g protein counter (EP-10) is a quick and valid clinical tool for dietary protein quantification. This study aims to assess the clinical effectiveness of the EP-10 in improving serum albumin and transferrin in chronic hemodialysis patients. Methods: Forty-five patients with low serum albumin (< 38 g /L) were enrolled in this study. Parameters measured included dry weight, height, dietary intake, and levels of serum albumin, transferrin, potassium, phosphate and kinetic modeling (Kt/v). The nutritional intervention incorporated the EP-10 in two ways (1)lto quantify protein intake of patients and (2)ito educate patients to meet their protein requirements. Mean values of the nutritional parameters before and after intervention were compared using paired t-test. Results: Three months after nutritional intervention, mean albumin levels increased significantly from 32.2+4.8g/L to 37.0+3.2g/L (p<0.001). Thirty-eight (84%) patients showed an increase in albumin levels while two (4%) maintained their levels. Of the thirty-six (80%) patients with low transferrin levels (<200 mg/dL), 28 (78%) had an increase and two maintained their levels post-intervention. Mean transferrin levels increased significantly from 169.4+39.9mg/dL to 180.9+38.1mg/dL (p< 0.05). Conclusion: Nutritional intervention incorporating the EP-10 method is able to make significant improvements to albumin and transferrin levels of chronic hemodialysis patients.

Evaluating stakeholder satisfaction during public participation in major infrastructure and construction projects : a fuzzy approach

Numerous different and sometimes discrepant interests can be affected, both positively and negatively, throughout the course of a major infrastructure and construction (MIC) project. Failing to address and meet the concerns and expectations of the stakeholders involved has resulted in many project failures. One way to address this issue is through a participatory approach to project decision making. Whether the participation mechanism is effective or not depends largely on the client/owner. This paper provides a means of systematically evaluating the effectiveness of the public participation exercise, or even the whole project, through the measurement of stakeholder satisfaction. Since the process of satisfaction measurement is complicated and uncertain, requiring approximate reasoning involving human intuition, a fuzzy approach is adopted. From this, a multi-factor hierarchical fuzzy comprehensive evaluation model is established to facilitate the evaluation of satisfaction in both single stakeholder group and overall MIC project stakeholders.

What are the key conditions associated with lower limb amputations in a major Australian teaching hospital?

Background Lower extremity amputation results in significant global morbidity and mortality. Australia appears to have a paucity of studies investigating lower extremity amputation. The primary aim of this retrospective study was to investigate key conditions associated with lower extremity amputations in an Australian population. Secondary objectives were to determine the influence of age and sex on lower extremity amputations, and the reliability of hospital coded amputations. Methods: Lower extremity amputation cases performed at the Princess Alexandra Hospital (Brisbane, Australia) between July 2006 and June 2007 were identified through the relevant hospital discharge dataset (n = 197). All eligible clinical records were interrogated for age, sex, key condition associated with amputation, amputation site, first ever amputation status and the accuracy of the original hospital coding. Exclusion criteria included records unavailable for audit and cases where the key condition was unable to be determined. Chi-squared, t-tests, ANOVA and post hoc tests were used to determine differences between groups. Kappa statistics were used to measure reliability between coded and audited amputations. A minimum significance level of p < 0.05 was used throughout. Results: One hundred and eighty-six cases were eligible and audited. Overall 69% were male, 56% were first amputations, 54% were major amputations, and mean age was 62 ± 16 years. Key conditions associated included type 2 diabetes (53%), peripheral arterial disease (non-diabetes) (18%), trauma (8%), type 1 diabetes (7%) and malignant tumours (5%). Differences in ages at amputation were associated with trauma 36 ± 10 years, type 1 diabetes 52 ± 12 years and type 2 diabetes 67 ± 10 years (p < 0.01). Reliability of original hospital coding was high with Kappa values over 0.8 for all variables. Conclusions: This study, the first in over 20 years to report on all levels of lower extremity amputations in Australia, found that people undergoing amputation are more likely to be older, male and have diabetes. It is recommended that large prospective studies are implemented and national lower extremity amputation rates are established to address the large preventable burden of lower extremity amputation in Australia.

