Investigation of a unique short open reading frame within the 3' untranslated region of the canine distemper virus matrix messenger RNA

Increasing evidence suggest that the long "untranslated" region (UTR) between the matrix (M) and the fusion (F) proteins of morbilliviruses has a functional role. In canine distemper virus (CDV), the F 5' UTR was recently shown to code for a long F signal peptide (Fsp). Subsequently, it was reported that the M/F UTRs combined with the long Fsp were synergistically regulating the F mRNA and protein expression, thereby modulating virulence. Unique to CDV, a short putative open reading frame (ORF) has been identified within the wild-type CDV-M 3' UTR (termed M2). Here, we investigated whether M2 was expressed from the genome of the virulent and demyelinating A75/17-CDV strain. An expression plasmid encoding the M2 ORF tagged both at its N-terminal (HA) and C-terminal domains (RFP), was first constructed. Then, a recombinant virus with its putative M2 ORF replaced by HA-M2-RFP was successfully recovered from cDNA (termed recA75/17(green)-HA-M2-RFP). M2 expression in cells transfected or infected with these mutants was studied by immunoprecipitation, immunofluorescence, immunoblot and flow cytometry analyses. Although fluorescence was readily detected in HA-M2-RFP-transfected cells, absence of red fluorescence emission in several recA75/17(green)-HA-M2-RFP-infected cell types suggested lack of M2 biosynthesis, which was confirmed by the other techniques. Consistent with these data, no functional role of the short polypeptide was revealed by infecting various cell types with HA-M2-RFP over-expressing or M2-knockout recombinant viruses. Thus, in sharp contrast to the CDV-F 5' UTR reported to translate a long Fsp, our data provided evidence that the CDV-M 3' UTR does not express any polypeptides.

Cancer classification using the Immunoscore: a worldwide task force

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the 'Immunoscore' into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

Evidence for chemokine-mediated coalescence of preformed flotillin hetero-oligomers in human T-cells

We have shown previously that endogenous flotillin-1 and -2, closely related proteins implicated in scaffolding of membrane microdomains, are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Coexpressed flotillin-1 and -2, but not singly expressed proteins, are also targeted to the uropod of T-cells and neutrophils. Biochemical studies suggest formation of flotillin homo- and hetero-oligomers in other cell types, but so far knowledge is lacking on in situ flotillin organization in leukocytes. We have now analyzed flotillin organization in human T-cells using fluorescence resonance energy transfer (FRET). Coexpressed C-terminally tagged flotillin-1-mCherry and flotillin-2-enhanced green fluorescent protein (EGFP) show significant FRET when analyzed in intact human T-cells in the absence and presence of chemokine. In contrast, little FRET was observed between coexpressed flotillin-1-mCherry and flotillin-1-EGFP before or after chemokine addition, indicating predominant formation of heterodimers and/or -oligomers. Interestingly coexpression of untagged flotillin-2 strongly enhanced FRET between differently tagged flotillin-1 molecules in resting and chemokine-stimulated cells, indicating that close contacts of flotillin-1 molecules only occur in flotillin-2-containing hetero-oligomers. Comparable results were obtained for tagged flotillin-2. We further show that disruption of the actin network, depletion of intracellular calcium, and inhibition of phospholipase C all result in suppression of chemokine-induced polarization and flotillin cap formation, but do not abolish FRET between tagged flotillin-1 and -2. Our results support predominant formation of flotillin-1 and -2 hetero-oligomers in resting and chemokine-stimulated human T-cells which may importantly contribute to structuring of the uropod.

The time window for generation of dendritic spikes by coincidence of action potentials and EPSPs is layer specific in somatosensory cortex

The precise timing of events in the brain has consequences for intracellular processes, synaptic plasticity, integration and network behaviour. Pyramidal neurons, the most widespread excitatory neuron of the neocortex have multiple spike initiation zones, which interact via dendritic and somatic spikes actively propagating in all directions within the dendritic tree. For these neurons, therefore, both the location and timing of synaptic inputs are critical. The time window for which the backpropagating action potential can influence dendritic spike generation has been extensively studied in layer 5 neocortical pyramidal neurons of rat somatosensory cortex. Here, we re-examine this coincidence detection window for pyramidal cell types across the rat somatosensory cortex in layers 2/3, 5 and 6. We find that the time-window for optimal interaction is widest and shifted in layer 5 pyramidal neurons relative to cells in layers 6 and 2/3. Inputs arriving at the same time and locations will therefore differentially affect spike-timing dependent processes in the different classes of pyramidal neurons.

