Psychosis is a severe mental condition that is characterized by a loss of contact with reality and is typically associated with hallucinations and delusional beliefs. There are numerous psychiatric conditions that present with psychotic symptoms, most importantly schizophrenia, bipolar affective disorder, and some forms of severe depression referred to as psychotic depression. The pathological mechanisms resulting in psychotic symptoms are not understood, nor is it understood whether the various psychotic illnesses are the result of similar biochemical disturbances. The identification of biological markers (so-called biomarkers) of psychosis is a fundamental step towards a better understanding of the pathogenesis of psychosis and holds the potential for more objective testing methods.

CD4+CD25+Foxp3+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance by inhibiting the expansion and function of conventional T cells. Treg development and homeostasis are regulated by the Ag receptor, costimulatory receptors such as CD28 and CTLA-4, and cytokines such as IL-2, IL-10, and TGF-beta. Here we show that the proportions of Tregs in the spleen and lymph nodes of mice with inactive p110delta PI3K (p110deltaD910A/D910A) are reduced despite enhanced Treg selection in the thymus. p110deltaD910A/D910A CD4+CD25+Foxp3+ Tregs showed attenuated suppressor function in vitro and failed to secrete IL-10. In adoptive transfer experiments, p110deltaD910A/D910A T cells failed to protect against experimental colitis. The identification of p110delta as an intracellular signaling protein that regulates the activity of CD4+CD25+Foxp3+ Tregs may facilitate the further elucidation of the molecular mechanisms responsible for Treg-mediated suppression.

Bowel cancer is common and is a major cause of death. Meta-analysis of randomised controlled trials estimates that screening for colorectal cancer using faecal occult blood (FOB) test reduces mortality from colorectal cancer by 16%. However, FOB testing has a low positive predictive value, with associated unnecessary cost, risk and anxiety from subsequent investigation, and is unacceptable to a proportion of the target population. Increased levels of an enzyme called matrix metalloproteinase 9 (MMP-9) have been found to be associated with colorectal cancer, and this can be measured from a blood sample. Serum MMP-9 is potentially an accurate, low risk and cost-effective population screening tool. This study aims to evaluate the accuracy of serum MMP-9 as a test for colorectal cancer in a primary care population.

The slow Wallerian degeneration gene (Wld(S)) delays Wallerian degeneration and axon pathology for several weeks in mice and rats. Interestingly, neuronal cell death is also delayed in some in vivo models, most strikingly in the progressive motoneuronopathy mouse. Here, we tested the hypothesis that Wld(S) has a direct protective effect on motoneurone cell bodies in vivo. Cell death was induced in rat L4 motoneurones by intravertebral avulsion of the corresponding ventral roots. This simultaneously removed most of the motor axon, minimizing the possibility that the protective effect toward axons could rescue cell bodies secondarily. There was no significant difference between the survival of motoneurones in control and Wld(S) rats, suggesting that the Wld(S) gene has no direct protective effect on cell bodies. We also tested for any delay in apoptotic motoneurone death following neonatal nerve injury in Wld(S) rats and found that, unlike Wld(S) mice, Wld(S) rats show no delay in cell death. However, the corresponding distal axons were preserved, confirming that motoneurone cell bodies and motor axons die by different mechanisms. Thus, Wld(S) does not directly prevent death of motoneurone cell bodies. It follows that the protection of neuronal cell bodies observed in several disease and injury models where axons or significant axonal stumps remain is most probably secondary to axonal protection.

