Reducing milking frequency from twice each day to three times each two days affected protein but not fat yield in a pasture-based dairy system.

Milking 3 times in 2 d (3-in-2) could enhance the attractiveness of the dairy workplace relative to twice-a-day milking (TAD) by reducing labor requirements for milking and increasing workforce flexibility. The objective of this study was to quantify the farm system interactions associated with milking 3-in-2 at 3 stages of lactation, with the aim of providing guidance to pasture-based dairy farmers and advisors on the likely consequences of adopting 3-in-2 milking on farm productivity and business performance. Seventy-nine multiparous and 37 primiparous cows were randomly allocated to 4 experimental farms stocked at 3.5 cows/ha. One herd was milked TAD for the whole lactation (August 2019 to May 2020), with the remaining 3 milked 3-in-2 for either the whole lactation, after December 1 when cows were an average of 101 d in milk, or after March 1 when days in milk averaged 189 d. Milking intervals over 48 h were 10-14-10-14 h for TAD and 12-18-18 h for 3-in-2. Animal, pasture, and farm system data were analyzed by linear regression, with the dependent variable being the annualized value of the performance metric of interest, and the number of days in the lactation milked 3-in-2 as the independent variable. For the proportion of the season milked 3-in-2, there was a significant effect on milk (-11%), protein (-8%), and lactose (-12%) yield per cow per year, but no effect of fat. Additionally, there was a positive effect (+6%) on body condition score before dry-off and the energy required for liveweight change (+26%), and a negative effect on the energy required for walking (-30%). There were no differences in estimated feed eaten, or pasture herbage accumulation, composition, or quality. Therefore, pasture management and feed allocation under 3-in-2 should be similar to TAD. On commercial farms, the degree to which reduced milk income can be offset by lower costs will be highly farm-specific, but opportunities for savings were identified in the results. The short walking distances on the research farm and potential to improve farm management using the time saved from fewer milkings suggests better production may be achieved with 3-in-2 milking on a commercial farm.

MUC13 protects colorectal cancer cells from death by activating the NF-κB pathway and is a potential therapeutic target.

MUC13 is a transmembrane mucin glycoprotein that is over produced by many cancers, although its functions are not fully understood. Nuclear factor-κB (NF-κB) is a key transcription factor promoting cancer cell survival, but therapeutically targeting this pathway has proved difficult because NF-κB has pleiotropic functions. Here, we report that MUC13 prevents colorectal cancer cell death by promoting two distinct pathways of NF-kB activation, consequently upregulating BCL-XL. MUC13 promoted tumor necrosis factor (TNF)-induced NF-κB activation by interacting with TNFR1 and the E3 ligase, cIAP1, to increase ubiquitination of RIPK1. MUC13 also promoted genotoxin-induced NF-κB activation by increasing phosphorylation of ATM and SUMOylation of NF-κB essential modulator. Moreover, elevated expression of cytoplasmic MUC13 and NF-κB correlated with colorectal cancer progression and metastases. Our demonstration that MUC13 enhances NF-κB signaling in response to both TNF and DNA-damaging agents provides a new molecular target for specific inhibition of NF-κB activation. As proof of principle, silencing MUC13 sensitized colorectal cancer cells to killing by cytotoxic drugs and inflammatory signals and abolished chemotherapy-induced enrichment of CD133+ CD44+ cancer stem cells, slowed xenograft growth in mice, and synergized with 5-fluourouracil to induce tumor regression. Therefore, these data indicate that combining chemotherapy and MUC13 antagonism could improve the treatment of metastatic cancers.

Mitigating road impacts on animals through learning principles.

