• Sonic Hedgehog regulates thymic epithelial cell differentiation

      Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L.; Ono, Masahiro; Holländer, Georg; Crompton, Tessa; University College London; et al. (Elsevier, 2016-01)
      Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus.
    • Tissue-derived hedgehog proteins modulate Th differentiation and disease

      Furmanski, Anna L.; Saldaña, José Ignacio; Ono, Masahiro; Sahni, Hemant; Paschalidis, Nikolaos; D'Acquisto, Fulvio; Crompton, Tessa; University College London (American Association for Immunologists, 2013-02-13)
      Genome-wide association studies of complex immune-mediated diseases have indicated that many genetic factors, each with individual low risk, contribute to overall disease. It is therefore timely and important to characterize how immune responses may be subtly modified by tissue context. In this article, we explore the role of tissue-derived molecules in influencing the function of T cells, which, owing to their migratory nature, come into contact with many different microenvironments through their lifespan. Hedgehog (Hh) proteins act as secreted morphogens, providing concentration-dependent positional and temporal cell-fate specification in solid tissues. Hh signaling is required for embryogenesis and is important in postnatal tissue renewal and in malignancy. However, the function of Hh in dynamic, fluid systems, such as in mammalian immunity, is largely unknown. In this article, we show that Hh-dependent transcription in T cells promoted Th2 transcriptional programs and differentiation, exacerbating allergic disease. Of interest, expression of Sonic Hh increased in lung epithelial cells following the induction of allergic disease, and lung T cells upregulated Hh target gene expression, indicating that T cells respond to locally secreted Hh ligands in vivo. We show that Il4, the key Th2 cytokine, is a novel transcriptional target of Hh signals in T cells, providing one mechanism for the role of Hh in Th differentiation. We propose that Hh, secreted from inflamed, remodeling, or malignant tissue, can modulate local T cell function. Our data present an unexpected and novel role for tissue-derived morphogens in the regulation of fluid immune responses, with implications for allergy and tumor responses, suggesting new uses for anti-Hh therapeutics.
    • Analogue peptides for the immunotherapy of human acute myeloid leukemia

      Hofmann, Susanne; Mead, Andrew; Malinovskis, Aleksandrs; Hardwick, Nicola R.; Guinn, Barbara-Ann; University of Bedfordshire (Springer, 2015-10-05)
      The use of peptide vaccines, enhanced by adjuvants, has shown some efficacy in clinical trials. However, responses are often short-lived and rarely induce notable memory responses. The reason is that self-antigens have already been presented to the immune system as the tumor develops, leading to tolerance or some degree of host tumor cell destruction. To try to break tolerance against self-antigens, one of the methods employed has been to modify peptides at the anchor residues to enhance their ability to bind major histocompatibility complex molecules, extending their exposure to the T-cell receptor. These modified or analogue peptides have been investigated as stimulators of the immune system in patients with different cancers with variable but sometimes notable success. In this review we describe the background and recent developments in the use of analogue peptides for the immunotherapy of acute myeloid leukemia describing knowledge useful for the application of analogue peptide treatments for other malignancies.
    • Application of the pMHC array to characterise tumour antigen specific T cell populations in leukaemia patients at disease diagnosis

      Brooks, Suzanne E.; Bonney, Stephanie A.; Lee, Cindy; Publicover, Amy; Khan, Ghazala; Smits, Evelien L.J.; Sigurdardottir, Dagmar; Arno, Matthew; Li, Demin; Mills, Ken I.; et al. (Public Library of Science, 2015-10-22)
      Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms’ Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8–1.4 x 106). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients.
    • Haemophilia A and cardiovascular morbidity in a female SHAM syndrome carrier due to skewed X chromosome inactivation