Genetic instability (GI) is a hallmark feature of tumor development. Securin, also known as pituitary tumor transforming gene (PTTG), is a mitotic checkpoint protein which is highly expressed in numerous cancers, is associated with tumor invasiveness, and induces GI in thyroid cells. We used fluorescence inter-simple sequence repeat PCR to assess GI caused primarily by DNA breakage events in 19 colorectal tumors. GI values ranged significantly, with Dukes' stage C&D colorectal tumors exhibiting greater GI and higher securin expression than Dukes' stage A&B tumors. Consistent with these findings, we observed a dose-dependent increase in GI in HCT116 cells in response to securin overexpression, as well as in non-transformed human fibroblasts. As securin has been implicated in a novel DNA repair pathway in fission yeast, we investigated its potential role in chemotoxic DNA damage response pathways in mammalian cells, using host cell reactivation assays. Securin overexpression in HCT116 cells inhibited etoposide-induced double-stranded DNA damage repair activity, and repressed Ku heterodimer function. Additionally, we observed that securin and Ku70 showed a reciprocal cytosol-nuclear translocation in response to etoposide-induced dsDNA damage. Our data suggest that, by repressing Ku70 activity and inhibiting the non-homologous end-joining dsDNA repair pathway, securin may be a critical gene in the development of GI in colorectal cancer.

Many models of autoimmunity are associated with lymphopenia. Most involve a T-helper cell (Th)1-type disease, including the diabetic BioBreeding (BB) rat. To investigate the roles of identified susceptibility loci in disease pathogenesis, we bred PVG-RT1(u), lymphopenia (lyp)/lyp rats, congenic for the iddm1 (RT1(u)) and iddm2 (lyp, Gimap5(-/-)) diabetes susceptibility loci on the PVG background. Surprisingly, these rats developed a spontaneous, progressive, inflammatory bowel disease. To understand the disease pathogenesis, we undertook investigations at the genetic, histologic, and cellular levels.

Bowel cancer is common and is a major cause of death. Most people with bowel symptoms who meet the criteria for urgent referral to secondary care will not be found to have bowel cancer, and some people who are found to have cancer will have been referred routinely rather than urgently. If general practitioners could better identify people who were likely to have bowel cancer or conditions that may lead to bowel cancer, the pressure on hospital clinics may be reduced, enabling these patients to be seen more quickly. Increased levels of an enzyme called matrix metalloproteinase 9 (MMP-9) have been found to be associated with such conditions, and this can be measured from a blood sample. This study aims to find out whether measuring MMP-9 levels could improve the appropriateness of urgent referrals for patients with bowel symptoms.

Vav proteins belong to the family of guanine-nucleotide-exchange factors for the Rho/Rac family of small G-proteins. In addition, they serve as important adapter proteins for the activation of PLCgamma (phospholipase Cgamma) isoforms by ITAM (immunoreceptor tyrosine-based activation motif) receptors, including the platelet collagen receptor GPVI (glycoprotein VI). Vav proteins are also regulated downstream of integrins, including the major platelet integrin alphaIIbbeta3, which has recently been shown to regulate PLCgamma2. In the present study, we have investigated the role of Vav family proteins in filopodia and lamellipodia formation on fibrinogen using platelets deficient in Vav1 and Vav3. Wild-type mouse platelets undergo a limited degree of spreading on fibrinogen, characterized by the formation of numerous filopodia and limited lamellipodia structures. Platelets deficient in Vav1 and Vav3 exhibit reduced filopodia and lamellipodia formation during spreading on fibrinogen. This is accompanied by reduced alphaIIbbeta3-mediated PLCgamma2 tyrosine phosphorylation and reduced Ca(2+) mobilization. In contrast, the G-protein agonist thrombin stimulates full spreading of control and Vav1/3-deficient platelets. Consistent with this, stimulation of F-actin (filamentous actin) formation and Rac activation by thrombin is not altered in Vav-deficient cells. These results demonstrate that Vav1 and Vav3 are required for optimal spreading and regulation of PLCgamma2 by integrin alphaIIbbeta3, but that their requirement is by-passed upon G-protein receptor activation.