GABAergic modification of myopic and hyperopic ocular tissue effects on scleral fibroblast growth

Purpose: Myopia is a common eye disorder affecting up to 90% of children in South East Asia and 30% of the population worldwide. Myopia of high severity is a leading cause of blindness around the world (4th to 5th most common). Changes and remodelling of the sclera i.e. increase cellular proliferation & increase protein synthesis within scleral cells (↑ scleral DNA) and thinning and lose of extracellular matrix of sclera (↓ scleral GAG synthesis) have been linked to myopic eye growth in animal models. Signals acting on the sclera are thought to originate in the retina, and are modulated by the retinal pigment epithelium (RPE) with limited evidence suggesting that the RPE can modify scleral cell growth in culture. However, the mechanism of retinal signal transmission and the role of posterior eye cup tissue, including the RPE, in mediating changes in scleral fibroblast growth during myopia development are unclear. Retinal transmitter systems are critically involved in pathways regulating eye growth, which ultimately lead to alterations in the sclera if eye size is to change. A dopaminergic agonist and muscarinic antagonists decrease the proliferation of scleral chondrocytes when co-cultured with chick’s retinal pigment epithelium (RPE). GABA receptors have recently been localised to chick sclera. We therefore hypothesised that posterior eye cup tissue from myopic eyes would stimulate and from hyperopic eyes would inhibit growth of scleral fibroblasts in vitro and that GABAergic agents could directly interact with scleral cells or indirectly modify the effects of myopic and hyperopic posterior eye cup tissue on scleral fibroblast growth. Method: Fibroblastic cells obtained from 8-day-old chick sclera were used to establish cell banks. Two major experiments were performed. Experiment 1: To determine if posterior eye cup tissues from myopic eye stimulates and hyperopic eye inhibits scleral cell proliferation, when co-cultured with scleral cells in vitro. This study comprised two linked experiments, i) monocular visual treatments of FDM (form-deprivation myopia), LIM (lens-induced myopia) and LIH (lens-induced hyperopia) with assessment of the effect of full punch eye cup tissue on DNA and GAG synthesis by cultured chick scleral fibroblasts, and ii) binocular visual treatments comprising LIM and LIH with assessment of the effect of individual layers of eye cup tissues (neural retina, RPE and choroid) on cultured chick scleral fibroblasts. Visual treatment was applied for 3 days. Experiment 2: To determine the direct interaction of GABA agents on scleral cell growth and to establish whether GABA agents modify the stimulatory/inhibitory effect of myopic and hyperopic posterior eye cup tissues on cultured scleral cell growth in vitro. Two linked experiments were performed. i) GABA agonists (muscimol and baclofen) and GABA antagonists (bicuculine (-), CGP46381 and TPMPA) were added to scleral cell culture medium to determine their direct effect on scleral cells. ii) GABAergic agents (agonists and antagonists) were administered to scleral fibroblasts co-cultured with posterior eye cup tissue (retina, RPE, retina/RPE, RPE/choroid). Ocular tissues were obtained from chick eyes wearing +15D (LIH) or -15D lenses (LIM) for 3 days. In both experiments, tissues were added to hanging cell culture insert (pore size 1.0ìm) placed over each well of 24 well plates while scleral cells were cultured in DMEM/F12, Glutamax (Gibco) plus 10% FBS and penicillin/streptomycin (50U/ml)) and fungizone (1.25ug/ml) (Gibco), at seeding density of 30,000 cells/well at the bottom of the well and allowed to grow for 3 days. Scleral cells proliferation rate throughout the study was evaluated by determining GAG and DNA content of scleral cells using Dimethylmethylene blue (DMMB) dye and Quant-iTTm Pico Green® dsDNA reagent respectively. Results and analysis: Based on DNA and GAG content, there was no significant difference in tissue effect of LIM and LIH eyes on scleral fibroblast growth (DNA: 8.4 ± 1.1μg versus 9.3 ± 2.3 μg, p=0.23; GAG: 10.13 ± 1.4 μg versus 12.67 ± 1.2 μg, F2,23=6.16, p=0.0005) when tissues were obtained from monocularly treated chick eyes (FDM or +15D lens or -15D lens over right eyes with left eyes untreated) and co-cultured as full punch. When chick eyes were treated binocularly with -15D lens (LIM) right eye and +15D lens (LIH) left eyes and tissue layers were separated, the retina from LIM eyes did not stimulate scleral cell proliferation compared to LIH eyes (DNA: 27.2 ± 6.7 μg versus 23.2 ± 1.5 μg, p=0.23; GAG: 28.1 ±3.7 μg versus 28.7 ± 4.2 μg, p=0.21). Similarly, the LIH and LIM choroid did not produce a differential effect based on DNA (LIM 46.9 ± 6.4 μg versus LIH 53.5 ± 4.7 μg, p=0.18), however the choroid from LIH eyes induced higher scleral GAG content than from LIM eyes (32.5 ± 6.7 μg versus 18.9 ± 1.2 μg, p=0.023). In contrast, the RPE from LIM eyes caused a significant increase in fibroblast proliferation whereas the RPE from LIH eyes was relatively inhibitory (72.4 ± 6.3 μg versus 27.9 ± 2.3 μg, F1, 6=69.99, p=0.0005). GAG data were opposite to DNA data e.g. the RPE from LIH eyes increased (33.7 ± 7.9 μg) while the RPE from LIM eyes decreased (28.2 ± 3.0 μg) scleral cell growth (F1, 6=13.99, p=0.010). Based on DNA content, GABA agents had a small direct effect on scleral cell growth; GABA agonists increased (21.4 ± 1.0% and 18.3 ± 1.0% with muscimol and baclofen, p=0.0021), whereas GABA antagonists decreased fibroblast proliferation (-23.7 ± 0.9% with bicuculine & CGP46381 and -28.1 ± 0.5% with TPMPA, p=0.0004). GABA agents also modified the effect of LIM and LIH tissues (p=0.0005).The increase in proliferation rate of scleral fibroblasts co-cultured with tissues (RPE, retina, RPE/retina and RPE/choroid) from LIM treated eyes was enhanced by GABA agonists (muscimol: 27.4 ± 1.2%, 35.8 ± 1.6%, 8.4 ± 0.3% and 11.9 ± 0.6%; baclofen: 27.0 ± 1.0%, 15.8 ± 1.5%, 16.8 ± 1.2% and 15.4 ± 0.4%, p=0.014) whereas GABA antagonists further reduced scleral fibroblasts growth (bicuculine: -52.5 ± 2.5%, -36.9 ± 1.4%, -37.5 ± 0.6% and -53.7 ± 0.9%; TPMPA: 57.3 ± 1.3%, -15.7 ± 1.2%, -33.5 ± 0.4% and -45.9 ± 1.5%; CGP46381: -51.9 ± 1.6%, -28.5 ± 1.5%, -25.4 ± 2.0% and -45.5 ± 1.9% respectively, p=0.0034). GAG data were opposite to DNA data throughout the experiment e.g. GABA agonists further inhibited while antagonists relatively enhanced scleral fibroblasts growth for both LIM and LIH tissue co-culture. The effect of GABA agents was relatively lower (p=0.0004) for tissue from LIH versus LIM eyes but was in a similar direction. There was a significant drug effect on all four tissue types e.g. RPE, retina, RPE/retina and RPE/choroid for both LIM and LIH tissue co-culture (F20,92=3.928, p=0.0005). However, the effect of GABA agents was greatest in co-culture with RPE tissue (F18,36=4.865, p=0.0005). Summary and Conclusion: 1) Retinal defocus signals are transferred to RPE and choroid which then exert their modifying effect on scleral GAG and DNA synthesis either through growth stimulating factors or directly interacting with scleral cells in process of scleral remodeling during LIM and LIH visual conditions. 2) GABAergic agents affect the proliferation of scleral fibroblasts both directly and when co-cultured with ocular tissues in vitro.