Roads are a nearly ubiquitous feature of the developed world, but their presence does not come without consequences. Many mammals, birds, reptiles, and amphibians suffer high rates of mortality through collision with motor vehicles, while other species treat roads as barriers that reduce gene flow between populations. Road effects extend beyond the pavement, where traffic noise is altering communities of songbirds, insects, and some mammals. Traditional methods of mitigation along roads include the creation of quieter pavement and tires and the construction of physical barriers to reduce sound transmission and movement. While effective, these forms of mitigation are costly and time-consuming. One alternative is the use of learning principles to create or extinguish aversive behaviors in animals living near roads. Classical and operant conditioning are well-documented techniques for altering behavior in response to novel cues and signals. Behavioral ecologists have used conditioning techniques to mitigate human-wildlife conflict challenges, alter predator-prey interactions, and facilitate reintroduction efforts. Yet, these principles have rarely been applied in the context of roads. We suggest that the field of road ecology is ripe with opportunity for experimentation with learning principles. We present tangible ways that learning techniques could be utilized to mitigate negative roadside behaviors, address the importance of evaluating fitness within these contexts, and evaluate the longevity of learned behaviors. This review serves as an invitation for empirical studies that test the effectiveness of learning paradigms as a mitigation tool in the context of roads.

Smart and green interfaces: from single bubbles/drops to industrial environmental and biomedical applications.

Interfaces can be called Smart and Green (S&G) when tailored such that the required technologies can be implemented with high efficiency, adaptability and selectivity. At the same time they also have to be eco-friendly, i.e. products must be biodegradable, reusable or simply more durable. Bubble and drop interfaces are in many of these smart technologies the fundamental entities and help develop smart products of the everyday life. Significant improvements of these processes and products can be achieved by implementing and manipulating specific properties of these interfaces in a simple and smart way, in order to accomplish specific tasks. The severe environmental issues require in addition attributing eco-friendly features to these interfaces, by incorporating innovative, or, sometimes, recycle materials and conceiving new production processes which minimize the use of natural resources and energy. Such concept can be extended to include important societal challenges related to support a sustainable development and a healthy population. The achievement of such ambitious targets requires the technology research to be supported by a robust development of theoretical and experimental tools, needed to understand in more details the behavior of complex interfaces. A wide but not exhaustive review of recent work concerned with green and smart interfaces is presented, addressing different scientific and technological fields. The presented approaches reveal a huge potential in relation to various technological fields, such as nanotechnologies, biotechnologies, medical diagnostics, and new or improved materials.

Silencing oncogene expression in cervical cancer stem-like cells inhibits their cell growth and self-renewal ability.

Accumulating evidence supports the concept that cancer stem cells (CSCs) are responsible for tumor initiation and maintenance. They are also considered as an attractive target for advanced cancer therapy. Using a sphere culture method that favors the growth of self-renewal cells, we have isolated sphere-forming cells (SFCs) from cervical cancer cell lines HeLa and SiHa. HeLa-SFCs were resistant to multiple chemotherapeutic drugs and were more tumorigenic, as evidenced by the growth of tumors following injection of immunodeficient mice with 1 × 10(4) cells, compared with 1 × 10(6) parental HeLa cells required to grow tumors of similar size in the same time frame. These cells showed an expression pattern of CD44(high)/CD24(low) that resembles the CSC surface biomarker of breast cancer. We further demonstrated that HeLa-SFCs expressed a higher level (6.9-fold) of the human papillomavirus oncogene E6, compared with that of parental HeLa cells. Gene silencing of E6 with a lentiviral-short-hairpin RNA (shRNA) profoundly inhibited HeLa-SFC sphere formation and cell growth. The inhibition of cell growth was even greater than that for sphere formation after E6 silence, suggesting that the loss of self-renewing ability may be more important. We then measured the expression of self-renewal genes, transformation growth factor-beta (TGF-ß) and leukemia-inhibitory factor (LIF), in shRNA-transduced HeLa-SFCs and found that expression of all three TGF-ß isoforms was significantly downregulated while LIF remained unchanged. Expression of the Ras gene (a downstream component of TGF-ß) was also markedly decreased, suggesting that the growth-inhibitory effect could be via the TGF-ß pathway. The above data indicate RNA interference-based therapy may offer a new approach for CSC-targeted cancer therapy.

Systemic delivery of E6/7 siRNA using novel lipidic particles and its application with cisplatin in cervical cancer mouse models.