      Janczar, Szymon; Kosinska, Joanna; Ploski, Rafal; Pastorczak, Agata; Wegner, Olga; Zalewska-Szewczyk, Beata; Paige, Adam J.W.; Borowiec, Maciej; Mlynarski, Wojciech; Medical University of Lodz; et al. (Elsevier, 2016-01)
      We have recently described a severe haemophilia A and moyamoya (SHAM) syndrome caused by Xq28 deletions encompassing F8 and the BRCC3 familial moyamoya gene. The phenotype includes haemophilia A, moyamoya angiopathy, dysmorphia and hypertension. The genetic analysis of the family of our SHAM patient demonstrated carrier state in proband's mother and sister. The patient's mother is apparently well, whereas his currently 18-years-old sister presents with mild haemophilia A, coarctation of the aorta, hypertension, and ventricular arrhythmia. We performed X chromosome inactivation assay based on HpaII methylation analysis of a polymorphic short tandem repeat (STR) in the X linked AR (androgen receptor) gene and used quantitative real-time RT PCR to measure the expression of genes from the deleted region in proband's family members. We found an extremely skewed X chromosome inactivation pattern in the female members of the family leading to preferential inactivation of the X chromosome without Xq28 deletion in patient's sister. We demonstrated differential expression of the genes from the deleted region in four members of the family, that tightly correlates with the clinical features. In conclusion, we show that the haematologic and cardiovascular morbidity and the discrepancy between patient's sister and mother despite the same genetic lesion are due to skewed X chromosome inactivation leading to clinically relevant differential expression of SHAM syndrome genes. This report highlights the role for BRCC3 in cardiovascular physiology and disease, and demonstrates that in some complex hereditary syndromes full diagnostics may require the examination of both genetic and epigenetic events.
    • Local understandings of conservation in southeastern Mexico and their implications for community-based conservation as an alternative paradigm

      Reyes-Garcia, Victoria; Ruiz-Mallen, Isabel; Porter-Bolland, Luciana; Garcia-Frapolli, Eduardo; Ellis, Edward A.; Mendez, Maria-Elena; Pritchard, Diana J.; Sanchez-Gonzalez, Maria-Consuelo; Institucio Catalana de Recerca i Estudis Avancats; Centro de Investigaciones en Ecosistemas; et al. (Wiley, 2013-08)
      Since the 1990s national and international programs have aimed to legitimize local conservation initiatives that might provide an alternative to the formal systems of state-managed or otherwise externally driven protected areas. We used discourse analysis (130 semistructured interviews with key informants) and descriptive statistics (679 surveys) to compare local perceptions of and experiences with state-driven versus community-driven conservation initiatives. We conducted our research in 6 communities in southeastern Mexico. Formalization of local conservation initiatives did not seem to be based on local knowledge and practices. Although interviewees thought community-based initiatives generated less conflict than statemanaged conservation initiatives, the community-based initiatives conformed to the biodiversity conservation paradigm that emphasizes restricted use of and access to resources. This restrictive approach to community-based conservation in Mexico, promoted through state and international conservation organizations, increased the area of protected land and had local support but was not built on locally relevant and multifunctional landscapes, a model that community-based conservation is assumed to advance.
    • Community based biodiversity monitoring in Mexico: current status, challenges, and future strategies for collaboration with scientists

      Pritchard, Diana J. (Springer, 2013-07-18)
      An array of social and political actors, from international to local levels, increasingly demand monitoring data on biodiversity and ecosystem functions. As elsewhere, prevalent approaches in Mexico emphasize the collection of scientific data regarding biological indicators, by professionals, for conservation planning, global targets, and biological inquiry. These are complicated, expensive, and dependent on external funding. They also fail to engage with communities, many of whom practice traditional forms of monitoring to manage their local environments and livelihoods. Community-based monitoring, an approach involving collaborations between scientists and communities, has the potential to contribute to sustainable forms of resource use management and as a cost-effective method. Efforts could develop once local rights of use and traditional knowledge systems are recognized, access to information is ensured, and a broad array of relevant environmental and social indicators are included.
    • Wnt-11 promotes neuroendocrine-like differentiation, survival and migration of prostate cancer cells

      Uysal-Onganer, Pinar; Kawano, Yoshiaki; Caro, Mercedes; Walker, Marjorie M.; Diez, Soraya; Darrington, R. Siobhan; Waxman, Jonathan; Kypta, Robert M. (BioMed Central, 2010)
      Wnt-11 is a secreted protein that modulates cell growth, differentiation and morphogenesis during development. We previously reported that Wnt-11 expression is elevated in hormone-independent prostate cancer and that the progression of prostate cancer from androgen-dependent to androgen-independent proliferation correlates with a loss of mutual inhibition between Wnt-11- and androgen receptor-dependent signals. However, the prevalence of increased expression of Wnt-11 in patient tumours and the functions of Wnt-11 in prostate cancer cells were not known.
    • TWIST1 regulates the activity of ubiquitin proteasome system via the miR-199/214 cluster in human end-stage dilated cardiomyopathy

