CD4 T Cell Responses to Theileria parva in Immune Cattle Recognize a Diverse Set of Parasite Antigens Presented on the Surface of Infected Lymphoblasts.

Parasite-specific CD8 T cell responses play a key role in mediating immunity against Theileria parva in cattle (Bos taurus), and there is evidence that efficient induction of these responses requires CD4 T cell responses. However, information on the antigenic specificity of the CD4 T cell response is lacking. The current study used a high-throughput system for Ag identification using CD4 T cells from immune animals to screen a library of ∼40,000 synthetic peptides representing 499 T. parva gene products. Use of CD4 T cells from 12 immune cattle, representing 12 MHC class II types, identified 26 Ags. Unlike CD8 T cell responses, which are focused on a few dominant Ags, multiple Ags were recognized by CD4 T cell responses of individual animals. The Ags had diverse properties, but included proteins encoded by two multimember gene families: five haloacid dehalogenases and five subtelomere-encoded variable secreted proteins. Most Ags had predicted signal peptides and/or were encoded by abundantly transcribed genes, but neither parameter on their own was reliable for predicting antigenicity. Mapping of the epitopes confirmed presentation by DR or DQ class II alleles and comparison of available T. parva genome sequences demonstrated that they included both conserved and polymorphic epitopes. Immunization of animals with vaccine vectors expressing two of the Ags demonstrated induction of CD4 T cell responses capable of recognizing parasitized cells. The results of this study provide detailed insight into the CD4 T cell responses induced by T. parva and identify Ags suitable for use in vaccine development.

Guanidinium-Functionalized Interpolyelectrolyte Complexes Enabling RNAi in Resistant Insect Pests.

RNAi-based technologies are ideal for pest control as they can provide species specificity and spare nontarget organisms. However, in some pests biological barriers prevent use of RNAi, and therefore broad application. In this study we tested the ability of a synthetic cationic polymer, poly-[ N-(3-guanidinopropyl)methacrylamide] (pGPMA), that mimics arginine-rich cell penetrating peptides to trigger RNAi in an insensitive animal- Spodoptera frugiperda. Polymer-dsRNA interpolyelectrolyte complexes (IPECs) were found to be efficiently taken up by cells, and to drive highly efficient gene knockdown. These IPECs could also trigger target gene knockdown and moderate larval mortality when fed to S. frugiperda larvae. This effect was sequence specific, which is consistent with the low toxicity we found to be associated with this polymer. A method for oral delivery of dsRNA is critical to development of RNAi-based insecticides. Thus, this technology has the potential to make RNAi-based pest control useful for targeting numerous species and facilitate use of RNAi in pest management practices.

Using Aldehyde Synergism To Direct the Design of Degradable Pro-Antimicrobial Networks.

We describe the design and synthesis of degradable, dual-release, pro-antimicrobial poly(thioether acetal) networks derived from synergistic pairs of aromatic terpene aldehydes. Initially, we identified pairs of aromatic terpene aldehyde derivatives exhibiting a synergistic antimicrobial activity against Pseudomonas aeruginosa by determining fractional inhibitory concentrations. Synergistic aldehydes were converted into dialkene acetal monomers and copolymerized at various ratios with a multifunctional thiol via thiol-ene photopolymerization. The step-growth nature of the thiol-ene polymerization ensures every cross-link junction contains a degradable acetal linkage enabling a fully cross-linked polymer network to revert into its small molecule constituents upon hydrolysis, releasing the synergistic aldehydes as active antimicrobial compounds. A three-pronged approach was used to characterize the poly(thioether acetal) materials: (i) determination of the degradation/aldehyde release behavior, (ii) evaluation of the antimicrobial activity, and (iii) identification of the cellular pathways impacted by the aldehydes on a library of mutated bacteria. From this approach, a polymer network derived from a 40:60 p-bromobenzaldehyde/p-anisaldehyde monomer ratio exhibited potent antimicrobial action against Pseudomonas aeruginosa, a common opportunistic human pathogen. From a transposon mutagenesis assay, we showed that these aldehydes target porins and multidrug efflux pumps. The aldehydes released from the poly(thioether acetal) networks exhibited negligible toxicity to mammalian tissue culture cells, supporting the potential development of these materials as dual-release antimicrobial biomaterial platforms.

Pro-Antimicrobial Networks via Degradable Acetals (PANDAs) Using Thiol-Ene Photopolymerization.

