Recombinant Kunjin virus replicon vaccines induce protective T-cell immunity against human papillomavirus 16 E7-expressing tumour

The persistence of the E7 oncoprotein in transformed cells in human papillomavirus (HPV)-associated cervical cancer provides a tumour-specific antigen to which immunotherapeutic strategies may be directed. Self-replicating RNA (replicon) vaccine vectors derived from the flavivirus Kunjin (KUN) have recently been reported to induce T-cell immunity. Here, we report that inclusion of a CTL epitope of HPV16 E7 protein into a polyepitope encoded by a KUN vector induced E7-directed T-cell responses and protected mice against challenge with an E7-expressing epithelial tumour. We found replicon RNA packaged into virus-like particles to be more effective than naked replicon RNA or plasmid DNA constructed to allow replicon RNA transcription in vivo. Protective immunity was induced although the E7 CTL epitope was subdominant in the context of other CTL epitopes in the polyepitope. The results demonstrate the efficacy of the KUN replicon vector system for inducing protective immunity directed towards a virally encoded human tumour-specific antigen, and for inducing multi-epitopic CTL responses. (C) 2004 Elsevier Inc. All rights reserved.

The relationship between the mismatch negativity (MMN) and psycholinguistic models of spoken word processing

Background: The results from previous studies have indicated that a pre-attentive component of the event-related potential (ERP), the mismatch negativity (MMN), may be an objective measure of the automatic auditory processing of phonemes and words. Aims: This article reviews the relationship between the MMN data and psycholinguistic models of spoken word processing, in order to determine whether the MMN may be used to objectively pinpoint spoken word processing deficits in individuals with aphasia. Main Contribution: This article outlines the ways in which the MMN data support psycholinguistic models currently used in the clinical management of aphasic individuals. Furthermore, the cell assembly model of the neurophysiological mechanisms underlying spoken word processing is discussed in relation to the MMN and psycholinguistic models. Conclusions: The MMN data support current theoretical psycholinguistic and neurophysiological models of spoken word processing. Future MMN studies that include normal and aphasic populations will further elucidate the role that the MMN may play in the clinical management of aphasic individuals.

Processing of English words with fine acoustic contrasts and simple tones: A mismatch negativity study

The purpose of this study was to compare the robustness of the event-related potential (ERP) response, called the mismatch negativity (MMN), when elicited by simple tone stimuli (differing in frequency, duration, or intensity) and speech stimuli (CV nonword contrast /de:/ vs. /ge:/ and CV word contrast /deI/ vs. /geI/). The study was conducted using 30 young adult subjects (Groups A and B; n = 15 each). The speech stimuli were presented to Group A at a stimulus onset asynchrony (SOA) of 610 msec and to Group B at an SOA of 900 msec. The tone stimuli were presented to both groups at an SOA of 610 msec. MMN responses were elicited by the simple tone stimuli (66.7%-96.7% of subjects with MMN "present," or significantly different from zero, p < 0.05) but not the speech stimuli (10% subjects with MMN present for nonwords, 10% for words). The length of the SOA (610 msec or 900 msec) had no effect on the ability to obtain consistent MMN responses to the speech stimuli. The results indicated a lack of robust MMN elicited by speech stimuli with fine acoustic contrasts under carefully controlled methodological conditions. The implications of these results are discussed in relation to conflicting reports in the literature of speech-elicited MMNs, and the importance of appropriate methodological design in MMN studies investigating speech processing in normal and pathological populations.

Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA

In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Silencing of integrated human papillomavirus type 18 oncogene transcription in cells expressing SerpinB2