      Baumgarten, Anna; Bang, Claudia; Tschirner, Anika; Engelmann, Anke; Adams, Volker; von Haehling, Stephan; Doehner, Wolfram; Pregla, Reinhard; Anker, Markus S.; Blecharz, Kinga; et al. (Elsevier, 2013-09-30)
      The transcription factor TWIST1 has been described to regulate the microRNA (miR)-199/214 cluster. Genetic disruption of TWIST1 resulted in a cachectic phenotype and early death of the knock-out mice. This might be connected to the activity of the ubiquitin-proteasome-system (UPS), as miR-199a has been suggested to regulate the ubiquitin E2 ligases Ube2i and Ube2g1.
    • Transferrin receptor 2 is crucial for iron sensing in human hepatocytes

      Rapisarda, Chiara; Puppi, Juliana; Hughes, Robin D.; Dhawan, Anil; Farnaud, Sébastien; Evans, Robert W.; Sharp, Paul A. (American Physiological Society, 2010-06-24)
      Hepcidin expression in vivo is regulated in proportion to iron status (i.e., increased by iron loading and decreased in iron deficiency). However, in vitro studies with hepatoma cell lines often show an inverse relationship between iron status and hepcidin expression. Here, we investigated possible molecular mechanisms responsible for the differences in iron sensing between hepatoma cell lines and human primary hepatocytes. RNA was collected from primary human hepatocytes, and HepG2 and HuH7 hepatoma cells were treated with either transferrin-bound and non-transferrin-bound iron. Expression of hepcidin, transferrin receptor 2, HFE, and hemojuvelin were quantified by real-time PCR. Hepcidin expression was increased in primary human hepatocytes following 24-h exposure to holoferric transferrin. In contrast, hepcidin mRNA levels in hepatoma cells were decreased by transferrin. Hepcidin expression was positively correlated with transferrin receptor 2 mRNA levels in primary human hepatocytes. Compared with primary hepatocytes, transferrin receptor 2 expression was significantly lower in hepatoma cell lines; furthermore, there was no correlation between transferrin receptor 2 and hepcidin mRNA levels in either HepG2 or HuH7 cells. Taken together our data suggest that transferrin receptor 2 is a likely candidate to explain the differences in iron sensing between hepatoma cell lines and primary human hepatocytes.
    • The interaction of arginine- and tryptophan-rich cyclic hexapeptides with Escherichia coli membranes

      Junkes, Christof; Wessolowski, Axel; Farnaud, Sébastien; Evans, Robert W.; Good, Liam; Bienert, Michael; Dathe, Margitta; Leibniz Institute of Molecular Pharmacology; Westminster University; King's College London (Wiley, 2008-04)
      Cyclization of R- and W-rich hexapeptides has been found to enhance specifically the antimicrobial activity against Gram-negative Escherichia coli. To gain insight into the role of the bacterial outer membrane in mediating selectivity, we assayed the activity of cyclic hexapeptides derived from the parent sequence c-(RRWWRF) against several E. coli strains and Bacillus subtilis, L-form bacteria, and E. coli lipopolysaccharide (LPS) mutant strains, and we also investigated the peptide-induced permeabilization of the outer and inner membrane of E. coli. Wall-deficient L-form bacteria were distinctly less susceptible than the wild type strain. The patterns of peptide-induced permeabilization of the outer and inner E. coli membranes correlated well with the antimicrobial activity, confirming that membrane permeabilization is a detrimental effect of the peptides upon bacteria. Truncation of LPS had no influence on the activity of the cyclic parent peptide, but the highly active c-(RRWFWR), with three adjacent aromatic residues, required the complete LPS for maximal activity. Furthermore, differences in the activity of the parent peptide and its all-D sequence indicated stereospecific interactions with the LPS mutant strains. We suggest that, depending on the primary sequence of the peptides, either hydrophobic interactions with the fatty acid chains of lipid A, or electrostatic interactions disturbing the polar core region and interference with saccharide-saccharide interactions prevail in the barrier-disturbing effect upon the outer membrane and thereby provide peptide accessibility to the inner membrane. The results underline the importance of tryptophan and arginine residues and their relative location for a high antimicrobial effect, and the activity-modulating function of the outer membrane of E. coli. In addition to membrane permeabilization, the data provided evidence for the involvement of other mechanisms in growth inhibition and killing of bacteria.
    • Intervention effects of Ganoderma lucidum spores on epileptiform discharge hippocampal neurons and expression of Neurotrophin-4 and N-Cadherin

