Hebbian analysis of the transformation of medial entorhinal grid-cell inputs to hippocampal place fields.

The discovery of grid cells in the medial entorhinal cortex (MEC) permits the characterization of hippocampal computation in much greater detail than previously possible. The present study addresses how an integrate-and-fire unit driven by grid-cell spike trains may transform the multipeaked, spatial firing pattern of grid cells into the single-peaked activity that is typical of hippocampal place cells. Previous studies have shown that in the absence of network interactions, this transformation can succeed only if the place cell receives inputs from grids with overlapping vertices at the location of the place cell's firing field. In our simulations, the selection of these inputs was accomplished by fast Hebbian plasticity alone. The resulting nonlinear process was acutely sensitive to small input variations. Simulations differing only in the exact spike timing of grid cells produced different field locations for the same place cells. Place fields became concentrated in areas that correlated with the initial trajectory of the animal; the introduction of feedback inhibitory cells reduced this bias. These results suggest distinct roles for plasticity of the perforant path synapses and for competition via feedback inhibition in the formation of place fields in a novel environment. Furthermore, they imply that variability in MEC spiking patterns or in the rat's trajectory is sufficient for generating a distinct population code in a novel environment and suggest that recalling this code in a familiar environment involves additional inputs and/or a different mode of operation of the network.

Dopamine-stimulated dephosphorylation of connexin 36 mediates AII amacrine cell uncoupling.

Gap junction proteins form the substrate for electrical coupling between neurons. These electrical synapses are widespread in the CNS and serve a variety of important functions. In the retina, connexin 36 (Cx36) gap junctions couple AII amacrine cells and are a requisite component of the high-sensitivity rod photoreceptor pathway. AII amacrine cell coupling strength is dynamically regulated by background light intensity, and uncoupling is thought to be mediated by dopamine signaling via D(1)-like receptors. One proposed mechanism for this uncoupling involves dopamine-stimulated phosphorylation of Cx36 at regulatory sites, mediated by protein kinase A. Here we provide evidence against this hypothesis and demonstrate a direct relationship between Cx36 phosphorylation and AII amacrine cell coupling strength. Dopamine receptor-driven uncoupling of the AII network results from protein kinase A activation of protein phosphatase 2A and subsequent dephosphorylation of Cx36. Protein phosphatase 1 activity negatively regulates this pathway. We also find that Cx36 gap junctions can exist in widely different phosphorylation states within a single neuron, implying that coupling is controlled at the level of individual gap junctions by locally assembled signaling complexes. This kind of synapse-by-synapse plasticity allows for precise control of neuronal coupling, as well as cell-type-specific responses dependent on the identity of the signaling complexes assembled.

Long-term injury induced plasticity in {\it Aplysia\/} neurons: Behavioral, electrophysiological, and morphological studies

Nerve injury is known to produce a variety of electrophysiological and morphological neuronal alterations (reviewed by Titmus and Faber, 1990; Bulloch and Ridgeway, 1989; Walters, 1994). Determining if these alterations are adaptive and how they are activated and maintained could provide important insight into basic cellular mechanisms of injury-induced plasticity. Furthermore, characterization of injury-induced plasticity provides a useful assay system for the identification of possible induction signals underlying these neuronal changes. Understanding fundamental mechanisms and underlying induction signals of injury-induced neuronal plasticity could facilitate development of treatment strategies for neural injury and neuropathic pain in humans.^ This dissertation characterizes long-lasting, injury-induced neuronal alterations using the nervous system of Aplysia californica as a model. These changes are examined at the behavioral, electrophysiological, and morphological levels. Injury-induced changes in the electrophysiological properties of neurons were found that increased the signaling effectiveness of the injured neurons. This increase in signalling effectiveness could act to compensate for partial destruction of the injured neuron's peripheral processes. Recovery of a defensive behavioral response which serves to protect the animal from further injury was found within 2 weeks of injury. For the behavioral recovery to occur, new neural pathways must have been formed between the denervated area and the CNS. This was found to be mediated at least in part by new axonal growth which extended from the injured cell back along the original pathway (i.e. into the injured nerve). In addition, injury produced central axonal sprouting into different nerves that do not usually contain the injured neuron's axons. This could be important for (i) finding alternative pathways to the periphery when the original pathways are impassable and (ii) the formation of additional synaptic connections with post-synaptic targets which would further enhance the signalling effectiveness of the injured cell. ^

Signals underlying the induction and expression of long-term, injury-related plasticity in sensory neurons of Aplysia