Expression of PTRF in PC3 cells modulates cholesterol dynamics and the actin cytoskeleton impacting secretion pathways

Expression of caveolin-1 is up-regulated in prostate cancer metastasis and is associated with aggressive recurrence of the disease. Intriguingly, caveolin-1 is also secreted from prostate cancer cell lines and has been identified in secreted prostasomes. Caveolin-1 is the major structural component of the plasma membrane invaginations called caveolae. Co-expression of the coat protein Polymerase I and transcript release factor (PTRF) is required for caveolae formation. We recently found that expression of caveolin-1 in the aggressive prostate cancer cell line PC-3 is not accompanied by PTRF, leading to noncaveolar caveolin-1 lipid rafts. Moreover, ectopic expression of PTRF in PC-3 cells sequesters caveolin-1 into caveolae. Here we quantitatively analyzed the effect of PTRF expression on the PC-3 proteome using stable isotope labeling by amino acids in culture and subcellular proteomics. We show that PTRF reduced the secretion of a subset of proteins including secreted proteases, cytokines, and growth regulatory proteins, partly via a reduction in prostasome secretion. To determine the cellular mechanism accounting for the observed reduction in secreted proteins we analyzed total membrane and the detergent-resistant membrane fractions. Our data show that PTRF expression selectively impaired the recruitment of actin cytoskeletal proteins to the detergent-resistant membrane, which correlated with altered cholesterol distribution in PC-3 cells expressing PTRF. Consistent with this, modulating cellular cholesterol altered the actin cytoskeleton and protein secretion in PC-3 cells. Intriguingly, several proteins that function in ER to Golgi trafficking were reduced by PTRF expression. Taken together, these results suggest that the noncaveolar caveolin-1 found in prostate cancer cells generates a lipid raft microenvironment that accentuates secretion pathways, possibly at the step of ER sorting/exit. Importantly, these effects could be modulated by PTRF expression.