      Wang, Shu-Qiu; Li, Xiao-Jie; Zhou, Shaobo; Sun, Di-Xiang; Wang, Hui; Cheng, Peng-Fei; Ma, Xiao-Ru; Liu, Lei; Liu, Jun-Xing; Wang, Fang-Fang; et al. (PLoS, 2013-04-24)
      Epilepsy can cause cerebral transient dysfunctions. Ganoderma lucidum spores (GLS), a traditional Chinese medicinal herb, has shown some antiepileptic effects in our previous studies. This was the first study of the effects of GLS on cultured primary hippocampal neurons, treated with Mg2+ free medium. This in vitro model of epileptiform discharge hippocampal neurons allowed us to investigate the anti-epileptic effects and mechanism of GLS activity. Primary hippocampal neurons from <1 day old rats were cultured and their morphologies observed under fluorescence microscope. Neurons were confirmed by immunofluorescent staining of neuron specific enolase (NSE). Sterile method for GLS generation was investigated and serial dilutions of GLS were used to test the maximum non-toxic concentration of GLS on hippocampal neurons. The optimized concentration of GLS of 0.122 mg/ml was identified and used for subsequent analysis. Using the in vitro model, hippocampal neurons were divided into 4 groups for subsequent treatment i) control, ii) model (incubated with Mg2+ free medium for 3 hours), iii) GLS group I (incubated with Mg2+ free medium containing GLS for 3 hours and replaced with normal medium and incubated for 6 hours) and iv) GLS group II (neurons incubated with Mg2+ free medium for 3 hours then replaced with a normal medium containing GLS for 6 hours). Neurotrophin-4 and N-Cadherin protein expression were detected using Western blot. The results showed that the number of normal hippocampal neurons increased and the morphologies of hippocampal neurons were well preserved after GLS treatment. Furthermore, the expression of neurotrophin-4 was significantly increased while the expression of N-Cadherin was decreased in the GLS treated group compared with the model group. This data indicates that GLS may protect hippocampal neurons by promoting neurotrophin-4 expression and inhibiting N-Cadherin expression.
    • Inhibition of xanthine oxidase reduces wasting and improves outcome in a rat model of cancer cachexia

      Springer, Jochen; Tschirner, Anika; Hartman, Kai; Palus, Sandra; Wirth, Eva K.; Ruis, Silvia Busquets; Möller, Nadine; von Haehling, Stephan; Argiles, Josep M.; Köhrle, Josef; et al. (Wiley, 2012-11-01)
      Cachexia is a common co-morbidity in cancer occurring in up to 80% of patients depending on the type of cancer. Uric acid (UA), the end-product of the purine metabolism, is elevated in cachexia due to tissue wasting and upregulated xanthine oxidase (XO) activity. High serum UA levels indicate increased XO-dependent production of oxygen free radicals (reactive oxygen species; ROS) and correlate with metabolic illness and poor survival. We hypothesized that XO-inhibition might reduce inflammatory signals accounting for tissue wasting and improve survival in experimental cancer cachexia. Animals were inoculated intraperitoneally with AH-130 hepatoma cells and treated with two XO-inhibitors: allopurinol [Allo, low (LD) and high dose (HD) 4 and 40 mg/kg/d] and its more effective active metabolite oxypurinol (Oxy, 4 and 40 mg/kg/d) or placebo for 15 days. Weight loss and tissue wasting of both fat and lean tissue (assessed by NMR-scanning) was reduced by both LD and HD Allo and LD-Oxy, but not by HD-Oxy. A robust induction of XO-activity for generation of reactive oxygen species was seen in the placebo group (assessed by electron paramagnetic spectroscopy), which was reduced by XO-inhibition. Increased ROS induced cytokine signaling, proteolytic activity and tissue degradation were all attenuated by XO inhibition. Survival was significantly and dose dependently improved. Food intake and spontaneous locomotor activity were higher, indicating a higher quality of life. Inhibition of XO can reduce tissue wasting and improve survival in cancer cachexia and clearly clinical studies are needed.
    • Identification of an iron-hepcidin complex