Small interfering RNA (siRNA) shows great promise in cancer therapy, but its effectiveness in vivo still remains a crucial issue for its transition into the clinics. Although the successful use of polyethylene glycol (PEG)ylated lipidic delivery systems have already been reported, most of the formulation procedures used are labour intensive and also result in unstable end products. We have previously developed a simple yet efficient hydration-of-freeze-dried-matrix (HFDM) method to entrap siRNA within lipid particles, in which the products exhibited superior stability. Here, we show that these HFDM-formulated particles are stable in the presence of serum and can deliver siRNA efficiently to tumours after intravenous administration. Using these particles, around 50% knockdown of the target gene expression was observed in tumours. With the use of siRNA targeting the E6/7 oncogenes expressed in cervical cancer, we showed a 50% reduction in tumour size. This level of tumour growth suppression was comparable to that achieved from cisplatin at the clinically used dose. Overall, our results demonstrate the feasibility of using HFDM-formulated particles to systematically administer E6/7-targeted siRNA for cervical cancer treatment. The simplicity of preparation procedure along with superior product stability obtained from our method offers an innovative approach for the in vivo delivery of siRNA.

Inhibition of cervical cancer cell growth in vitro and in vivo with dual shRNAs.

RNA interference (RNAi)-based gene silencing is widely used in laboratories for gene function studies and also holds a great promise for developing treatments for diseases. However, in vivo delivery of RNAi therapy remains a key issue. Lentiviral vectors have been employed for stable gene transfer and gene therapy and therefore are expected to deliver a stable and durable RNAi therapy. But this does not seem to be true in some disease models. Here, we showed that lentivirus delivered short-hairpin RNA (shRNA) against human papillomavirus (HPV) E6/E7 oncogenes were effective for only 2 weeks in a cervical cancer model. However, using this vector to carry two copies of the same shRNA or two shRNAs targeting at two different but closely related genes (HPV E6 and vascular endothelial growth factor) was more effective at silencing the gene targets and inhibiting cell or even tumor growth than their single shRNA counterparts. The cancer cells treated with dual shRNA were also more sensitive to chemotherapeutic drugs than single shRNA-treated cells. These results suggest that a multi-shRNA strategy may be a more attractive approach for developing an RNAi therapy for this cancer.

Shared and persistent asymptomatic cutaneous human papillomavirus infections in healthy skin.

Cutaneous human papillomavirus (HPV) types are commonly found in normal skin, and some of them have been suspected to play a role in the development of non-melanoma skin cancer. This present study is divided into three sections, the aims of this study were to examine if certain HPV-types persist over time and if HPV-types are shared within families. From the first part of the study, swab samples from foreheads were collected for three longitudinal studies from one family with a newborn baby. Five specific HPV-types were isolated from the family with a newborn, with HPV-5 and FA67 being found at various time points and prevalence rates in all four members of the family. Part 2 consisted of a followed up study from two families with a 6 years interval. Six of the family members were found to have at least one of the HPV-types identified in the family 6 years earlier. Many of the HPV-types identified were shared within the families studied. Part 3 of this study involved weekly samples from four healthy females for 4 months. Among the four healthy individuals, 11%, 65%, and 56% of the weekly samples were HPV-DNA positive with one individual HPV-negative. All specimens were tested for HPV-DNA by PCR using the broad range HPV-type primer pair FAP59/64. The positive samples were HPV-type determined by cloning and sequencing. Specific cutaneous HPV-types persist over long periods of time in healthy skin in most individuals investigated and certain HPVs are shared between family members.

Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes.

In this study, we investigated the suppressive effect of a short hairpin RNA delivered by a lentiviral vector (LV-shRNA) against human papillomavirus (HPV) type 18 E6 on the expression of the oncogenes E6 and E7 in cervical cancer HeLa cells both in vitro and in vivo. The LV-shRNA effectively delivered the shRNA to HeLa cells and lead to a dose-dependent reduction of E7 protein and the stabilization of E6 target proteins, p53 and p21. Low-dose infection of HeLa cells with LV-shRNA caused reduced cell growth and the induction of senescence, whereas a high-dose infection resulted in specific cell death via apoptosis. Transplant of HeLa cells infected with a low dose of LV-shRNA into Rag-/- mice significantly reduced the tumor weight, whereas transplant of cells infected with a high dose resulted in a complete loss of tumor growth. Systemic delivery of LV-shRNA into mice with established HeLa cell lung metastases led to a significant reduction in the number of tumor nodules. Our data collectively suggest that lentiviral delivery is an effective way to achieve stable suppression of E6/E7 oncogene expression and induce inhibition of tumor growth both in vitro and in vivo. These results encourage further investigation of this form of RNA interference as a promising treatment for cervical cancer.