The duration of ERK1/2 activation influences the nature of the biological response to agonist. Members of the AP-1 transcription factor family are well known targets of the ERK1/2 pathway and are expressed in a temporally coordinated fashion during cell cycle re-entry. In CCl39 fibroblasts, sustained ERK1/2 activation is required for the expression of Fra-1, Fra-2, c-Jun and JunB, whereas expression of c-Fos is still strongly induced even in response to transient ERK activation. However, the significance of this pattern of expression for AP-1 activity has not been addressed. Here we show that growth factor stimulated activation of the C-terminal c-Fos transactivation domain (TAD) serves as a sensor for ERK1/2 signal duration whereas the c-JunTAD is not responsive to growth factors. In addition, sustained ERK1/2 activation determines the duration of increases in AP-1 DNA binding complexes as well as their qualitative make up. Finally, this is reflected in both the duration and quantitative transcriptional output of stably integrated AP-1 reporter constructs, indicating that AP-1 activity is finely tuned to ERK1/2 signal duration. These results provide new insights into the importance of ERK1/2 signal duration in the regulation of AP-1 and provide an explanation for how differences in signal duration can lead to both quantitative and qualitative changes in gene expression.

We have developed a spike sorting method, using a combination of various machine learning algorithms, to analyse electrophysiological data and automatically determine the number of sampled neurons from an individual electrode, and discriminate their activities. We discuss extensions to a standard unsupervised learning algorithm (Kohonen), as using a simple application of this technique would only identify a known number of clusters. Our extra techniques automatically identify the number of clusters within the dataset, and their sizes, thereby reducing the chance of misclassification. We also discuss a new pre-processing technique, which transforms the data into a higher dimensional feature space revealing separable clusters. Using principal component analysis (PCA) alone may not achieve this. Our new approach appends the features acquired using PCA with features describing the geometric shapes that constitute a spike waveform. To validate our new spike sorting approach, we have applied it to multi-electrode array datasets acquired from the rat olfactory bulb, and from the sheep infero-temporal cortex, and using simulated data. The SOMA sofware is available at http://www.sussex.ac.uk/Users/pmh20/spikes.

Through their ability to regulate production of the key lipid messenger PtdIns(3,4,5)P(3), the class I phosphatidylinositol-3-OH kinases (PI(3)Ks) support many critical cell responses. They, in turn, can be regulated by cell-surface receptors through signals acting on either their adaptor subunits (for example, through phosphotyrosine or Gbetagammas) or their catalytic subunits (for example, through GTP-Ras). The relative significance of these controlling inputs is undefined in vivo. Here, we have studied the roles of Gbetagammas, the adaptor p101, Ras and the Ras binding domain (RBD) in the control of the class I PI(3)K, PI(3)Kgamma, in mouse neutrophils. Loss of p101 leads to major reductions in the accumulation of PtdIns(3,4,5)P(3), activation of protein kinase B (PKB) and in migration towards G-protein activating ligands in vitro, and to an aseptically inflamed peritoneum in vivo. Loss of sensitivity of PI(3)Kgamma to Ras unexpectedly caused similar reductions, but additionally caused a substantial loss in production of reactive oxygen species (ROS). We conclude that Gbetagammas, p101 and the Ras-RBD interaction all have important roles in the regulation of PI(3)Kgamma in vivo and that they can simultaneously, but differentially, control distinct PI(3)Kgamma effectors.

The mouse Kcnq1 imprinted domain is located on distal chromosome 7 and contains several imprinted genes that are paternally repressed. Repression of these genes is regulated by a non-coding antisense transcript, Kcnq1ot1, which is paternally expressed. Maternal repression of Kcnq1ot1 is controlled by DNA methylation originating in the oocyte. Some genes in the region are imprinted only in the placenta, whereas others are imprinted in both extra-embryonic and embryonic lineages. Here, we show that Kcnq1ot1 is paternally expressed in preimplantation embryos from the two-cell stage, and that ubiquitously imprinted genes proximal to Kcnq1ot1 are already repressed in blastocysts, ES cells and TS cells. Repressive histone marks such as H3K27me3 are present on the paternal allele of these genes in both ES and TS cells. Placentally imprinted genes that are distal to Kcnq1ot1, by contrast, are not imprinted in blastocysts, ES or TS cells. In these genes, paternal silencing and differential histone marks arise during differentiation of the trophoblast lineage between E4.5 and E7.5. Our findings show that the dynamics during preimplantation development of gene inactivation and acquisition of repressive histone marks in ubiquitously imprinted genes of the Kcnq1 domain are very similar to those of imprinted X inactivation. By contrast, genes that are only imprinted in the placenta, while regulated by the same non-coding RNA transcript Kcnq1ot1, undergo epigenetic inactivation during differentiation of the trophoblast lineage. Our findings establish a model for how epigenetic gene silencing by non-coding RNA may depend on distance from the non-coding RNA and on lineage and differentiation specific factors.