An important goal in the study of long-term memory is to understand the signals that induce and maintain the underlying neural alterations. In Aplysia, long-term sensitization of defensive reflexes has been examined in depth as a simple model of memory. Extensive studies of sensory neurons (SNs) in Aplysia have led to a cellular and molecular model of long-term memory that has greatly influenced memory research. According to this model, induction of long-term memory in Aplysia depends upon serotonin (5-HT) release and subsequent activation of the cAMP-PKA pathway in SNs. The evidence supporting this model mainly came from studies of long-term synaptic facilitation (LTF) using dissociated (and therefore axotomized) cells growing in culture. However, studies in more intact preparations have produced complex and discrepant results. Because these SNs function as nociceptors, and display similar alterations (long-term hyperexcitability [LTH], LTF, and growth) in models of memory and nerve injury, this study examined the roles of 5-HT and the cAMP-PKA pathway in the induction and expression of long-term, injury-related LTH and LTF in Aplysia SNs. ^ The results presented here suggest that 5-HT is not a primary signal for inducing LTH (and perhaps LTF) in Aplysia SNs. Prolonged treatment with 5-HT failed to induce LTH of Aplysia SNs in either ganglia or dissociated-cell preparations. Treatment with a 5-HT antagonist, methiothepin, during noxious nerve stimulation failed to reduce 24 hr LTH. Furthermore, while 5-HT can induce LTF of SN synapses, this LTF appears to be an indirect effect of 5-HT on other cells. When neural activity was suppressed by elevating divalent cations or by using tetrodotoxin (TTX), 5-HT failed to induce LTF. Unlike LTF, LTH of the SNs could not be produced, even when 5-HT treatment occurred in normal artificial sea water (ASW), suggesting that LTH and LTF are likely to depend on different signals for induction. However, methiothepin reduced the later expression of LTH induced by nerve stimulation, suggesting that 5-HT contributes to the maintenance of LTH in Aplysia SNs.n of somata from the ganglion (which axotomizes SNs) or crushing peripheral n. ^ In summary, this study found that 5-HT and the cAMP-PKA pathway are not involved in the induction of long-term, injury-related LTH of Aplysia SNs, but persistent release of 5-HT and persistent PKA activity contribute to the maintenance of LTH induced by injury. (Abstract shortened by UMI.)^

Increased expression of putative cancer stem cell markers in the bone marrow of prostate cancer patients is associated with bone metastasis progression.

BACKGROUND The number of cells positive for the α-6 and α-2 integrin subunits and the c-Met receptor in primary tumors and bone biopsies from prostate cancer patients has been correlated with metastasis and disease progression. The objective of this study was to quantify disseminated tumour cells present in bone marrow in prostate cancer patients using specific markers and determine their correlation with metastasis and survival. METHODS Patients were included at different stage of prostate cancer disease, from localised to metastatic castration-resistant prostate cancer. Healthy men were used as a control group. Bone marrow samples were collected and nucleated cells separated. These were stained for CD45, α-2, α-6 integrin subunits and c-Met and samples were processed for analysis and quantification of CD45-/α2+/α6+/c-met + cells using flow cytometry. Clinical and pathological parameters were assessed and survival measured. Statistical analyses were made of associations between disease specific parameters, bone marrow flow cytometry data, prostate-specific antigen (PSA) progression free survival and bone metastases progression free survival. RESULTS For all markers, the presence of more than 0.1% positive cells in bone marrow aspirates was significantly associated with the risk of biochemical progression, the risk of developing metastasis and death from prostate cancer. CONCLUSIONS Quantification of cells carrying putative stem cell markers in bone marrow is a potential indicator of disease progression. Functional studies on isolated cells are needed to show more specifically their property for metastatic spread in prostate cancer.

High expression of FOXP3 in primary melanoma is associated with tumour progression.

BACKGROUND The antitumour immune response plays an important role in the prognosis of melanoma. High numbers of circulating regulatory T cells have been associated with rapid disease progression. OBJECTIVES To assess the influence of forkhead box protein (FOXP)3, CD1a and langerin expression on the prognosis of primary melanoma. METHODS We analysed 185 primary melanomas by immunohistochemical staining for expression of the regulatory T-cell marker FOXP3 and the dendritic cell markers langerin and CD1a, and correlated marker expression with clinical outcome. RESULTS Disease-free survival and overall survival were significantly longer in patients expressing low levels of FOXP3 in the primary melanoma, whereas they were associated with high expression of CD1a. The negative prognostic value of FOXP3 expression was independent of the Breslow tumour thickness. Langerin expression did not correlate with the clinical outcome. CONCLUSIONS High expression of FOXP3 in the primary melanoma may be used as an additional independent prognostic marker for early tumour progression in patients with melanoma.