Evaluation of the effect of uterine artery embolisation on menstrual blood loss and uterine volume.

OBJECTIVE: To evaluate the effect of uterine artery embolisation (UAE) on menstrual blood loss (MBL) and uterine volume in women with symptomatic uterine fibroids. DESIGN: Prospective observational study. SETTING: West of Scotland gynaecology and radiology departments. POPULATION: Fifty women (mean age 43 years) with symptomatic fibroids undergoing UAE between January 1999 and June 2003. METHODS: Women collected sanitary protection from one menses pre-embolisation and at regular intervals thereafter. This allowed objective measurement of MBL using the alkaline haematin technique. Uterine volume was calculated using magnetic resonance imaging (MRI) before and six months following embolisation. Interventional radiologists performed bilateral UAE. The Wilcoxon's signed rank test was used for statistical analysis of data. MAIN OUTCOME MEASURES: Post-embolisation MBL and uterine volume changes. RESULTS: Median pretreatment MBL was 162 mL (mean 234, range 9-1339). The median MBL decreased to 60 mL at 3 months (n= 34, range 0-767, P < 0.001), 70 mL at 6-9 months (n= 34, range 0-1283, P < 0.001), 37 mL at 12-24 months (n= 25, range 0-265, P < 0.001), 18 mL at 24-36 months (n= 17, range 0-205, P < 0.001) and 41 mL at 36-48 months (n= 6, range 0-66, P < 0.05). The median reduction in uterine volume was 40% (n= 46, 95% CI 33.0-49.7, P < 0.001). CONCLUSIONS: UAE causes a statistically significant reduction in objectively measured MBL. UAE is also associated with a statistically significant reduction in uterine volume at six months. There was no relationship between the changes in uterine volume and MBL.

Don't wait for a sensory level--listen to the symptoms: a prospective audit of the delays in diagnosis of malignant cord compression.

AIM: To report details concerning symptoms (especially pain) preceding the development of malignant cord compression (MCC); delays between onset/reporting of symptoms and confirmed diagnosis of MCC; accuracy of investigations carried out. METHODS: A prospective observational study examined the diagnosis, management and outcome of 319 patients diagnosed with MCC at three Scottish cancer centres between January 1998-April 1999. The process was considered from the perspectives of the patient, the GP and the hospital doctor. RESULTS: At diagnosis, most patients (82%) were either unable to walk or only able to do so with help. Pain was reported by nearly all patients interviewed (94%) and had been present for approximately 3 months (median=90 days). It was severe in 84% of cases, with the distribution and characteristics of nerve root pain in 79%. The site of pain did not correspond to the site of compression. Where reported, weakness and/or sensory problems had been noticed by the patient for some time before diagnosis (median intervals 20 and 12 days, respectively). Most patients reported early symptoms to their General Practitioner (GP) and diagnosis was established, following referral and investigation, approximately 2 months (median=66 days) later. CONCLUSION: Patients who develop spinal metastases are at risk of irreversible spinal cord damage. Weakness and sensory abnormalities are reported late and identified even later, despite patients having reported pain for a considerable time. Patients with cancer who describe severe back or spinal nerve root pain need urgent assessment on the basis of their symptoms, as signs may occur too late. Plain films and bone scans requested for patients in this audit predicted accurately the level of compression in only 21% and 19% of cases, respectively. The only accurate investigation to establish the presence and site of a compressive lesion is magnetic resonance imaging (MRI). A referral guideline based on suspicious symptoms in addition to suspicious signs is suggested.

Efficiency of delivery of DNA to cells by bovine papillomavirus type-1 L1/L2 pseudovirions.