Dopamine deficiency, caused by the degeneration of nigrostriatal dopaminergic neurons, is the cause of the major clinical motor symptoms of Parkinson's disease. These symptoms can be treated successfully with a range of drugs that include levodopa, inhibitors of the enzymatic breakdown of levodopa and dopamine agonists delivered by oral, subcutaneous, transcutaneous, intravenous or intra-duodenal routes. However, Parkinson's disease involves degeneration of non-dopaminergic neurons and the treatment of the resulting predominantly non-motor features remains a challenge. This review describes the important recent advances that underlie the development of novel dopaminergic and non-dopaminergic drugs for Parkinson's disease, and also for the motor complications that arise from the use of existing therapies.

The role of PI3K in T cell activation and costimulation has been controversial. We previously reported that a kinase-inactivating mutation (D910A) in the p110delta isoform of PI3K results in normal T cell development, but impaired TCR-stimulated cell proliferation in vitro. This proliferative defect can be overcome by providing CD28 costimulation, which raises the question as to whether p110delta activity plays a role in T cell activation in vivo, which occurs primarily in the context of costimulation. In this study, we show that the PI3K signaling pathway in CD28-costimulated p110delta D910A/D910A T cells is impaired, but that ERK phosphorylation and NF-kappaB nuclear translocation are unaffected. Under in vitro conditions of physiological Ag presentation and costimulation, p110delta D910A/D910A T cells showed normal survival, but underwent fewer divisions. Differentiation along the Th1 and Th2 lineages was impaired in p110delta D910A/D910A T cells and could not be rescued by exogenous cytokines in vitro. Adoptive transfer and immunization experiments in mice revealed that clonal expansion and differentiation in response to Ag and physiological costimulation were also compromised. Thus, p110delta contributes significantly to Th cell expansion and differentiation in vitro and in vivo, also in the context of CD28 costimulation.

The Zfp36l1 gene encodes a zinc finger-containing mRNA binding protein implicated in the posttranscriptional control of gene expression. Mouse embryos homozygous for a targeted mutation in the Zfp36l1 locus died mid-gestation and exhibited extraembryonic and intraembryonic vascular abnormalities and heart defects. In the developing placenta, there was a failure of the extraembryonic mesoderm to invaginate the trophoblast layer. The phenotype was associated with an elevated expression of vascular endothelial growth factor (VEGF)-A in the embryos and in embryonic fibroblasts cultured under conditions of both normoxia and hypoxia. VEGF-A overproduction by embryonic fibroblasts was not a consequence of changes in Vegf-a mRNA stability; instead, we observed enhanced association with polyribosomes, suggesting Zfp36l1 influences translational regulation. These data implicate Zfp36l1as a negative regulator of Vegf-a gene activity during development.