The serine protease inhibitor SerpinB2 (PAI-2), a major product of differentiating squamous epithelial cells, has recently been shown to bind and protect the retinoblastoma protein (Rb) from degradation. In human papillomavirus type 18 (HPV-18) -transformed epithelial cells the expression of the E6 and E7 oncoproteins is controlled by the HPV-18 upstream regulatory region (URR). Here we illustrate that PAI-2 expression in the HPV-18-transformed cervical carcinoma line HeLa resulted in the restoration of Rb expression, which led to the functional silencing of transcription from the HPV-18 URR. This caused loss of E7 protein expression and restoration of multiple E6- and E7-targeted host proteins, including p53, c-Myc, and c-Jun. Rb expression emerged as sufficient for the transcriptional repression of the URR, with repression mediated via the C/EB beta-YY1 binding site (URR 7709 to 7719). In contrast to HeLa cells, where the C/EBP beta-YY1 dimer binds this site, in PAI-2- and/or Rb-expressing cells the site was occupied by the dominant-negative C/EBP beta isoform liver-enriched transcriptional inhibitory protein (LIP). PAI-2 expression thus has a potent suppressive effect on HPV-18 oncogene transcription mediated by Rb and LIP, a finding with potential implications for prognosis and treatment of HPV-transformed lesions.

A polytope DNA vaccine elicits multiple effector and memory CTL responses and protects against human papillomavirus 16 E7-expressing tumour

Vaccine-induced CD8 T cells directed to tumourspecific antigens are recognised as important components of protective and therapeutic immunity against tumours. Where tumour antigens have pathogenic potential or where immunogenic epitopes are lost from tumours, development of subunit vaccines consisting of multiple individual epitopes is an attractive alternative to immunising with whole tumour antigen. In the present study we investigate the efficacy of two DNA-based multiepitope('polytope') vaccines containing murine (H-2(b)) and human (HLA-A* 0201)-restricted epitopes of the E7 oncoprotein of human papillomavirus type 16, in eliciting tumour-protective cytotoxic T-lymphocyte (CTL) responses. We show that the first of these polytopes elicited powerful effector CTL responses ( measured by IFN-gamma ELISpot) and long-lived memory CTL responses ( measured by functional CTL assay and tetramers) in immunised mice. The responses could be boosted by immunisation with a recombinant vaccinia virus expressing the polytope. Responses induced by immunisation with polytope DNA alone partially protected against infection with recombinant vaccinia virus expressing the polytope. Complete protection was afforded against challenge with an E7-expressing tumour, and reduced growth of nascent tumours was observed. A second polytope differing in the exact composition and order of CTL epitopes, and lacking an inserted endoplasmic reticulum targeting sequence and T-helper epitope, induced much poorer CTL responses and failed to protect against tumour challenge. These observations indicate the validity of a DNA polytope vaccine approach to human papillomavirus E7 - associated carcinoma, and underscore the importance of design in polytope vaccine construction.

The mismatch negativity (MMN) response to complex tones and spoken words in individuals with aphasia

Background: The mismatch negativity (MMN) is a fronto-centrally distributed event-related potential (ERP) that is elicited by any discriminable auditory change. It is an ideal neurophysiological tool for measuring the auditory processing skills of individuals with aphasia because it can be elicited even in the absence of attention. Previous MMN studies have shown that acoustic processing of tone or pitch deviance is relatively preserved in aphasia, whereas the basic acoustic processing of speech stimuli can be impaired (e.g., auditory discrimination). However, no MMN study has yet investigated the higher levels of auditory processing, such as language-specific phonological and/or lexical processing, in individuals with aphasia. Aims: The aim of the current study was to investigate the MMN response of normal and language-disordered subjects to tone stimuli and speech stimuli that incorporate the basic auditory processing (acoustic, acoustic-phonetic) levels of non-speech and speech sound processing, and also the language-specific phonological and lexical levels of spoken word processing. Furthermore, this study aimed to correlate the aphasic MMN data with language performance on a variety of tasks specifically targeted at the different levels of spoken word processing. Methods M Procedures: Six adults with aphasia (71.7 years +/- 3.0) and six healthy age-, gender-, and education-matched controls (72.2 years +/- 5.4) participated in the study. All subjects were right-handed and native speakers of English. Each subject was presented with complex harmonic tone stimuli, differing in pitch or duration, and consonant-vowel (CV) speech stimuli (non-word /de:/versus real world/deI/). The probability of the deviant for each tone or speech contrast was 10%. The subjects were also presented with the same stimuli in behavioural discrimination tasks, and were administered a language assessment battery to measure their auditory comprehension skills. Outcomes O Results: The aphasic subjects demonstrated attenuated MMN responses to complex tone duration deviance and to speech stimuli (words and non-words), and their responses to the frequency, duration, and real word deviant stimuli were found to strongly correlate with performance on the auditory comprehension section of the Western Aphasia Battery (WAB). Furthermore, deficits in attentional lexical decision skills demonstrated by the aphasic subjects correlated with a word-related enhancement demonstrated during the automatic MMN paradigm, providing evidence to support the word advantage effect, thought to reflect the activation of language-specific memory traces in the brain for words. Conclusions: These results indicate that the MMN may be used as a technique for investigating general and more specific auditory comprehension skills of individuals with aphasia, using speech and/or non-speech stimuli, independent of the individual's attention. The combined use of the objective MMN technique and current clinical language assessments may result in improved rehabilitative management of aphasic individuals.

