CD40 Triggering of Heterodimeric IL-12 p70 Production by Dendritic Cells In Vivo Requires a Microbial Priming Signal

CD40 ligation triggers IL-12 production by dendritic cells (DC) in vitro. Here, we demonstrate that CD40 cross-linking alone is not sufficient to induce IL-12 production by DC in vivo. Indeed, resting DC make neither the IL-12 p35 nor IL-12 p40 subunits and express only low levels of CD40. Nevertheless, after DC activation by microbial stimuli that primarily upregulate IL-12 p40 and augment CD40 expression, CD40 ligation induces a significant increase in IL-12 p35 and IL-12 p70 heterodimer production. Similarly, IL-12 p70 is produced during T cell activation in the presence but not in the absence of microbial stimuli. Thus, production of bioactive IL-12 by DC can be amplified by T cell–derived signals but must be initiated by innate signals.

Myocyte remodeling in response to hypertrophic stimuli requires nucleocytoplasmic shuttling of muscle LIM protein

CSRP3 or muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein and a mechanosensor in cardiac myocytes. MLP regulation and function was studied in cultured neonatal rat myocytes treated with pharmacological or mechanical stimuli. Either verapamil or BDM decreased nuclear MLP while phenylephrine and cyclic strain increased it. These results suggest that myocyte contractility regulates MLP subcellular localization. When RNA polymerase II was inhibited with alpha-amanitin, nuclear MLP was reduced by 30%. However, when both RNA polymerase I and II were inhibited with actinomycin D, there was a 90% decrease in nuclear MLP suggesting that its nuclear translocation is regulated by both nuclear and nucleolar transcriptional activity. Using cell permeable synthetic peptides containing the putative nuclear localization signal (NLS) of MLP, nuclear import of the protein in cultured rat neonatal myocytes was inhibited. The NLS of MLP also localizes to the nucleolus. Inhibition of nuclear translocation prevented the increased protein accumulation in response to phenylephrine. Furthermore, cyclic strain of myocytes after prior NLS treatment to remove nuclear MLP resulted in disarrayed sarcomeres. Increased protein synthesis and brain natriuretic peptide expression were also prevented suggesting that MLP is required for remodeling of the myo filaments and gene expression. These findings suggest that nucleocytoplasmic shuttling MLP plays an important role in the regulation of the myocyte remodeling and hypertrophy and is required for adaptation to hypertrophic stimuli. (C) 2009 Elsevier Inc. All rights reserved.

Self-organisation in the assembly of gels from mixtures of different dendritic peptide building blocks

This paper investigates dendritic peptides capable of assembling into nanostructured gels, and explores the effect on self-assembly of mixing different molecular building blocks. Thermal measurements, small angle Xray scattering (SAXS) and circular dichroism (CD) spectroscopy are used to probe these materials on macroscopic, nanoscopic and molecular length scales. The results from these investigations demonstrate that in this case, systems with different "size" and "chirality" factors can self-organise, whilst systems with different "shape" factors cannot. The "size" and "chirality" factors are directly connected with the molecular information programmed into the dendritic peptides, whilst the shape factor depends on the group linking these peptides together-this is consistent with molecular recognition hydrogen bond pathways between the peptidic building blocks controlling the ability of these systems to self-recognise. These results demonstrate that mixtures of relatively complex peptides, with only subtle differences on the molecular scale, can self-organise into nanoscale structures, an important step in the spontaneous assembly of ordered systems from complex mixtures.

A direct comparison of one- and two-component dendritic self-assembled materials: elucidating molecular recognition pathways

This paper compares and contrasts, for the first time, one- and two-component gelation systems that are direct structural analogues and draws conclusions about the molecular recognition pathways that underpin fibrillar self-assembly. The new one-component systems comprise L-lysine-based dendritic headgroups covalently connected to an aliphatic diamine spacer chain via an amide bond, One-component gelators with different generations of headgroup (from first to third generation) and different length spacer chains are reported. The self-assembly of these dendrimers in toluene was elucidated using thermal measurements, circular dichroism (CD) and NMR spectroscopies, scanning electron microscopy (SEM), and small-angle X-ray scattering (SAXS). The observations are compared with previous results for the analogous two-component gelation system in which the dendritic headgroups are bound to the aliphatic spacer chain noncovalently via acid-amine interactions. The one-component system is inherently a more effective gelator, partly as a consequence of the additional covalent amide groups that provide a new hydrogen bonding molecular recognition pathway, whereas the two-component analogue relies solely on intermolecular hydrogen bond interactions between the chiral dendritic headgroups. Furthermore, because these amide groups are important in the assembly process for the one-component system, the chiral information preset in the dendritic headgroups is not always transcribed into the nanoscale assembly, whereas for the two-component system, fiber formation is always accompanied by chiral ordering because the molecular recognition pathway is completely dependent on hydrogen bond interactions between well-organized chiral dendritic headgroups.