Transmission at the parallel fibre-Purkinje neurone synapse of the cerebellum can be depressed by a number of presynaptic receptors: endocannabinoid (CB1), metabotropic glutamate (mGluR4), adenosine (A1) and GABA (GABA(B)), which have been implicated in both short- and long-term synaptic plasticity. Stimulation of parallel fibres also activates glutamate receptors and transporters on the Bergmann glial cell that forms a sheath around the synapse. The resulting glial extrasynaptic currents (ESC) exhibit short- and long-term plasticity, which differs from the plasticity of adjacent synapses. This functional independence could arise from differential modulation of presynaptic release sites targeted to synapses or glia, but the sensitivity of glial ESC to these inhibitory pathways is unknown. Here I show that all four presynaptic receptors depress parallel fibre-Bergmann glial cell signalling with similar potency to synaptic transmission. Depression of glial ESC is accompanied by a decrease in paired pulse ratio. However, application of receptor antagonists had no effect on ESC amplitude, indicating that tonic activation of these pathways does not occur, and antagonists failed to block the activity-dependent depression of glial ESC observed during tetanic or low frequency stimulation. These data suggest that modulation of presynaptic glutamate release does not underlie glial plasticity.

Microglia are associated with neuritic plaques in Alzheimer disease (AD) and serve as a primary component of the innate immune response in the brain. Neuritic plaques are fibrous deposits composed of the amyloid beta-peptide fragments (Abeta) of the amyloid precursor protein (APP). Numerous studies have shown that the immune cells in the vicinity of amyloid deposits in AD express mRNA and proteins for pro-inflammatory cytokines, leading to the hypothesis that microglia demonstrate classical (Th-1) immune activation in AD. Nonetheless, the complex role of microglial activation has yet to be fully explored since recent studies show that peripheral macrophages enter an "alternative" activation state.

It is unclear whether TGF-beta, a critical differentiation factor for T cells producing interleukin 17 (T(H)-17 cells), is required for the initiation of experimental autoimmune encephalomyelitis (EAE) in vivo. Here we show that mice whose T cells cannot respond to TGF-beta signaling lack T(H)-17 cells and do not develop EAE despite the presence of T helper cell type 1 infiltrates in the spinal cord. Local but not systemic antibody blockade of TGF-beta prevented T(H)-17 cell differentiation and the onset of EAE. The pathogen stimulus zymosan, like mycobacterium, induced T(H)-17 cells and initiated EAE, but the disease was transient and correlated with reduced production of interleukin 23. These data show that TGF-beta is essential for the initiation of EAE and suggest that disease progression may require ongoing chronic inflammation and production of interleukin 23.

The chromosome conformation capture technique is used to monitor intra- and intermolecular chromosomal associations. By introducing an adaptation of this technique, Ling and colleagues have identified an unexpected coassociation between two loci on separate chromosomes in mouse nuclei, the imprinted Igf2-H19 locus of chromosome 7 and the Wsb1-Nf1 locus of chromosome 11. Strikingly, this interaction is CCCTC-binding factor (CTCF)-dependent and strictly allele specific. These findings extend our appreciation for genome organization and its influence on gene expression and imprinting.

The production of reactive oxygen species by the NADPH oxidase complex of phagocytes plays a critical role in our defence against bacterial and fungal infections. The PX domains of two oxidase components, p47(phox) and p40(phox), are known to bind phosphoinositide products of PI3Ks but the physiological roles of these interactions are unclear. We have created mice which carry an R58A mutation in the PX domain of their p40(phox) gene, which selectively prevents binding to PtdIns3P. p40(phoxR58A/R58A) embryos do not develop normally but p40(phoxR58A/-) mice are viable and neutrophils from these animals exhibit significantly reduced oxidase responses compared to those from their p40(phox+/-) siblings (e.g. 60% reduced in response to phagocytosis of Staphylococcus aureus). Wortmannin inhibition of the S. aureus oxidase response correlates with inhibition of phagosomal PtdIns3P accumulation and overlaps with the reduction in this response caused by the R58A mutation, suggesting PI3K regulation of this response is substantially dependent on PtdIns3P-binding to p40(phox). p40(phoxR58A/-) mice are significantly compromised in their ability to kill S. aureus in vivo, defining the physiological importance of this interaction.