We describe the synthesis of pro-antimicrobial networks via degradable acetals (PANDAs) as a new paradigm for sequestration and triggered release of volatile, bioactive aldehydes. PANDAs derived from diallyl p-chlorobenzaldehyde acetal degrade and release p-chlorobenzaldehyde as an antibacterial and antifungal agent under mild conditions (pH 7.4/high humidity). We show that PANDAs enable facile access to materials with tunable release profiles, potent antimicrobial activity without triggering antimicrobial resistance, and minimal cytotoxicity.

Block ionomer complexes consisting of siRNA and aRAFT-synthesized hydrophilic-block-cationic copolymers II: The influence of cationic block charge density on gene suppression.

Block ionomer complex (BIC)-siRNA interactions and effectiveness in cell transfection are reported. Aqueous RAFT polymerization was used to prepare a series of hydrophilic-block-cationic copolymers in which the cationic block statistically incorporates increasing amounts of neutral, hydrophilic monomer such that the number of cationic groups remains unchanged but the cationic charge density is diluted along the polymer backbone. Reduced charge density decreases the electrostatic binding strength between copolymers and siRNA with the goal of improving siRNA release after targeted cellular delivery. However, lower binding strength resulted in decreased transfection and RNA interference pathway activation, leading to reduced gene knockdown. Enzymatic siRNA degradation studies with BICs indicated lowered binding strength increases susceptibility to RNases, which is the likely cause for poor gene knockdown.

Senescence induction in renal carcinoma cells by Nutlin-3: a potential therapeutic strategy based on MDM2 antagonism.

Although the role of p53 as a tumour suppressor in renal cell carcinoma (RCC) is unclear, our recent analysis suggests that increased wild-type p53 protein expression is associated with poor outcome. A growing body of evidence also suggests that p53 expression and increased co-expression of MDM2 are linked with poor prognosis in RCC. We have therefore examined whether an MDM2 antagonist; Nutlin-3, might rescue/increase p53 expression and induce growth inhibition or apoptosis in RCC cells that retain wild-type p53. We show that inhibition of p53 suppression by MDM2 in RCC cells promotes growth arrest and p53-dependent senescence - phenotypes known to mediate p53 tumour suppression in vivo. We propose that future investigations of therapeutic strategies for RCC should incorporate MDM2 antagonism as part of strategies aimed at rescuing/augmenting p53 tumour suppressor function.

EAU policy on live surgery events.

CONTEXT: Live surgery is an important part of surgical education, with an increase in the number of live surgery events (LSEs) at meetings despite controversy about their real educational value, risks to patient safety, and conflicts of interest. OBJECTIVE: To provide a European Association of Urology (EAU) policy on LSEs to regulate their organisation during urologic meetings. EVIDENCE ACQUISITION: The project was carried out in phases: a systematic literature review generating key questions, surveys sent to Live Surgery Panel members, and Internet- and panel-based consensus finding using the Delphi process to agree on and formulate a policy. EVIDENCE SYNTHESIS: The EAU will endorse LSEs, provided that the EAU Code of Conduct for live surgery and all organisational requirements are followed. Outcome data must be submitted to an EAU Web-based registry and complications reported using the revised Martin criteria. Regular audits will take place to evaluate compliance as well as the educational role of live surgery. CONCLUSIONS: This policy represents the consensus view of an expert panel established to advise the EAU. The EAU recognises the educational role of live surgery and endorses live case demonstration at urologic meetings that are conducted within a clearly defined regulatory framework. The overriding principle is that patient safety must take priority over all other considerations in the conduct of live surgery. PATIENT SUMMARY: Controversy exists regarding the true educational value of live surgical demonstrations on patients at surgical meetings. An EAU committee of experts developed a policy on how best to conduct live surgery at urologic meetings. The key principle is to ensure safety for every patient, including a code of conduct and checklist for live surgery, specific rules for how the surgery is organised and performed, and how each patient's results are reported to the EAU. For detailed information, please visit www.uroweb.org.

Combined p53 and MDM2 biomarker analysis shows a unique pattern of expression associated with poor prognosis in patients with renal cell carcinoma undergoing radical nephrectomy.