To investigate the efficiency of encapsidation of plasmid by papillomavirus virus-like particles (PV VLPs), and the infectivity of the resultant PV pseudovirions, Cos-1 cells were transfected with an 8-kb plasmid incorporating a green fluorescent protein (GFP) reporter gene (pGSV), and infected with bovine PV (BPV-1) L1/L2 recombinant vaccinia virus to produce BPV1 pseudovirions. Approximately 1 in 1.5 x 10(4) of dense (1.35 g/ml) PV pseudovirions and 0.3 in 10(4) of less-dense (1.29 g/ml) pseudovirions packaged an intact pGSV plasmid. The majority (>75%) of packaged plasmids contained deletions, and the deletions affected all tested genes. After exposure of Cos-1 cells to BPV-1 pseudovirions at an MOI of 40,000:1, 6% of cells expressed GFP, giving a calculated efficiency of delivery of the pGSV plasmid, by pseudovirions which had packaged an intact plasmid, of approximately 5%. Plasmid delivery was not effected by purified pGSV plasmid, was blocked by antiserum against BPV-1, and was not blocked by DNase treatment of pseudovirions, confirming that delivery was mediated by DNA within the pseudovirion. We conclude that a major limitation to the use of PV pseudovirions as a gene delivery system is that intact plasmid DNA is not efficiently selected for packaging by VLPs in cell-based pseudovirions production systems.

Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation.

The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. Here we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in cell proliferation, suggesting involvement of the Ras-MAP kinase pathway. Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein Shc to beta4. Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min. These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

The human papillomavirus E7 protein is able to inhibit the antiviral and anti-growth functions of interferon-alpha.

It is now well recognized that cervical cancer is caused by infection with certain human papillomavirus (HPV) subtypes and while interferon-alpha (IFN-alpha) is used to treat HPV-infected lesions, HPV appears to have developed a means to avoid the effects of IFN-alpha. Clinically, resistance appears to be associated with the expression of the E7 oncoprotein. Here we investigated the effects of expression in cells of the E7 protein from high- and low-risk papillomavirus subtypes on a range of responses to IFN-alpha. 2fTGH, a cell line dependent on IFN-alpha for growth in selection medium, grew significantly less well in the presence of E7, and the antiproliferative effects of IFN-alpha upon epithelial cells was lost upon E7 expression. The antiviral effects of IFN-alpha were abrogated in E7-expressing cells. Loss of response to IFN-alpha was found to occur in both high- and low-risk papillomaviruses. Finally, deletion of amino acids 21-24 of HPV type 16 E7 protein partially reversed repression. We conclude that E7 inhibits the functional effects of IFN-alpha and that this property is shared by all HPV subtypes tested.

BPV1 E2 protein enhances packaging of full-length plasmid DNA in BPV1 pseudovirions.

We studied determinants of efficient encapsidation of circular DNA, incorporating a PV early region DNA sequence (nt 584-1978) previously shown to enhance packaging of DNA within papillomavirus (PV)-like particles (VLPs). Insect coelomic cells (Sf-9) and cultured monkey kidney cells (Cos-1) were transfected with an 8-kb reporter plasmid incorporating the putative BPV packaging sequence and infected with BPV1 L1 and L2 recombinant baculovirus or vaccinia virus. Heavy (1.34 g/ml) and light (1.30 g/ml) VLPs were produced, and each packaged some of the input plasmid. In light VLPs, truncated plasmids, which nevertheless incorporated the PV-derived DNA packaging sequence, were more common than full-length plasmids. Packaging efficiency of the plasmid was estimated at 1 plasmid per 10(4) VLPs in both Cos-1 and Sf-9 cells. In each cell type, expression of the BPV1 early region protein E2 in trans doubled the quantity of heavy but not light VLPs and also increased the packaging efficiency of full-length circular plasmids by threefold in heavy VLPs. The resultant pseudovirions incorporated significant amounts of E2 protein. Pseudovirions, comprising plasmids packaged within heavy VLPs, mediated the delivery of packaged plasmid into Cos-1 cells, whereby "infectivity" was blocked by antisera to BPV1 L1, but not antisera to BPV1 E4. We conclude that (a) packaging of DNA within PV L1+L2 pseudovirions is enhanced by BPV1 E2 acting in trans, (b) E2 may be packaged with the pseudovirion, and (c) E2-mediated enhancement of packaging favors 8-kb plasmid incorporation over incorporation of shorter DNA sequences.