Creating Queensland, creative communities partnership - major Brisbane festival & Australia Council for the Arts

In March 2010, Brisbane Festival commissioned a Research Team, led by Dr Bree Hadley and Dr Sandra Gattenhof, Creative Industries Faculty, Queensland University of Technology, to conduct an evaluation of the Creating Queensland program, a new Creative Communities partnership between Brisbane Festival and the Australia Council for the Arts. This Final Report reviews and reports on the effectiveness of the program gathered during three phases throughout 2010: Phase 1, in which the research team analysed Brisbane Festival’s pre-existing data on the Creating Queensland events in 2009; Phase 2, in which the research team designed a new suite of instruments to gather data from producers, producing partners, artists and attendees involved in the Creating Queensland events in 2010; and Phase 3, in which the research team used content analysis of the narratives emerging in the data to establish how Brisbane Festival has adopted processes, activities or engagement protocols to operate as catalysts that produce experiences with specific impacts on individuals and communities. The Final Report finds that the Creating Queensland events concentrate on developing specific experiences for those involved – usually associated with storytelling, showcasing, and the valorisation or re-valorisation of neglected or forgotten cultural forms – in order to give communities a voice. It finds that the events prioritise accessibility – usually associated with allowing specific local communities or local artists to present material that is meaningful to them – and inclusivity – usually associated with using connections with producing partners (such as the Multicultural Development Association) to bring more and more people into the program. It finds that the events have a capacity-building effect, which allows local communities to increase their capacity to launch their own ideas, initiatives or events, allows individuals to increase their employability, or allows communities and individuals to increase their visibility within mainstream cultural practices and infrastructure. The Final Report further finds that Brisbane Festival has, throughout its years of commitment to community programming, developed specific techniques to enable events in the Creating Queensland program to have these effects, that these can be tracked, and, as a result, deployed or redeployed both by Brisbane Festival and other community arts organisations in the development of effective community arts programs. The data demonstrates that Creating Queensland is, by and large, having the desired effect on communities – people are actually participating, presenting work, and increasing their personal, professional and social skills in various ways, and this is valued by all stakeholders. The data also demonstrates that, as would be expected with any community arts program – particularly programs of this size and complexity – there are areas in which Creating Queensland is functioning exceptionally well and areas in which continuous improvement processes should be continued. Areas of excellence relate to Brisbane Festival’s longstanding commitment to community arts, and active community participation in the arts, as well as its ability to create well-known and loved programs that use effective techniques to have a positive impact on communities. Areas for improvement relate to Brisbane Festival’s potential to benefit from the following: clarifying relationships between community participants and professionals; increasing mentoring relationships between these groups; consolidating the discourses it uses to describe event aims across strategic, production, and publicity documents across the years; and re-considering the number of small events inside the larger Creating Queensland program.

Transient expression of Human papillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious tobamovirus vector

A Tobacco mosaic virus (TMV)-derived vector was used to express a native Human papillomavirus type 16 (HPV-16) L1 gene in Nicotiana benthamiana by means of infectious in vitro RNA transcripts inoculated onto N. benthamiana plants. HPV-16 L1 protein expression was quantitated by enzyme-linked immunosorbent assays (ELISA) after concentration of the plant extract. We estimated that the L1 product yield was 20-37 μg/kg of fresh leaf material. The L1 protein in the concentrated extract was antigenically characterised using the neutralising and conformation-specific Mabs H16:V5 and H16:E70, which bound to the plant-produced protein. Particles observed by transmission electron microscopy were mainly capsomers but virus-like particles (VLPs) similar to those produced in other systems were also present. Immunisation of rabbits with the concentrated plant extract induced a weak immune response. This is the first report of the successful expression of an HPV L1 gene in plants using a plant virus vector. © 2006 Elsevier B.V. All rights reserved.

Plant-produced cottontail rabbit papillomavirus L1 protein protects against tumor challenge : A proof-of-concept study

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A highly divergent South African geminivirus species illuminates the ancient evolutionary history of this family