Atrial cardiomyocytes make an important contribution to the refilling of ventricles with blood, which enhances the subsequent ejection of blood from the heart. The dependence of cardiac function on the contribution of atria becomes increasingly important with age and exercise. We know much less about the calcium signals that link electrical depolarisation to contraction within atrial myocytes in comparison with ventricular myocytes. Nevertheless, recent work has shed new light on calcium signalling in atrial cells. At an ultrastructural level, atrial and ventricular myocytes have many similarities. However, a few key structural differences, in particular the lack of transverse tubules (;T-tubules') in atrial myocytes, make these two cell types display vastly different calcium patterns in response to depolarisation. The lack of T-tubules in atrial myocytes means that depolarisation provokes calcium signals that largely originate around the periphery of the cells. To engage the contractile machinery, the calcium signal must propagate centripetally deeper into the cells. This inward movement of calcium is ultimately controlled by hormones that can promote or decrease calcium release within the myocytes. Enhanced centripetal movement of calcium in atrial myocytes leads to increased contraction and a more substantial contribution to blood pumping. The calcium signalling paradigm within atrial cells applies to other cardiac cell types that also do not express T-tubules, such as neonatal ventricular myocytes, and Purkinje cells that aid in the spread of electrical depolarisation. Furthermore, during heart failure ventricular myocytes progressively lose their regular T-tubule expression, and their pattern of response resembles that of atrial cells.

Abnormal clearance by the mononuclear phagocytic system of immune complexes (IC) is important in the pathogenesis of systemic lupus erythematosus (SLE). We have developed an in vitro model to investigate the cellular mechanisms involved in the transfer of soluble IC from erythrocytes to human macrophages under physiological flow conditions. In this assay, erythrocytes bearing fluorescently labelled IC are perfused over monolayers of human monocytes or monocyte-derived macrophages in a parallel-plate flow chamber, and transfer quantified using confocal microscopy and flow cytometry. Using aggregated human IgG as a model IC, we have been able to demonstrate transfer of IC from erythrocytes to macrophages. Blocking studies with specific neutralizing antibodies have shown that both complement and Fcgamma receptors are required for IC transfer. Blockade of CR4 (alpha(x)beta(2) integrin), FcgammaRIIa or FcgammaRIII reduced transfer, while anti-CR3 (alpha(m)beta(2) integrin) had no effect. Blockade of CR3, FcgammaRIIa or FcgammaRIII also reduced the number of adhesive interactions between fluorescently labelled IC-bearing erythrocytes and macrophage monolayers. Taken together with the transfer data, this suggests differing roles for these receptors in the human IC transfer reaction that includes an adhesive function which facilitates IC processing by mononuclear phagocytes. Finally, a functional effect of the FcgammaRIIa R131/H131 polymorphism, important in susceptibility to SLE, has also been demonstrated using this model. Uptake of IgG(2) but not IgG(1)-containing soluble IC was reduced by macrophages from individuals homozygous for the R131 allelic variant of the receptor.

The p53 protein has a highly evolutionarily conserved role in metazoans as 'guardian of the genome', mediating cell-cycle arrest and apoptosis in response to genotoxic injury. In large, long-lived animals with substantial somatic regenerative capacity, such as vertebrates, p53 is an important tumour suppressor--an attribute thought to stem directly from its induction of death or arrest in mutant cells with damaged or unstable genomes. Chemotherapy and radiation exposure both induce widespread p53-dependent DNA damage. This triggers potentially lethal pathologies that are generally deemed an unfortunate but unavoidable consequence of the role p53 has in tumour suppression. Here we show, using a mouse model in which p53 status can be reversibly switched in vivo between functional and inactive states, that the p53-mediated pathological response to whole-body irradiation, a prototypical genotoxic carcinogen, is irrelevant for suppression of radiation-induced lymphoma. In contrast, delaying the restoration of p53 function until the acute radiation response has subsided abrogates all of the radiation-induced pathology yet preserves much of the protection from lymphoma. Such protection is absolutely dependent on p19(ARF)--a tumour suppressor induced not by DNA damage, but by oncogenic disruption of the cell cycle.