Utilisation of dendritic architectures in molecular recognition and self-assembly processes

This mini-review outlines recent key developments in the use of dendritic architectures in self-assembly processes via utilisation of molecular recognition motifs.

Towards the automatic reconstruction of dendritic trees using particle filters

The 3D reconstruction of a Golgi-stained dendritic tree from a serial stack of images captured with a transmitted light bright-field microscope is investigated. Modifications to the bootstrap filter are discussed such that the tree structure may be estimated recursively as a series of connected segments. The tracking performance of the bootstrap particle filter is compared against Differential Evolution, an evolutionary global optimisation method, both in terms of robustness and accuracy. It is found that the particle filtering approach is significantly more robust and accurate for the data considered.

Methods for comparing and constraining models of dendritic spines

Physiological evidence using Infrared Video Microscopy during the uncaging of glutamate has proven the existence of excitable calcium ion channels in spine heads, highlighting the need for reliable models of spines. In this study we compare the three main methods of simulating excitable spines: Baer & Rinzel's Continuum (B&R) model, Coombes' Spike-Diffuse-Spike (SDS) model and paired cable and ion channel equations (Cable model). Tests are done to determine how well the models approximate each other in terms of speed and heights of travelling waves. Significant quantitative differences are found between the models: travelling waves in the SDS model in particular are found to travel at much lower speeds and sometimes much higher voltages than in the Cable or B&R models. Meanwhile qualitative differences are found between the B&R and SDS models over realistic parameter ranges. The cause of these differences is investigated and potential solutions proposed.

Generation, description and storage of dendritic morphology data

It is generally assumed that the variability of neuronal morphology has an important effect on both the connectivity and the activity of the nervous system, but this effect has not been thoroughly investigated. Neuroanatomical archives represent a crucial tool to explore structure–function relationships in the brain. We are developing computational tools to describe, generate, store and render large sets of three–dimensional neuronal structures in a format that is compact, quantitative, accurate and readily accessible to the neuroscientist. Single–cell neuroanatomy can be characterized quantitatively at several levels. In computer–aided neuronal tracing files, a dendritic tree is described as a series of cylinders, each represented by diameter, spatial coordinates and the connectivity to other cylinders in the tree. This ‘Cartesian’ description constitutes a completely accurate mapping of dendritic morphology but it bears little intuitive information for the neuroscientist. In contrast, a classical neuroanatomical analysis characterizes neuronal dendrites on the basis of the statistical distributions of morphological parameters, e.g. maximum branching order or bifurcation asymmetry. This description is intuitively more accessible, but it only yields information on the collective anatomy of a group of dendrites, i.e. it is not complete enough to provide a precise ‘blueprint’ of the original data. We are adopting a third, intermediate level of description, which consists of the algorithmic generation of neuronal structures within a certain morphological class based on a set of ‘fundamental’, measured parameters. This description is as intuitive as a classical neuroanatomical analysis (parameters have an intuitive interpretation), and as complete as a Cartesian file (the algorithms generate and display complete neurons). The advantages of the algorithmic description of neuronal structure are immense. If an algorithm can measure the values of a handful of parameters from an experimental database and generate virtual neurons whose anatomy is statistically indistinguishable from that of their real counterparts, a great deal of data compression and amplification can be achieved. Data compression results from the quantitative and complete description of thousands of neurons with a handful of statistical distributions of parameters. Data amplification is possible because, from a set of experimental neurons, many more virtual analogues can be generated. This approach could allow one, in principle, to create and store a neuroanatomical database containing data for an entire human brain in a personal computer. We are using two programs, L–NEURON and ARBORVITAE, to investigate systematically the potential of several different algorithms for the generation of virtual neurons. Using these programs, we have generated anatomically plausible virtual neurons for several morphological classes, including guinea pig cerebellar Purkinje cells and cat spinal cord motor neurons. These virtual neurons are stored in an online electronic archive of dendritic morphology. This process highlights the potential and the limitations of the ‘computational neuroanatomy’ strategy for neuroscience databases.