      Farnaud, Sébastien; Rapisarda, Chiara; Bui, Tam; Drake, Alex F.; Cammack, Richard; Evans, Robert W; University of Westminster (Portland Press, 2008-08-01)
      Following its identification as a liver-expressed antimicrobial peptide, the hepcidin peptide was later shown to be a key player in iron homoeostasis. It is now proposed to be the 'iron hormone' which, by interacting with the iron transporter ferroportin, prevents further iron import into the circulatory system. This conclusion was reached using the corresponding synthetic peptide, emphasizing the functional importance of the mature 25-mer peptide, but omitting the possible functionality of its maturation. From urine-purified native hepcidin, we recently demonstrated that a proportion of the purified hepcidin had formed iron-hepcidin complexes. This interaction was investigated further by computer modelling and, based on the sequence similarity of hepcidin with metallothionein, a three-dimensional model of hepcidin, containing one atom of iron, was constructed. To characterize these complexes further, the interaction with iron was analysed using different spectroscopic methods. Monoferric hepcidin was identified by MS, as were possibly other complexes containing two and three atoms of iron respectively, although these were present only in minor amounts. UV/visible absorbance and CD studies identified the iron-binding events which were facilitated at a physiological pH. EPR spectroscopy identified the ferric state of the bound metal, and indicated that the iron-hepcidin complex shares some similarities with the rubredoxin iron-sulfur complex, suggesting the presence of Fe(3+) in a tetrahedral sulfur co-ordination. The potential roles of iron binding for hepcidin are discussed, and we propose either a regulatory function in the maturation of pro-hepcidin into active hepcidin or as the necessary link in the interaction between hepcidin and ferroportin.
    • Gli2, hedgehog and TCR signalling

      Furmanski, Anna L.; Crompton, Tessa (Impact Journals, 2015-07)
    • Preserved global histone H4 acetylation linked to ETV6-RUNX1 fusion and PAX5 deletions is associated with favorable outcome in pediatric B-cell progenitor acute lymphoblastic leukemia.

      Janczar, Karolina; Janczar, Szymon; Pastorczak, Agata; Mycko, K.; Paige, Adam J.W.; Zalewska-Szewczyk, Beata; Wagrowska-Danilewicz, M.; Danilewicz, Marian; Mlynarski, Wojciech; Medical University of Lodz; et al. (Elsevier, 2015-10-20)
      Epigenetic dysregulation is a hallmark of cancer executed by a number of complex processes the most important of which converge on DNA methylation and histone protein modifications. Epigenetic marks are potentially reversible and thus promising drug targets. In the setting of acute lymphoblastic leukemia (ALL) they have been associated with clinicopathological features including risk of relapse or molecular subgroups of the disease. Here, using immunocytochemistry of bone marrow smears from diagnosis, we studied global histone H4 acetylation, whose loss was previously linked to treatment failure in adults with ALL, in pediatric patients. We demonstrate that preserved global histone H4 acetylation is significantly associated with favorable outcome (RFS, EFS, OS) in children with B cell progenitor (BCP) ALL, recapitulating the findings from adult populations. Further, for the first time we demonstrate differential histone H4 acetylation in molecular subclasses of BCP-ALL including cases with ETV6-RUNX1 fusion gene or PAX5 deletion or deletions in genes linked to B cell development. We conclude global histone H4 acetylation is a prognostic marker and a potential therapeutic target in ALL.
    • A genome wide transcriptional model of the complex response to pre-TCR signalling during thymocyte differentiation