Biological response of broiler chickens fed peas (Pisum sativum L.) expressing the bean (Phaseolus vulgaris L.) alpha-amylase inhibitor transgene

The nutritive value of transgenic peas expressing an a-amylase inhibitor (alpha-Ail) was evaluated with broiler chickens. The effects of feeding transgenic peas on the development of visceral organs associated with digestion and nutrient absorption were also examined. The chemical composition of the conventional and the transgenic peas used in this study were similar. In the two feeding trials, that were conducted normal and transgenic peas were incorporated into a maize-soybean diet at concentrations up to 500 g kg(-1). The diets were balanced to contain similar levels of apparent metabolisable energy (AME) and amino acids. In the first trial, the birds were fed the diets from 3 to 17days post-hatching and with levels of transgenic peas at 250 g kg(-1) or greater there was a significant reduction in body weight but an increase in feed intake resulting in deceased feed conversion efficiency. In the second trial, in which the birds were fed diets containing 300 g kg(-1) transgenic peas until 40 days of age, growth performance was significantly reduced. It was also demonstrated that the ileal starch digestibility coefficient (0.80 vs 0.42) was significantly reduced in the birds fed transgenic peas. Determination of AME and ileal digestibility of amino acids in 5-week-old broilers demonstrated a significant reduction in AME (12.12 vs 5.08 MJ kg(-1) DM) in the birds fed the transgenic peas. The AME value recorded for transgenic peas reflected the lower starch digestibility of this line. Real digestion of protein and amino acids was unaffected by treatment. Expression of a-Ail in peas did not appear to affect bird health or the utilisation of dietary protein. However, the significant reduction in ileal digestion of starch in transgenic peas does reduce the utility of this feedstuff in monogastric diets where efficient energy utilisation is required. (c) 2006 Society of Chemical Industry.

Characterization of neogenin-expressing neural progenitor populations and migrating neuroblasts in the embryonic mouse forebrain

Many studies have demonstrated a role for netrin-1-deleted in colorectal cancer (DCC) interactions in both axon guidance and neuronal migration. Neogenin, a member of the DCC receptor family, has recently been shown to be a chemorepulsive axon guidance receptor for the repulsive guidance molecule (RGM) family of guidance cues [Rajagopalan S, Deitinghoff L, Davis D, Conrad S, Skutella T, Chedotal A, Mueller B, Strittmatter S (2004) Neogenin mediates the action of repulsive guidance molecule. Nat Cell Biol 6:755-762]. Here we show that neogenin is present on neural progenitors, including neurogenic radial glia, in the embryonic mouse forebrain suggesting that neogenin expression is a hallmark of neural progenitor populations. Neogenin-positive progenitors were isolated from embryonic day 14.5 forebrain using flow cytometry and cultured as neurospheres. Neogenin-positive progenitors gave rise to neurospheres displaying a high proliferative and neurogenic potential. In contrast, neogenin-negative forebrain cells did not produce long-term neurosphere cultures and did not possess a significant neurogenic potential. These observations argue strongly for a role for neogenin in neural progenitor biology. In addition, we also observed neogenin on parvalbumin- and calbindin-positive interneuron neuroblasts that were migrating through the medial and lateral ganglionic eminences, suggesting a role for neogenin in tangential migration. Therefore, neogenin may be a multi-functional receptor regulating both progenitor activity and neuroblast migration in the embryonic forebrain. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.