Effects of dendritic morphology on CA3 pyramidal cell electrophysiology: a simulation study

We investigated the effect of morphological differences on neuronal firing behavior within the hippocampal CA3 pyramidal cell family by using three-dimensional reconstructions of dendritic morphology in computational simulations of electrophysiology. In this paper, we report for the first time that differences in dendritic structure within the same morphological class can have a dramatic influence on the firing rate and firing mode (spiking versus bursting and type of bursting). Our method consisted of converting morphological measurements from three-dimensional neuroanatomical data of CA3 pyramidal cells into a computational simulator format. In the simulation, active channels were distributed evenly across the cells so that the electrophysiological differences observed in the neurons would only be due to morphological differences. We found that differences in the size of the dendritic tree of CA3 pyramidal cells had a significant qualitative and quantitative effect on the electrophysiological response. Cells with larger dendritic trees: (1) had a lower burst rate, but a higher spike rate within a burst, (2) had higher thresholds for transitions from quiescent to bursting and from bursting to regular spiking and (3) tended to burst with a plateau. Dendritic tree size alone did not account for all the differences in electrophysiological responses. Differences in apical branching, such as the distribution of branch points and terminations per branch order, appear to effect the duration of a burst. These results highlight the importance of considering the contribution of morphology in electrophysiological and simulation studies.

The role of myeloid receptors on murine plasmacytoid dendritic cells in induction of type I interferon

This study tested the hypothesis that a set of predominantly myeloid restricted receptors (F4/80, CD36, Dectin-1, CD200 receptor and mannan binding lectins) and the broadly expressed CD200 played a role in a key function of plasmacytoid DC (pDC), virally induced type I interferon (IFN) production. The Dectin-1 ligands zymosan, glucan phosphate and the anti-Dectin-1 monoclonal antibody (mAb) 2A11 had no effect on influenza virus induced IFNα/β production by murine splenic pDC. However, mannan, a broad blocking reagent against mannose specific receptors, inhibited IFNα/β production by pDC in response to inactivated influenza virus. Moreover, viral glycoproteins (influenza virus haemagglutinin and HIV-1 gp120) stimulated IFNα/β production by splenocytes in a mannan-inhibitable manner, implicating the function of a lectin in glycoprotein induced IFN production. Lastly, the effect of CD200 on IFN induction was investigated. CD200 knock-out macrophages produced more IFNα than wild-type macrophages in response to polyI:C, a MyD88-independent stimulus, consistent with CD200's known inhibitory effect on myeloid cells. In contrast, blocking CD200 with an anti-CD200 mAb resulted in reduced IFNα production by pDC-containing splenocytes in response to CpG and influenza virus (MyD88-dependent stimuli). This suggests there could be a differential effect of CD200 on MyD88 dependent and independent IFN induction pathways in pDC and macrophages. This study supports the hypothesis that a mannan-inhibitable lectin and CD200 are involved in virally induced type I IFN induction.

Effects of oxidised low density lipoprotein on dendritic cells: a possible immunoregulatory component of the atherogenic micro-environment?

Objective: The objective of this study was to explore the relationship between low density lipoprotein (LDL) and dendritic cell (DC) activation, based upon the hypothesis that reactive oxygen species (ROS)-mediated modification of proteins that may be present in local DC microenvironments could be important as mediators of this activation. Although LDL are known to be oxidised in vivo, and taken up by macrophages during atherogenesis; their effect on DC has not been explored previously. Methods: Human DCs were prepared from peripheral blood monocytes using GM-CSF and IL-4. Plasma LDLs were isolated by sequential gradient centrifugation, oxidised in CuSO4, and oxidation arrested to yield mild, moderate and highly oxidised LDL forms. DCs exposed to these LDLs were investigated using combined phenotypic, functional (autologous T cell activation), morphological and viability assays. Results: Highly-oxidised LDL increased DC HLA-DR, CD40 and CD86 expression, corroborated by increased DC-induced T cell proliferation. Both native and oxidised LDL induced prominent DC clustering. However, high concentrations of highly-oxidised LDL inhibited DC function, due to increased DC apoptosis. Conclusions: This study supports the hypothesis that oxidised LDL are capable of triggering the transition from sentinel to messenger DC. Furthermore, the DC clustering–activation–apoptosis sequence in the presence of different LDL forms is consistent with a regulatory DC role in immunopathogenesis of atheroma. A sequence of initial accumulation of DC, increasing LDL oxidation, and DC-induced T cell activation, may explain why local breach of tolerance can occur. Above a threshold level, however, supervening DC apoptosis limits this, contributing instead to the central plaque core.