The Emerging Role of TLR and Innate Immunity in Cardiovascular Disease

Cardiovascular disease is a complex disorder involving multiple pathophysiological processes, several of which involve activation of toll-like receptors (TLRs) of the innate immune system. As sentinels of innate immunity TLRs are nonclonally germline-encoded molecular pattern recognition receptors that recognize exogenous as well as tissue-derived molecular dangers signals promoting inflammation. In addition to their expression in immune cells, TLRs are found in other tissues and cell types including cardiomyocytes, endothelial and vascular smooth muscle cells. TLRs are differentially regulated in various cell types by several cardiovascular risk factors such as hypercholesterolemia, hyperlipidemia, and hyperglycemia and may represent a key mechanism linking chronic inflammation, cardiovascular disease progression, and activation of the immune system. Modulation of TLR signaling by specific TLR agonists or antagonists, alone or in combination, may be a useful therapeutic approach to treat various cardiovascular inflammatory conditions such as atherosclerosis, peripheral arterial disease, secondary microvascular complications of diabetes, autoimmune disease, and ischemia reperfusion injury. In this paper we discuss recent developments and current evidence for the role of TLR in cardiovascular disease as well as the therapeutic potential of various compounds on inhibition of TLR-mediated inflammatory responses.

Inhibition of the human epithelial calcium channel TRPV6 by 2-aminoethoxydiphenyl borate (2-APB)

TRPV6, a highly calcium-selective member of the transient receptor potential (TRP) channel superfamily, is a major pathway for calcium absorption in the fetal and adult body. It is expressed abundantly in the duodenum, the placenta and exocrine tissues. TRVP6 was postulated to contribute to store-operated calcium channel (SOC) activity in certain cell types such as exocrine cells. In this study, we tested 2-APB, a widely used SOC inhibitor on human TRPV6 (hTRPV6) activity using fluorescence imaging, patch clamp and radioactive tracer techniques in transiently and stably transfected HEK293 cells. We found that the basal calcium and cadmium influx was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells. 2-APB inhibited hTRPV6 activity in both transient and stably transfected cells. This effect was slightly sensitive toward extracellular calcium. The extracellular sodium concentration did not affect the inhibition of hTRPV6 by 2-APB. However, N-methyl-d-glucamine significantly diminished the inhibitory effect of 2-APB presumably through direct interaction with this compound. Furthermore, 2-APB inhibited the activity of TRPV6 orthologs but not human TRPV5. 2-APB may serve as a parental compound for the development of therapeutic strategies specifically targeting the hTRPV6 calcium channel.

Angiotensin Ii As A General Growth Factor

Angiotensin II (Ang II), a key protein in the renin-angiotensin system, can induce cardiac hypertrophy through an intracrine system as well as affect gene transcription. The receptor to Ang II responsible for this effect, AT1, has been localized to the nucleus of cell types in addition to cardiomyocytes. In this study, we induced expression of Ang II in MC3T3 osteoblasts and K7M2 osteosarcomas and measured changes in protein expression of Annexin V and matrix metalloproteinase 2 (MMP2), proteins identified previously through mass spectrometry analysis as being regulated by Ang II. Annexin V is downregulated in both immortalized murine bone (MC3T3) cells and in cancerous immortalized murine (K7M2) cells induced to express Ang II. MC3T3 cells which express Ang II show a downregulation of MMP2 expression, but Ang II-expressing K7M2 cells show an upregulation of MMP2. The differential regulation of MMP2 between the cancerous cells and noncancerous cells implicates a role for Ang in in tumor metastasis, as MMP2 is a metastatic protein. Annexin V is used as a marker for apoptosis, but nothing is known of the function of the endogenous protein. That Annexin V is potentially regulated by Ang II provides more information with which to characterize the protein and could suggest a function for Annexin V as part of a signal transduction pathway inside of the cell.