Multiple Symmetric Lipomatosis--MR appearances.

Multiple Symmetric Lipomatosis (MSL) is a rare disorder of lipid metabolism which is mainly seen in Mediterranean and eastern European populations, which results in massive fat accumulation mainly around the neck and back. The main differential diagnosis lies between MSL and the fat accumulation of Cushing's disease, and liposarcoma. This case demonstrates that MR imaging is a valuable aid to the diagnosis and treatment of this disease by giving excellent definition of soft tissue and vascular structures, allowing accurate assessment and preoperative planning of the disease.

Expression of the alpha6 integrin confers papillomavirus binding upon receptor-negative B-cells.

Papillomaviruses (PV) bind to a wide range of cell lines in a specific and saturable manner. We have recently identified a candidate receptor for papillomavirus as the alpha6 integrin (Evander et al., J. Virol. 71, 2449-2456, 1997). We have further investigated the role the alpha6 integrin plays in PV binding. Here we show that the cells expressing the alpha6 integrin, partnered with either the beta4 integrin or the beta1 integrin, are equally able to bind PV HPV6b L1 virus-like particles, indicating that the beta partner does not play a major role in virus binding. In order to provide definitive evidence that the alpha6 integrin is required for PV binding we undertook to genetically complement the receptor-negative B-cell line DG75 by expressing the human alpha6A gene. The transduction of the alpha6 integrin gene into DG75 cells results in the cell surface expression of the alpha6 protein and this expression confers upon DG75 cells the ability to bind laminin, a normal ligand for alpha6 integrin. Furthermore, the alpha6 protein is partnered with the beta1 integrin in DG75 cells. Finally, we show that the DG75-alpha6 cells were able to bind papillomavirus VLPs and this binding was inhibited by a functionally blocking anti-alpha6 antibody. Together these data indicate that the alpha6 integrin is a primary cell receptor for papillomaviruses and is both necessary and sufficient for PV binding.

The human papillomavirus E7 oncoprotein abrogates signaling mediated by interferon-alpha.

Greater than 95% of all cervical carcinomas have been found to be associated with "high-risk" human papillomavirus (mainly types 16 and 18) infections, with the viral E6 and E7 oncoproteins essential for neoplastic development and maintenance. Interferon-alpha (IFNalpha) is used in the treatment of HPV infections yet both in vivo and in vitro data suggest that the virus has developed mechanisms to avoid the effects of interferon. Here we show that the HPV16 E7 oncoprotein is able to inhibit the induction of IFNalpha-inducible genes but has no effect of IFNgamma-inducible genes. Expression of E7 correlates with the loss of formation of the interferon-stimulated gene factor 3 (ISGF3) transcription complex. Moreover, in the presence of E7, p48, the DNA-binding component of ISGF3, was unable to translocate to the nucleus upon IFNalpha stimulation. A direct protein-protein interaction was identified between E7 and p48 with the site of interaction within E7 defined as the region between amino acids 17-37, a domain that includes the binding site for the retinoblastoma protein, pRb. These results suggest that HPV, via E7, targets p48, resulting in the loss of IFNalpha-mediated signal transduction and may provide a means by which HPV can avoid the innate immune system.

Potential strategies utilised by papillomavirus to evade host immunity.

The co-evolution of papillomaviruses (PV) and their mammalian hosts has produced mechanisms by which PV might avoid specific and non-specific host immune responses. Low level expression of PV proteins in infected basal epithelial cells, together with an absence of inflammation and of virus-induced cell lysis, restricts the opportunity for effective PV protein presentation to immunocytes by dendritic cells. Additionally, PV early proteins, by a range of mechanisms, may restrict the efficacy of antigen presentation by these cells. Should an immune response be induced to PV antigens, resting keratinocytes (KC) appear resistant to interferon-gamma-enhanced mechanisms of cytotoxic T-lymphocyte (CTL)-mediated lysis, and expression of PV antigens by resting KC can tolerise PV-specific CTL. Thus, KC, in the absence of inflammation, may represent an immunologically privileged site for PV infection. Together, these mechanisms play a part in allowing persistence of PV-induced proliferative skin lesions for months to years, even in immunocompetent hosts.