What have dendritic cells ever done for adjuvant design? Cellular and molecular methods for the rational development of vaccine adjuvants

Our new molecular understanding of immune priming states that dendritic cell activation is absolutely pivotal for expansion and differentiation of naïve T lymphocytes, and it follows that understanding DC activation is essential to understand and design vaccine adjuvants. This chapter describes how dendritic cells can be used as a core tool to provide detailed quantitative and predictive immunomics information about how adjuvants function. The role of distinct antigen, costimulation, and differentiation signals from activated DC in priming is explained. Four categories of input signals which control DC activation – direct pathogen detection, sensing of injury or cell death, indirect activation via endogenous proinflammatory mediators, and feedback from activated T cells – are compared and contrasted. Practical methods for studying adjuvants using DC are summarised and the importance of DC subset choice, simulating T cell feedback, and use of knockout cells is highlighted. Finally, five case studies are examined that illustrate the benefit of DC activation analysis for understanding vaccine adjuvant function.

Ageing impairs the T cell response to dendritic cells

Dendritic cells (DCs) are critical in priming adaptive T-cell responses, but the effects of ageing on interactions between DCs and T cells are unclear. This study investigated the influence of ageing on the maturation of and cytokine production by human blood-enriched DCs, and the impact on T cell responses in an allogeneic mixed leucocyte reaction (MLR). DCs from old subjects (65-75y) produced significantly less TNF-α and IFN-γ than young subjects (20-30y) in response to lipopolysaccharide (LPS), but expression of maturation markers and co-stimulatory molecules was preserved. In the MLR, DCs from older subjects induced significantly restricted proliferation of young T cells, activation of CD8+ T cells and expression of IL-12 and IFN-γ in T cells compared with young DCs. T cells from older subjects responded more weakly to DC stimulation compared with young T cells, regardless of whether the DCs were derived from young or older subjects. In conclusion, the capacity of DCs to induce T cell activation is significantly impaired by ageing.

Dendritic cells interact with CD4 T cells in intestinal mucosa

Absence of lymph nodes in nonmammalian species, expression of MHCII by APCs in the periphery, and the recent findings that T cells can change their polarization status after presentation in the lymph nodes imply a role for MHCII-mediated presentation outside the organized lymphoid tissue. This study shows that MHCII+ ECs and DCs from the intestinal mucosa of the pig can present antigen to T cells in vitro. In vivo, APCs colocalize with T cells in pig and mouse intestinal mucosa. In the pig, endothelium is involved in these interactions in neonates but not in adults, indicating different roles for stromal and professional APCs in the neonate compared with the adult. The ratio of expression of DQ and DR MHCII locus products was lower on ECs than on other mucosal APCs, indicating that the two types of cells present different peptide sets. Adult nonendothelial APCs expressed a higher ratio of DQ/DR than in neonates. These results suggest that mucosal DCs can present antigen locally to primed T cells and that stromal APCs are recruited to these interactions in some cases. This raises the possibility that local presentation may influence T cell responses at the effector stage after initial presentation in the lymph node.

Modulation of stretch-induced myocyte remodeling and gene expression by nitric oxide: a novel role for lipoma preferred partner in myofibrillogenesis

Prolonged hemodynamic load as a result of hypertension eventually leads to maladaptive cardiac adaptation and heart failure. The signalling pathways that underlie these changes are still poorly understood. The adaptive response to mechanical load is mediated by mechanosensors which convert the mechanical stimuli into a biological response. We examined the effect of cyclic mechanical stretch on myocyte adaptation using neonatal rat ventricular myocytes with 10% (adaptive) or 20% (maladaptive) maximum strain, 1Hz for 48 hours to mimic in vivo mechanical stress. Cells were also treated with and without L-NAME, a general nitric oxide synthase (NOS) inhibitor to suppress NO production. Maladaptive 20% mechanical stretch led to a significant loss of intact sarcomeres which was rescued by LNAME (P<0.05, n≥5 cultures). We hypothesized that the mechanism was through NOinduced alteration of myocyte gene expression. L-NAME up-regulated the mechanosensing proteins Muscle LIM protein (MLP (by 100%, p<0.05, n=4 cultures)) and lipoma preferred partner, a novel cardiac protein (LPP (by 80%, p<0.05, n=4 cultures)). L-NAME also significantly altered the subcellular localisation of LPP and MLP in a manner that favoured growth and adaptation. These findings suggest that NO participates in stretch-mediated adaptation. The use of isoform selective NOS inhibitors indicated a complex interaction between iNOS and nNOS isoforms regulate gene expression. LPP knockdown by siRNA led to formation of α-actinin aggregates and Z-bodies showing that myofibrillogenesis was impaired. There was an up-regulation of E3 ubiquitin ligase (MUL1) by 75% (P<0.05, n=5 cultures). This indicates that NO contributes to stretch-mediated adaptation via the upregulation of proteins associated mechansensing and myofibrillogenesis, thereby presenting potential therapeutic targets during the progression of heart failure. Keywords: Mechanotransduction, heart failure, stretch, heart, hypertrophy