OBJECTIVE: To resolve much debated issues surrounding p53 function, expression and mutation in renal cell carcinoma (RCC), we performed the first study to simultaneously determine p53/MDM2 expression, TP53 mutational status (in p53-positive patients) and outcome in RCC. PATIENTS AND METHODS: In total, 90 specimens obtained from patients with RCC, who were treated by radical nephrectomy, were analyzed by immunohistochemistry for p53 and MDM2 on a tissue microarray, and p53 was functionally and genetically analyzed in p53 positive samples. Outcome analysis was by the Kaplan-Meier method and univariate analysis was used to identify variables for subsequent multivariate analysis of correlations between clinical parameters and biomarker expression. RESULTS: Up-regulation of p53 in RCC is strongly linked with MDM2 up-regulation (P < 0.001). Increased coexpression of p53 and MDM2 identifies those patients with a significantly reduced disease-specific survival by univariate (P= 0.036) and Cox multiple regression analysis (P= 0.027; relative risk, 3.20). Functional (i.e. functional analysis of separated alleles in yeast) and genetic analysis of tumours with increased p53 expression shows that most patients (86%) retain wild-type p53. CONCLUSIONS: Coexpression of p53/MDM2 identifies a subset of patients with poor prognosis, despite all of them having organ-confined disease. Up-regulated p53 is typically wild-type and thus provides a mechanistic explanation for the association between p53 and MDM2 expression: up-regulated wild-type p53 likely promotes the observed MDM2 coexpression. The results obtained in the present study suggest that the p53 pathway is altered in a tissue/disease-specific manner and that therapeutic strategies targeting this pathway should be investigated to determine whether the tumour suppressive function of p53 can be rescued in RCC.

Emission characteristics of photonic crystal light-emitting diodes.

Experimentally measured optical properties of photonic crystal LEDs are reported here. Photonic crystal and photonic quasi-crystal structures were fabricated on GaN epilayer LED wafer material using both direct-write electron beam lithography and nanoimprint lithography. Some of these structures were processed to make finished LEDs. Both electroluminescence and photoluminescence measurements were performed on these structures. Devices were characterized for their current-voltage characteristics, emission spectra, far-field emission pattern, and angular emission pattern. These results are useful for fabricating photonic crystal LEDs and assessing their operational properties.

Characterization of age-related changes in bovine CD8+ T-cells.

Evaluation of the changes induced by immunological interventions requires a baseline against which to compare those changes. The age-related changes in the CD8(+) T-cell population of cattle were studied. The results indicate that CD8(+) T-cells could be divided into γ/δ TCR1(+) and γ/δ TCR1(-) according to their expression of the γ/δ T-cell receptor. As a proportion, the CD8(+) γ/δ TCR1(+) population appears to increase with age. Within the CD8(+)γ/δ TCR1(-) a population of cells expressing a profile of surface molecules previously associated with effector memory T cells (CD45RO(+), CD62L(-), CD27(-), CD45RA(-) and CD28(-)) increases with age. Furthermore, a parallel increase with age in the proportion of CD8(+)CD45RO(+) T cells that express the cytotoxic granule protein perforin was observed. In peripheral tissues, namely lungs, it was found that the majority of CD8(+) T cells present expressed a phenotype indicative of previously primed T cells (high expression of CD45RO and perforin). In contrast, only a small population of memory CD8(+) T cells was present in lymphoid tissue where most of the CD8(+) T cells expressed a naïve phenotype. In conclusion, in cattle, like in human, CD8(+) T cells that express a phenotype associated with antigen experience accumulate with age that may play a role in immunocompetence as the individual ages.

The fabrication of mono-domain highly ordered nanoporous alumina on a wafer scale by a guided electric field.

This paper describes the formation of mono-domain highly ordered nanoporous alumina on the scale of a 2 inch diameter silicon wafer by anodization of aluminium evaporated on a patterned SiO(2) mask on a silicon substrate. The position of the ordered pores correlates with holes in the SiO(2) mask, which guide the electric field during anodization and initiates pore nucleation. The technique is suitable for the production of ordered nanoporous alumina on a wafer scale and overcomes the time, cost and scale limitations of existing processes.

p53 and MDM2 in renal cell carcinoma: biomarkers for disease progression and future therapeutic targets?

Renal cell carcinoma (RCC) is the most common type of kidney cancer and follows an unpredictable disease course. To improve prognostication, a better understanding of critical genes associated with disease progression is required. The objective of this review was to focus attention on 2 such genes, p53 and murine double minute 2 (MDM2), and to provide a comprehensive summary and critical analysis of the literature regarding these genes in RCC. Information was compiled by searching the PubMed database for articles that were published or e-published up to April 1, 2009. Search terms included renal cancer, renal cell carcinoma, p53, and MDM2. Full articles and any supplementary data were examined; and, when appropriate, references were checked for additional material. All studies that described assessment of p53 and/or MDM2 in renal cancer were included. The authors concluded that increased p53 expression, but not p53 mutation, is associated with reduced overall survival/more rapid disease progression in RCC. There also was evidence that MDM2 up-regulation is associated with decreased disease-specific survival. Two features of RCC stood out as unusual and will require further investigation. First, increased p53 expression is tightly linked with increased MDM2 expression; and, second, patients who have tumors that display increased p53 and MDM2 expression may have the poorest overall survival. Because there was no evidence to support the conclusion that p53 mutation is associated with poorer survival, it seemed clear that increased p53 expression in RCC occurs independent of mutation. Further investigation of the mechanisms leading to increased p53/MDM2 expression in RCC may lead to improved prognostication and to the identification of novel therapeutic interventions.