Magnetic resonance imaging features of an extranodal T-cell rich B-cell lymphoma in the pharyngeal mucosa in a horse

An 11-year-old Warmblood gelding was presented for inspiratory stridor and dysphagia. Based on history and clinical examination, a solitary mass localised in the oropharynx was suspected. Due to its inaccessibility and defensive behaviour of the horse, it was difficult to visualise this mass either by upper airway endoscopy or by oral examination and the conventional imaging methods (radiology and ultrasound) provided only limited information. Fine needle aspiration cytology was suggestive of lymphoma, but the exact localisation and the extent of tissue infiltration of the tumour could only be defined by magnetic resonance imaging (MRI). MRI has proved to be a very useful diagnostic tool in equine lameness investigation and, as this case illustrates, it has considerable diagnostic potential for soft tissue examination of the equine head.

Radiotherapy for renal-cell carcinoma

Renal-cell carcinoma is considered to be a radioresistant tumour, but this notion might be wrong. If given in a few (even single) fractions, but at a high fraction dose, stereotactic body radiotherapy becomes increasingly important in the management of renal-cell carcinoma, both in primary settings and in treatment of oligometastatic disease. There is an established biological rationale for the radiosensitivity of renal-cell carcinoma to stereotactic body radiotherapy based on the ceramide pathway, which is activated only when a high dose per fraction is given. Apart from the direct effect of stereotactic body radiotherapy on renal-cell carcinoma, stereotactic body radiotherapy can also induce an abscopal effect. This effect, caused by immunological processes, might be enhanced when targeted drugs and stereotactic body radiotherapy are combined. Therefore, rigorous, prospective randomised trials involving a multidisciplinary scientific panel are needed urgently.

Combination of expression levels of miR-21 and miR-126 is associated with cancer-specific survival in clear-cell renal cell carcinoma

BACKGROUND Renal cell carcinoma (RCC) is marked by high mortality rate. To date, no robust risk stratification by clinical or molecular prognosticators of cancer-specific survival (CSS) has been established for early stages. Transcriptional profiling of small non-coding RNA gene products (miRNAs) seems promising for prognostic stratification. The expression of miR-21 and miR-126 was analysed in a large cohort of RCC patients; a combined risk score (CRS)-model was constructed based on expression levels of both miRNAs. METHODS Expression of miR-21 and miR-126 was evaluated by qRT-PCR in tumour and adjacent non-neoplastic tissue in n = 139 clear cell RCC patients. Relation of miR-21 and miR-126 expression with various clinical parameters was assessed. Parameters were analysed by uni- and multivariate COX regression. A factor derived from the z-score resulting from the COX model was determined for both miRs separately and a combined risk score (CRS) was calculated multiplying the relative expression of miR-21 and miR-126 by this factor. The best fitting COX model was selected by relative goodness-of-fit with the Akaike information criterion (AIC). RESULTS RCC with and without miR-21 up- and miR-126 downregulation differed significantly in synchronous metastatic status and CSS. Upregulation of miR-21 and downregulation of miR-126 were independently prognostic. A combined risk score (CRS) based on the expression of both miRs showed high sensitivity and specificity in predicting CSS and prediction was independent from any other clinico-pathological parameter. Association of CRS with CSS was successfully validated in a testing cohort containing patients with high and low risk for progressive disease. CONCLUSIONS A combined expression level of miR-21 and miR-126 accurately predicted CSS in two independent RCC cohorts and seems feasible for clinical application in assessing prognosis.

Prognostic value of matrix metalloproteinases in oral squamous cell carcinoma

The aim of this study was to investigate whether there is a correlation between the expressions of four matrix metalloproteinases (MMPs): MMP-2, MMP-7, MMP-9 and MMP-13, and the TNM (tumour-node-metastasis) stages of oral squamous cell carcinoma (OSCC); and to explore the implication of these MMPs in OSCC dissemination. Samples from 61 patients diagnosed with oropharyngeal tumour were studied by immunohistochemistry against MMP-2, MMP-7, MMP-9 and MMP-13. The assessment of immunoreactivity was semi-quantitative. The results showed that MMP-2 and MMP-9 had similar expression patterns in the tumour cells with no changes in the immunoreactivity during tumour progression. MMP-9 always had the highest expression, whereas that of MMP-2 was moderate. MMP-7 showed a significant decrease in expression levels during tumour evolution. MMP-13 had constant expression levels within stage T2 and T3, but showed a remarkable decline in immunoreactivity in stage T4. No significant differences in the MMPs immunoreactivity between tumour cells and stroma were observed. Although strong evidence for the application of MMPs as reliable predictive markers for node metastasis was not acquired, we believe that combining patients' MMPs expression intensity and clinical features may improve the diagnosis and prognosis. Strong evidence for the application of MMPs as reliable predictive markers for node metastasis was not acquired. Application of MMPs as prognostic indicators for the malignancy potential of OSCC might be considered in every case of tumour examination. We believe that combining patients' MMPs expression intensity and clinical features may improve the process of making diagnosis and prognosis.