Background. We have characterised a new highly divergent geminivirus species, Eragrostis curvula streak virus (ECSV), found infecting a hardy perennial South African wild grass. ECSV represents a new genus-level geminivirus lineage, and has a mixture of features normally associated with other specific geminivirus genera. Results. Whereas the ECSV genome is predicted to express a replication associated protein (Rep) from an unspliced complementary strand transcript that is most similar to those of begomoviruses, curtoviruses and topocuviruses, its Rep also contains what is apparently a canonical retinoblastoma related protein interaction motif such as that found in mastreviruses. Similarly, while ECSV has the same unusual TAAGATTCC virion strand replication origin nonanucleotide found in another recently described divergent geminivirus, Beet curly top Iran virus (BCTIV), the rest of the transcription and replication origin is structurally more similar to those found in begomoviruses and curtoviruses than it is to those found in BCTIV and mastreviruses. ECSV also has what might be a homologue of the begomovirus transcription activator protein gene found in begomoviruses, a mastrevirus-like coat protein gene and two intergenic regions. Conclusion. Although it superficially resembles a chimaera of geminiviruses from different genera, the ECSV genome is not obviously recombinant, implying that the features it shares with other geminiviruses are those that were probably present within the last common ancestor of these viruses. In addition to inferring how the ancestral geminivirus genome may have looked, we use the discovery of ECSV to refine various hypotheses regarding the recombinant origins of the major geminivirus lineages. © 2009 Varsani et al; licensee BioMed Central Ltd.

The capsid protein of beak and feather disease virus binds to the viral DNA and is responsible for transporting the replication-associated protein into the nucleus

Circoviruses lack an autonomous DNA polymerase and are dependent on the replication machinery of the host cell for de novo DNA synthesis. Accordingly, the viral DNA needs to cross both the plasma membrane and the nuclear envelope before replication can occur. Here we report on the subcellular distribution of the beak and feather disease virus (BFDV) capsid protein (CP) and replication-associated protein (Rep) expressed via recombinant baculoviruses in an insect cell system and test the hypothesis that the CP is responsible for transporting the viral genome, as well as Rep, across the nuclear envelope. The intracellular localization of the BFDV CP was found to be directed by three partially overlapping bipartite nuclear localization signals (NLSs) situated between residues 16 and 56 at the N terminus of the protein. Moreover, a DNA binding region was also mapped to the N terminus of the protein and falls within the region containing the three putative NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome. Interestingly, whereas Rep expressed on its own in insect cells is restricted to the cytoplasm, coexpression with CP alters the subcellular localization of Rep to the nucleus, strongly suggesting that an interaction with CP facilitates movement of Rep into the nucleus. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Comparison of cervical and blood T-cell responses to human papillomavirus-16 in women with human papillomavirus-associated cervical intraepithelial neoplasia

Human papillomaviruses (HPVs) are obligate epithelial pathogens and typically cause localized mucosal infections. We therefore hypothesized that T-cell responses to HPV antigens would be greater at sites of pathology than in the blood. Focusing on HPV-16 because of its association with cervical cancer, the magnitude of HPV-specific T-cell responses at the cervix was compared with those in the peripheral blood by intracellular cytokine staining following direct ex vivo stimulation with both virus-like particles assembled from the major capsid protein L1, and the major HPV oncoprotein, E7. We show that both CD4 + and CD8 + T cells from the cervix responded to the HPV-16 antigens and that interferon-γ (IFN-γ) production was HPV type-specific. Comparing HPV-specific T-cell IFN-γ responses at the cervix with those in the blood, we found that while CD4 + and CD8 + T-cell responses to L1 were significantly correlated between compartments (P = 0.02 and P = 0.05, respectively), IFN-γ responses in both T-cell subsets were significantly greater in magnitude at the cervix than in peripheral blood (P = 0.02 and P = 0.003, respectively). In contrast, both CD4 + and CD8 + T-cell IFN-γ responses to E7 were of similar magnitude in both compartments and CD8 + responses were significantly correlated between these distinct immunological compartments (P = 0.04). We therefore show that inflammatory T-cell responses against L1 (but not E7) demonstrate clear compartmental bias and the magnitude of these responses do reflect local viral replication but that correlation of HPV-specific responses between compartments indicates their linkage.

Strategies for the prevention of cervical cancer by human papillomavirus vaccination

As cervical cancer is causally associated with 14 high-risk types of human papillomavirus (HPV), a successful HPV vaccine will have a major impact on this disease. Although some persistent HPV infections progress to cervical cancer, host immunity is generally able to clear most HPV infections. Both cell-mediated and antibody responses have been implicated in influencing the susceptibility, persistence or clearance of genital HPV infection. There have been two clinical trials that show that vaccines based on virus-like particles (VLPs) made from the major capsid protein, L1, are able to type specifically protect against cervical intra-epithelial neoplasia and infection. However, there is no evidence that even a mixed VLP vaccine will protect against types not included in the vaccine, and a major challenge that remains is how to engineer protection across a broader spectrum of viruses. Strategies for production of HPV vaccines using different vaccine vectors and different production systems are also reviewed. © 2005 Elsevier Ltd. All rights reserved.