Altering the sphingosine-1-phosphate/ceramide balance: a promising approach for tumor therapy

In recent years sphingolipids have emerged as important signaling molecules regulating fundamental cell responses such as cell death and differentiation, proliferation and aspects of inflammation. Especially ceramide has been a main focus of research since it possesses pro-apoptotic capacity in many cell types. A counterplayer of ceramide was found in sphingosine-1-phosphate (S1P), which is generated from ceramide by the consecutive actions of ceramidase and sphingosine kinase. S1P can potently induce cell proliferation via binding to and activation of the Edg family of receptors which have now been renamed as S1P receptors. Obviously, a delicate balance between ceramide and sphingosine-1-phosphate determines whether cells undergo apoptosis or proliferate, two cell responses that are critically involved in tumor development. Directing the balance in favor of ceramide, i.e. by inhibiting ceramidase or sphingosine kinase activities may support the pro-apoptotic action of ceramide and thus may have beneficial effects in cancer therapy. This review will summarize novel insights into the regulation of sphingolipid formation and their potential involvement in tumor development. Finally, we will pinpoint potential new targets for tumor therapy.

Equine peripheral blood-derived progenitors in comparison to bone marrow-derived mesenchymal stem cells

Fibroblast-like cells isolated from peripheral blood of human, canine, guinea pig, and rat have been demonstrated to possess the capacity to differentiate into several mesenchymal lineages. The aim of this work was to investigate the possibility of isolating pluripotent precursor cells from equine peripheral blood and compare them with equine bone marrow-derived mesenchymal stem cells. Human mesenchymal stem cells (MSCs) were used as a control for cell multipotency assessment. Venous blood (n = 33) and bone marrow (n = 5) were obtained from adult horses. Mononuclear cells were obtained by Ficoll gradient centrifugation and cultured in monolayer, and adherent fibroblast-like cells were tested for their differentiation potential. Chondrogenic differentiation was performed in serum-free medium in pellet cultures as a three-dimensional model, whereas osteogenic and adipogenic differentiation were induced in monolayer culture. Evidence for differentiation was made via biochemical, histological, and reverse transcription-polymerase chain reaction evaluations. Fibroblast-like cells were observed on day 10 in 12 out of 33 samples and were allowed to proliferate until confluence. Equine peripheral blood-derived cells had osteogenic and adipogenic differentiation capacities comparable to cells derived from bone marrow. Both cell types showed a limited capacity to produce lipid droplets compared to human MSCs. This result may be due to the assay conditions, which are established for human MSCs from bone marrow and may not be optimal for equine progenitor cells. Bone marrow-derived equine and human MSCs could be induced to develop cartilage, whereas equine peripheral blood progenitors did not show any capacity to produce cartilage at the histological level. In conclusion, equine peripheral blood-derived fibroblast-like cells can differentiate into distinct mesenchymal lineages but have less multipotency than bone marrow-derived MSCs under the conditions used in this study.

Gelatinase B [matrix metalloproteinase (MMP)-9] and collagenases (MMP-8/-13) are upregulated in cerebrospinal fluid during aseptic and bacterial meningitis in children

We investigated the protein expression of gelatinases [matrix metalloproteinase (MMP)-2 and -9] and collagenases (MMP-8 and -13) in cerebrospinal fluid (CSF) from patients with bacterial (BM, n = 17) and aseptic (AM, n = 14) meningitis. In both, MMP-8 and -9 were increased in 100% of patients, whereas MMP-13 was detectable in 53% and 82% respectively. Three patients with clinical signs of meningitis, without CSF pleocytosis, scored positive for all three MMPs. MMP-8 appeared in two isoforms, granulocyte-type [polymorphonuclear cell (PMN)] and fibroblast/macrophage (F/M) MMP-8. Analysis of kinetic changes from serial lumbar punctures showed that these MMPs are independently regulated, and correlate only partly with CSF cytosis or levels of the endogenous inhibitor, tissue inhibitor of matrix metalloproteinase-1. In vitro, T cells, peripheral blood mononuclear cells (PBMCs) and granulocytes (PMN) release MMP-8 and -9, whereas MMP-13 could be found only in the former two cell types. Using models of exogenous (n-formyl-Met-Leu-Phe, T cell receptor cross-linking) and host-derived stimuli (interleukin-2), the kinetics and the release of the MMP-8, -9 and -13 showed strong variation between these immune cells and suggest release from preformed stocks. In addition, MMP-9 is also synthesized de novo in PBMCs and T cells. In conclusion, invading immune cells contribute only partially to MMPs in CSF during meningitis, and parenchymal cells are an equally relevant source. In this context, in patients with clinical signs of meningitis, but without CSF pleocytosis, MMPs seem to be a highly sensitive marker for intrathecal inflammation. The present data support the concept that broad-spectrum enzyme inhibition targeting gelatinases and collagenases is a potential strategy for adjunctive therapy in infectious meningitis.