      Sahni, Hemant; Ross, Susan; Barbarulo, Alessandro; Solanki, Anisha; Lau, Ching-In; Furmanski, Anna L.; Saldaña, José Ignacio; Ono, Masahiro; Hubank, Mike; Barenco, Martino; et al. (Impact Journals, 2015-09-20)
      Developing thymocytes require pre-TCR signalling to differentiate from CD4-CD8- double negative to CD4+CD8+ double positive cell. Here we followed the transcriptional response to pre-TCR signalling in a synchronised population of differentiating double negative thymocytes. This time series analysis revealed a complex transcriptional response, in which thousands of genes were up and down-regulated before changes in cell surface phenotype were detected. Genome-wide measurement of RNA degradation of individual genes showed great heterogeneity in the rate of degradation between different genes. We therefore used time course expression and degradation data and a genome wide transcriptional modelling (GWTM) strategy to model the transcriptional response of genes up-regulated on pre-TCR signal transduction. This analysis revealed five major temporally distinct transcriptional activities that up regulate transcription through time, whereas down-regulation of expression occurred in three waves. Our model thus placed known regulators in a temporal perspective, and in addition identified novel candidate regulators of thymocyte differentiation.
    • An analogue peptide from the cancer testis antigen, PASD1, induces CD8+ T cell-responses against naturally processed peptide.

      Hardwick, Nicola R.; Buchan, Sarah; Ingram, Wendy; Khan, Ghazala; Vittes, Gisella; Rice, Jason; Pulford, Karen; Mufti, Ghulam J.; Stevenson, Freda; Guinn, Barbara-Ann; et al. (Cancer Research Institute, 2013)
      We have previously identified the novel Cancer/Testis antigen PASD1 by immunoscreening a testis library with pooled acute myeloid leukemia (AML) patient sera. To develop a cytotoxic T lymphocyte (CTL)-inducing vaccine, we have now investigated the carboxy-terminal region, known to contain serological determinants, for MHC class I (HLA-A⋆0201)-binding peptides. Algorithm-selected natural peptides failed to show detectable HLA-A⋆0201 binding in T2 assays. However, anchor-modified analogue peptides showed enhanced binding, with decreased off-rates. Analogue peptide-loaded antigen-presenting cells (APCs) induced IFN-γ production by T cells from normal donors and patients. In addition, peptide-specific T cells could be expanded from cancer patients by stimulation with the PASD1 analogue peptide Pa14. For clinical application, a DNA fusion gene vaccine encoding Pa14 was designed and tested in "humanized" mice. Splenocytes from vaccinated mice showed in vitro cytotoxicity against tumour cells, either exogenously loaded with the corresponding wild-type peptide (Pw8) or expressing endogenously processed PASD1 protein. We show for the first time that a DNA vaccine encoding an altered PASD1 epitope can induce CTLs to target the natural peptide expressed by human tumour cells.
    • Predicting ecological regime shift under climate change: new modelling techniques and potential of molecular-based approaches

      Stafford, Richard; Smith, V.Anne; Husmeier, Dirk; Grima, Thomas; Guinn, Barbara-Ann; Bournemouth University; University of St Andrews; University of Glasgow; University of Bedfordshire (Current Zoology, 2013)
      Ecological regime shift is the rapid transition from one stable community structure to another, often ecologically inferior, stable community. Such regime shifts are especially common in shallow marine communities, such as the transition of kelp forests to algal turfs that harbour far lower biodiversity. Stable regimes in communities are a result of balanced interactions between species, and predicting new regimes therefore requires an evaluation of new species interactions, as well as the resilience of the ‘stable’ position. While computational optimisation techniques can predict new potential regimes, predicting the most likely community state of the various options produced is currently educated guess work. In this study we integrate a stable regime optimisation approach with a Bayesian network used to infer prior knowledge of the likely stress of climate change (or, in practice, any other disturbance) on each component species of a representative rocky shore community model. Combining the results, by calculating the product of the match between resilient computational predictions and the posterior probabilities of the Bayesian network, gives a refined set of model predictors, and demonstrates the use of the process in determining community changes, as might occur through processes such as climate change. To inform Bayesian priors, we conduct a review of molecular approaches applied to the analysis of the transcriptome of rocky shore organisms, and show how such an approach could be linked to measureable stress variables in the field. Hence species-specific microarrays could be designed as biomarkers of in situ stress, and used to inform predictive modelling approaches such as those described here.
    • High and low, but not intermediate, PRAME expression levels are poor prognostic markers in myelodysplastic syndrome at disease presentation

      Liberante, Fabio G.; Pellagatti, Andrea; Boncheva, Viktoriya Bogdanova; Bowen, David T.; Mills, Ken I.; Boultwood, Jacqueline; Guinn, Barbara-Ann; University of Bedfordshire; Leeds Teaching Hospitals NHS Trust; Queens University Belfast; et al. (Wiley, 2013-07)