Current developments in the use of stem cell for therapeutic neovascularisation: is the future therapy "cell-free"?

The plasticity and self-regenerative properties of stem cells have opened new avenues in regenerative medicine. Greater understanding of the biology of stem cells is followed by growing expectations of a rapid translation into alternative therapeutic options. Recent preclinical studies and clinical trials employing stem and progenitor cells from different sources have shown encouraging results. However, their underlying mechanisms are still poorly understood, the potential adverse effects and the discrepancy in efficacy remain to be further investigated. Their essential role in vessel regeneration has made endothelial progenitor cells (EPC) a suitable candidate for therapeutic applications aiming at tissue revascularisation. Recent evidence suggests that EPC contribute to neovascularisation not only by direct participation in tissue homeostasis but mainly via paracrine mechanisms. In future, novel therapeutic strategies could be based on EPC paracrine factors or synthetic factors, and replace cell transplantation.

Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer

BACKGROUND The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated. OBJECTIVE To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells. METHODS IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments. RESULTS IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival. CONCLUSIONS Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.

The liver stage of Plasmodium berghei inhibits host cell apoptosis.

Plasmodium berghei is the causative agent of rodent malaria and is widely used as a model system to study the liver stage of Plasmodium parasites. The entry of P. berghei sporozoites into hepatocytes has extensively been studied, but little is known about parasite-host interaction during later developmental stages of the intracellular parasite. Growth of the parasite far beyond the normal size of the host cell is an important stress factor for the infected cell. Cell stress is known to trigger programmed cell death (apoptosis) and we examined several apoptotic markers in P. berghei-infected cells and compared their level of expression and their distribution to that of non-infected cells. As none of the apoptotic markers investigated were found altered in infected cells, we hypothesized that parasite infection might confer resistance to apoptosis of the host cell. Treatment with peroxide or serum deprivation induced apoptosis in non-infected HepG2 cells, whereas P. berghei-infected cells appeared protected, indicating that the parasite interferes indeed with the apoptotic machinery of the host cell. To prove the physiological relevance of these results, mice were infected with high numbers of P. berghei sporozoites and treated with tumour necrosis factor (TNF)-alpha/D-galactosamine to induce massive liver apoptosis. Liver sections of these mice, stained for degraded DNA, confirmed that infected cells containing viable parasites were protected from programmed cell death. However, in non-treated control mice as well as in TNF-alpha-treated mice a small proportion of dead intracellular parasites with degraded DNA were detected. Most hepatocytes containing dead parasites provoked an infiltration of immunocompetent cells, indicating that these cells are no longer protected from cell death.

Cell mixing induced by myc is required for competitive tissue invasion and destruction

Cell-cell intercalation is used in several developmental processes to shape the normal body plan. There is no clear evidence that intercalation is involved in pathologies. Here we use the proto-oncogene myc to study a process analogous to early phase of tumour expansion: myc-induced cell competition. Cell competition is a conserved mechanism driving the elimination of slow-proliferating cells (so-called 'losers') by faster-proliferating neighbours (so-called 'winners') through apoptosis and is important in preventing developmental malformations and maintain tissue fitness. Here we show, using long-term live imaging of myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser-winner interface morphology can modulate the strength of competition. We further show that elimination of loser clones requires winner-loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilization of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference in F-actin levels between loser and winner junctions, induced by differential levels of the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate. Our results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues.

Morphological plasticity of the coral skeleton under CO2-driven seawater acidification

Ocean acidification causes corals to calcify at reduced rates, but current understanding of the underlying processes is limited. Here, we conduct a mechanistic study into how seawater acidification alters skeletal growth of the coral Stylophora pistillata. Reductions in colony calcification rates are manifested as increases in skeletal porosity at lower pH, while linear extension of skeletons remains unchanged. Inspection of the microstructure of skeletons and measurements of pH at the site of calcification indicate that dissolution is not responsible for changes in skeletal porosity. Instead, changes occur by enlargement of corallite-calyxes and thinning of associated skeletal elements, constituting a modification in skeleton architecture. We also detect increases in the organic matrix protein content of skeletons formed under lower pH. Overall, our study reveals that seawater acidification not only causes decreases in calcification, but can also cause morphological change of the coral skeleton to a more porous and potentially fragile phenotype.