Response to Innes and Mattsson: Neospora caninum emerges from the shadow of Toxoplasma gondii

It is tempting to extrapolate research findings regarding the intensively studied Toxoplasma gondii to Neospora caninum. This is based on morphological and ultrastructural studies, the molecular phylogeny of both parasites, their wide host ranges in nature, their ability to invade many different cell types in vitro and the occurrence of homologous proteins in both species. However, as Innes and Mattsson point out, T. gondii is the most successful parasite worldwide, whereas N. caninum has a more limited host range. Thus, some of the most challenging questions are: (i) what is T. gondii doing that N. caninum is not doing, or is doing differently, that renders the former so much more successful? And (ii) can some of these features be exploited for the development of interventional tools to limit infection and pathology caused by N. caninum?

Differential regulation of metzincins in experimental chronic renal allograft rejection: potential markers and novel therapeutic targets

Chronic renal allograft rejection is characterized by alterations in the extracellular matrix compartment and in the proliferation of various cell types. These features are controlled, in part by the metzincin superfamily of metallo-endopeptidases, including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM) and meprin. Therefore, we investigated the regulation of metzincins in the established Fisher to Lewis rat kidney transplant model. Studies were performed using frozen homogenates and paraffin sections of rat kidneys at day 0 (healthy controls) and during periods of chronic rejection at day +60 and day +100 following transplantation. The messenger RNA (mRNA) expression was examined by Affymetrix Rat Expression Array 230A GeneChip and by real-time Taqman polymerase chain reaction analyses. Protein expression was studied by zymography, Western blot analyses, and immunohistology. mRNA levels of MMPs (MMP-2/-11/-12/-14), of their inhibitors (tissue inhibitors of metalloproteinase (TIMP)-1/-2), ADAM-17 and transforming growth factor (TGF)-beta1 significantly increased during chronic renal allograft rejection. MMP-2 activity and immunohistological staining were augmented accordingly. The most important mRNA elevation was observed in the case of MMP-12. As expected, Western blot analyses also demonstrated increased production of MMP-12, MMP-14, and TIMP-2 (in the latter two cases as individual proteins and as complexes). In contrast, mRNA levels of MMP-9/-24 and meprin alpha/beta had decreased. Accordingly, MMP-9 protein levels and meprin alpha/beta synthesis and activity were downregulated significantly. Members of metzincin families (MMP, ADAM, and meprin) and of TIMPs are differentially regulated in chronic renal allograft rejection. Thus, an altered pattern of metzincins may represent novel diagnostic markers and possibly may provide novel targets for future therapeutic interventions.

Functional properties of multiple isoforms of human divalent metal-ion transporter 1 (DMT1)