The European Association of Urology (EAU) guidelines methodology: a critical evaluation.

OBJECTIVES: Guidelines can be produced and written in numerous ways. The aim of the present article is to describe and evaluate the method currently used to produce the European Association of Urology (EAU) guidelines. DESIGN, SETTING, AND PARTICIPANTS: The methodology is described in detail, compared to other urologic guidelines by members of the EAU Guidelines Office Board. MEASUREMENTS: The new methodology is evaluated by the Appraisal of Guidelines for Research and Evaluation (AGREE) instrument. RESULTS AND LIMITATIONS: The currently used methodology is adapted to the aims and objectives as established by the EAU for their guidelines; wide coverage (essentially all fields of urology) and useful to urologists all over Europe. The frequent updates are easily accessible in a printed and electronic format. The AGREE instrument supports these strong points, but also identifies potentially weak points, such as no patient involvement, no formal validation of the guidelines texts prior to publication, and lack of discussion of organisational barriers and cost implications. CONCLUSION: The currently used methodology for the production of EAU guidelines fulfils the association's main objectives related to their guidelines, but the texts will benefit from the inclusion of country-specific cost and organisational data. For the practising clinician, these guidelines will help to take science into clinical practice.

Implications of computer tomography measurement in the management of renal tumours.

BACKGROUND: To compare radiographic measurement and pathological measurement of renal tumours to see if there was a significant difference between the two as this may have implications in the management. METHODS: We retrospectively analyzed CT measurements of 106 consecutive patients who underwent either radical or nephron sparing surgery in our institution and compared this to the actual measurement of the surgical specimen. The largest axial measurement was compared as this is the primary consideration before offering either treatment modality. RESULTS: The mean age of the patients was 64 years (range 31-92). There were 76 males and 30 females. The median tumour size was 70 mm (range 16-175) on CT and 65 mm (range 15-90) on pathological measurement. 25 patients had a CT size < or = 40 mm. CT tended to overestimate the size of tumours in 41 patients, underestimate in 45 and agree with surgical size in 20 patients. Statistically there was no significant difference between the two measurements (p = 0.7, Wilcoxon sign ranked test). When subdivided into tumours less than 40 mm (p = 0.7) and more than 40 mm (p = 0.09) again there was no statistically significant difference between the two measurements. However in 5(5%) patients who were not offered nephron sparing surgery based on CT findings (size > 40 mm) the pathological size was < or = 40 mm (p = < 0.001, Fishers Exact test). Pathologically the tumours were classified as renal cell carcinoma (n = 98), angiomyolipoma (3), and oncocytoma (5). CONCLUSION: CT measurement of renal tumour size correlates well with the actual size of the tumour. However CT does tend to overestimate the size in a small number of patients which may have a bearing on the modality of treatment offered.

Routine use of magnetic resonance imaging in the management of T(1c) carcinoma of the prostate: is it necessary?

PURPOSE: To assess the role and implications of MRI in the management of patients with stage T(1c) prostate cancer. PATIENTS AND METHODS: Data were collected from our oncology database, where all new prostate cancers are recorded, for a period of 3 years ending December 2005. A total of 915 patients were found to have prostate cancer. Of the 204 patients with stage T(1c) disease, 144 were considered eligible for radical treatment and underwent cross-sectional imaging in the form of an MRI scan. Gleason grade, clinical stage, cross-sectional imaging results, and subsequent treatment were recorded. The results were analyzed to see whether the MRI findings altered the modality of treatment offered to the patient. RESULTS: Of the 144 patients, 137 had scans that showed no extracapsular invasion, while five scans were equivocal. All five patients had further investigation, either by CT scanning or targeted biopsies, which confirmed the cancer to be localized. In the remaining two cases, the MRI findings upstaged T(1c) disease to T(3) disease, as there was evidence of extracapsular involvement. The imaging result therefore affected treatment choice in only two patients in that radical surgery was not offered because of the scan findings. CONCLUSIONS: The role of MRI in the management of clinical stage T(1c) prostate cancer is limited, as it altered the management of only 1.3% of our patients. The cost v the value of this study should be discussed with the patient before MRI is prescribed.