DMT1 (divalent metal-ion transporter 1) is a widely expressed metal-ion transporter that is vital for intestinal iron absorption and iron utilization by most cell types throughout the body, including erythroid precursors. Mutations in DMT1 cause severe microcytic anaemia in animal models. Four DMT1 isoforms that differ in their N- and C-termini arise from mRNA transcripts that vary both at their 5'-ends (starting in exon 1A or exon 1B) and at their 3'-ends giving rise to mRNAs containing (+) or lacking (-) the 3'-IRE (iron-responsive element) and resulting in altered C-terminal coding sequences. To determine whether these variations result in functional differences between isoforms, we explored the functional properties of each isoform using the voltage clamp and radiotracer assays in cRNA-injected Xenopus oocytes. 1A/IRE+-DMT1 mediated Fe2+-evoked currents that were saturable (K(0.5)(Fe) approximately 1-2 microM), temperature-dependent (Q10 approximately 2), H+-dependent (K(0.5)(H) approximately 1 muM) and voltage-dependent. 1A/IRE+-DMT1 exhibited the provisional substrate profile (ranked on currents) Cd2+, Co2+, Fe2+, Mn2+>Ni2+, V3+>>Pb2+. Zn2+ also evoked large currents; however, the zinc-evoked current was accounted for by H+ and Cl- conductances and was not associated with significant Zn2+ transport. 1B/IRE+-DMT1 exhibited the same substrate profile, Fe2+ affinity and dependence on the H+ electrochemical gradient. Each isoform mediated 55Fe2+ uptake and Fe2+-evoked currents at low extracellular pH. Whereas iron transport activity varied markedly between the four isoforms, the activity for each correlated with the density of anti-DMT1 immunostaining in the plasma membrane, and the turnover rate of the Fe2+ transport cycle did not differ between isoforms. Therefore all four isoforms of human DMT1 function as metal-ion transporters of equivalent efficiency. Our results reveal that the N- and C-terminal sequence variations among the DMT1 isoforms do not alter DMT1 functional properties. We therefore propose that these variations serve as tissue-specific signals or cues to direct DMT1 to the appropriate subcellular compartments (e.g. in erythroid cells) or the plasma membrane (e.g. in intestine).

Targeting the conversion of ceramide to sphingosine 1-phosphate as a novel strategy for cancer therapy

Sphingolipids not only function as structural components of cell membranes but also act as signaling molecules to regulate fundamental cellular responses, such as cell death and differentiation, proliferation and certain types of inflammation. Particularly the cellular balance between ceramide and sphingosine 1-phosphate seems to be crucial for a cell's decision to either undergo apoptosis or proliferate, two events which are implicated in tumor development and growth. Whereas ceramide possesses proapoptotic capacity in many cell types, sphingosine 1-phosphate acts as a counterplayer able to induce cell proliferation and protect cells from undergoing apoptosis. Therefore, tipping the balance in favour of ceramide production, i.e. by inhibiting ceramidase or sphingosine kinase activities has potential to support its proapoptotic action and hence represents a promising rational approach to effective cancer therapy. This review highlights most recent data on the regulation of cellular sphingolipid formation and their potential implication in tumor development, and provides perspectives for their use as targets in molecular intervention therapy.

Translocation of particles and inflammatory responses after exposure to fine particles and nanoparticles in an epithelial airway model

ABSTRACT: BACKGROUND: Experimental studies provide evidence that inhaled nanoparticles may translocate over the airspace epithelium and cause increased cellular inflammation. Little is known, however, about the dependence of particle size or material on translocation characteristics, inflammatory response and intracellular localization. RESULTS: Using a triple cell co-culture model of the human airway wall composed of epithelial cells, macrophages and dendritic cells we quantified the entering of fine (1 mum) and nano-sized (0.078 mum) polystyrene particles by laser scanning microscopy. The number distribution of particles within the cell types was significantly different between fine and nano-sized particles suggesting different translocation characteristics. Analysis of the intracellular localization of gold (0.025 mum) and titanium dioxide (0.02-0.03 mum) nanoparticles by energy filtering transmission electron microscopy showed differences in intracellular localization depending on particle composition. Titanium dioxide nanoparticles were detected as single particles without membranes as well as in membrane-bound agglomerations. Gold nanoparticles were found inside the cells as free particles only. The potential of the different particle types (different sizes and different materials) to induce a cellular response was determined by measurements of the tumour necrosis factor-alpha in the supernatants. We measured a 2-3 fold increase of tumour necrosis factor-alpha in the supernatants after applying 1 mum polystyrene particles, gold nanoparticles, but not with polystyrene and titanium dioxide nanoparticles. CONCLUSION: Quantitative laser scanning microscopy provided evidence that the translocation and entering characteristics of particles are size-dependent. Energy filtering transmission electron microscopy showed that the intracellular localization of nanoparticles depends on the particle material. Both particle size and material affect the cellular responses to particle exposure as measured by the generation of tumour necrosis factor-alpha.